Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†
Identifieur interne : 002772 ( Istex/Corpus ); précédent : 002771; suivant : 002773Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†
Auteurs : Dmitrii V. Bugreev ; Svetlana Baranova ; Olga D. Zakharova ; Vincent Parissi ; Cécile Desjobert ; Enzo Sottofattori ; Alessandro Balbi ; Simon Litvak ; Laura Tarrago-Litvak ; Georgy A. NevinskySource :
- Biochemistry [ 0006-2960 ] ; 2003.
Abstract
Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼1.5 orders of magnitude (ΔG° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the kcat term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.
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DOI: 10.1021/bi0300480
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Bugreev</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation></author><author><name sortKey="Baranova, Svetlana" sort="Baranova, Svetlana" uniqKey="Baranova S" first="Svetlana" last="Baranova">Svetlana Baranova</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation></author><author><name sortKey="Zakharova, Olga D" sort="Zakharova, Olga D" uniqKey="Zakharova O" first="Olga D." last="Zakharova">Olga D. Zakharova</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation></author><author><name sortKey="Parissi, Vincent" sort="Parissi, Vincent" uniqKey="Parissi V" first="Vincent" last="Parissi">Vincent Parissi</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation></author><author><name sortKey="Desjobert, Cecile" sort="Desjobert, Cecile" uniqKey="Desjobert C" first="Cécile" last="Desjobert">Cécile Desjobert</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation></author><author><name sortKey="Sottofattori, Enzo" sort="Sottofattori, Enzo" uniqKey="Sottofattori E" first="Enzo" last="Sottofattori">Enzo Sottofattori</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Genoa.</mods:affiliation></affiliation></author><author><name sortKey="Balbi, Alessandro" sort="Balbi, Alessandro" uniqKey="Balbi A" first="Alessandro" last="Balbi">Alessandro Balbi</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Genoa.</mods:affiliation></affiliation></author><author><name sortKey="Litvak, Simon" sort="Litvak, Simon" uniqKey="Litvak S" first="Simon" last="Litvak">Simon Litvak</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation></author><author><name sortKey="Tarrago Litvak, Laura" sort="Tarrago Litvak, Laura" uniqKey="Tarrago Litvak L" first="Laura" last="Tarrago-Litvak">Laura Tarrago-Litvak</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation><affiliation><mods:affiliation> Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</mods:affiliation></affiliation></author><author><name sortKey="Nevinsky, Georgy A" sort="Nevinsky, Georgy A" uniqKey="Nevinsky G" first="Georgy A." last="Nevinsky">Georgy A. Nevinsky</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation><affiliation><mods:affiliation> Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:CA832659F002CBA73B2DAFEEEC4EBAAB31AE80B5</idno><date when="2003" year="2003">2003</date><idno type="doi">10.1021/bi0300480</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002772</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002772</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation
of DNA by Human Immunodeficiency Virus Type 1 Integrase<ref type="bib" target="#bi0300480AF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Bugreev, Dmitrii V" sort="Bugreev, Dmitrii V" uniqKey="Bugreev D" first="Dmitrii V." last="Bugreev">Dmitrii V. Bugreev</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation></author><author><name sortKey="Baranova, Svetlana" sort="Baranova, Svetlana" uniqKey="Baranova S" first="Svetlana" last="Baranova">Svetlana Baranova</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation></author><author><name sortKey="Zakharova, Olga D" sort="Zakharova, Olga D" uniqKey="Zakharova O" first="Olga D." last="Zakharova">Olga D. Zakharova</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation></author><author><name sortKey="Parissi, Vincent" sort="Parissi, Vincent" uniqKey="Parissi V" first="Vincent" last="Parissi">Vincent Parissi</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation></author><author><name sortKey="Desjobert, Cecile" sort="Desjobert, Cecile" uniqKey="Desjobert C" first="Cécile" last="Desjobert">Cécile Desjobert</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation></author><author><name sortKey="Sottofattori, Enzo" sort="Sottofattori, Enzo" uniqKey="Sottofattori E" first="Enzo" last="Sottofattori">Enzo Sottofattori</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Genoa.</mods:affiliation></affiliation></author><author><name sortKey="Balbi, Alessandro" sort="Balbi, Alessandro" uniqKey="Balbi A" first="Alessandro" last="Balbi">Alessandro Balbi</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Genoa.</mods:affiliation></affiliation></author><author><name sortKey="Litvak, Simon" sort="Litvak, Simon" uniqKey="Litvak S" first="Simon" last="Litvak">Simon Litvak</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation></author><author><name sortKey="Tarrago Litvak, Laura" sort="Tarrago Litvak, Laura" uniqKey="Tarrago Litvak L" first="Laura" last="Tarrago-Litvak">Laura Tarrago-Litvak</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</mods:affiliation></affiliation><affiliation><mods:affiliation> Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</mods:affiliation></affiliation></author><author><name sortKey="Nevinsky, Georgy A" sort="Nevinsky, Georgy A" uniqKey="Nevinsky G" first="Georgy A." last="Nevinsky">Georgy A. Nevinsky</name><affiliation><mods:affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</mods:affiliation></affiliation><affiliation><mods:affiliation> Siberian Division of Russian Academy of Sciences.</mods:affiliation></affiliation><affiliation><mods:affiliation> Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2003</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">30</biblScope><biblScope unit="page" from="9235">9235</biblScope><biblScope unit="page" to="9247">9247</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼1.5 orders of magnitude (ΔG° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the kcat term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>integrase</json:string><json:string>odns</json:string><json:string>oligonucleotides</json:string><json:string>viral</json:string><json:string>biochemistry</json:string><json:string>specific odns</json:string><json:string>dinucleotide</json:string><json:string>second strand</json:string><json:string>relative contribution</json:string><json:string>preincubation</json:string><json:string>nonspecific oligonucleotides</json:string><json:string>retroviral</json:string><json:string>catalytically</json:string><json:string>affinity</json:string><json:string>nucleotide</json:string><json:string>duplex</json:string><json:string>reaction mixture</json:string><json:string>bugreev</json:string><json:string>processing reaction</json:string><json:string>deoxyribose</json:string><json:string>biol</json:string><json:string>total affinity</json:string><json:string>mncl2</json:string><json:string>thermodynamic model</json:string><json:string>oligomers</json:string><json:string>complex formation</json:string><json:string>catalysis</json:string><json:string>nucleotide unit</json:string><json:string>virol</json:string><json:string>oligomeric</json:string><json:string>enzyme</json:string><json:string>substrate specificity</json:string><json:string>datum</json:string><json:string>important role</json:string><json:string>complementary strand</json:string><json:string>nonlinear regression analysis</json:string><json:string>same length</json:string><json:string>specific interaction</json:string><json:string>ligand</json:string><json:string>different concentration</json:string><json:string>specific binding</json:string><json:string>high affinity</json:string><json:string>internucleotide phosphate group</json:string><json:string>relative activity</json:string><json:string>independent determination</json:string><json:string>specific oligonucleotides</json:string><json:string>deoxyribose phosphate</json:string><json:string>nonspecific odns</json:string><json:string>weak contact</json:string><json:string>odns corresponding</json:string><json:string>restriction enzyme</json:string><json:string>analogue</json:string><json:string>specific substrate</json:string><json:string>conformational change</json:string><json:string>major contribution</json:string><json:string>experimental procedure</json:string><json:string>enzyme affinity</json:string><json:string>silc approach</json:string><json:string>nonspecific binding</json:string><json:string>specific contact</json:string><json:string>escherichia coli</json:string><json:string>integrase biochemistry</json:string><json:string>individual contact</json:string><json:string>ligand complexity</json:string><json:string>human immunodeficiency virus type</json:string><json:string>siberian division</json:string><json:string>relative hydrophobicity</json:string><json:string>noncleaved strand</json:string><json:string>catalytically active oligomers</json:string><json:string>crystal structure</json:string><json:string>active site</json:string><json:string>hydrogen bond</json:string><json:string>russian academy</json:string><json:string>strand</json:string><json:string>thermodynamic</json:string><json:string>conformation</json:string><json:string>interaction</json:string><json:string>catalytic</json:string><json:string>average result</json:string><json:string>strand transfer</json:string><json:string>energetic basis</json:string><json:string>long terminal</json:string><json:string>reaction product</json:string><json:string>transition state</json:string><json:string>substrate concentration</json:string><json:string>substrate recognition</json:string><json:string>alternative conformation</json:string><json:string>retroviral integrase</json:string><json:string>attachment site</json:string><json:string>structural element</json:string><json:string>stable analogue</json:string><json:string>nonspecific interaction</json:string><json:string>enzyme action</json:string><json:string>optimal conformation</json:string><json:string>mononucleotide unit</json:string><json:string>monotonic increase</json:string><json:string>oligomeric form</json:string><json:string>enzyme interaction</json:string><json:string>other nucleotide</json:string><json:string>enzyme specificity</json:string><json:string>waals interaction</json:string><json:string>sequence specificity</json:string><json:string>flexible structure</json:string><json:string>rigid nonflexible structure</json:string><json:string>ethylated analogue</json:string><json:string>high specificity</json:string><json:string>structural characteristic</json:string><json:string>integrase activity</json:string><json:string>electrostatic interaction</json:string><json:string>free energy</json:string><json:string>integrase catalytic core</json:string><json:string>acid interaction</json:string><json:string>nonspecific contact</json:string><json:string>victor segalen bordeaux</json:string><json:string>enzyme structure</json:string><json:string>different condition</json:string><json:string>much lower</json:string><json:string>first strand</json:string><json:string>better activator</json:string><json:string>linear viral</json:string><json:string>total concentration</json:string><json:string>specific activity</json:string><json:string>relative amount</json:string><json:string>dynamic equilibrium</json:string><json:string>vmax value</json:string><json:string>catalytically active oligomeric form</json:string><json:string>high efficiency</json:string><json:string>preformed catalytically active oligomeric form</json:string><json:string>several factor</json:string><json:string>different mode</json:string><json:string>complementary interaction</json:string><json:string>different contact</json:string><json:string>overall total</json:string><json:string>phosphate backbone</json:string><json:string>fraction composition</json:string><json:string>weak interaction</json:string><json:string>reaction rate</json:string><json:string>integration reaction</json:string><json:string>catalytic step</json:string><json:string>minimal ligand</json:string><json:string>integrase protein</json:string><json:string>complete reaction mixture</json:string></teeft></keywords><author><json:item><name>BUGREEV Dmitrii V.</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>Siberian Division of Russian Academy of Sciences.</json:string></affiliations></json:item><json:item><name>BARANOVA Svetlana</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>Siberian Division of Russian Academy of Sciences.</json:string></affiliations></json:item><json:item><name>ZAKHAROVA Olga D.</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>Siberian Division of Russian Academy of Sciences.</json:string></affiliations></json:item><json:item><name>PARISSI Vincent</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</json:string></affiliations></json:item><json:item><name>DESJOBERT Cécile</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</json:string></affiliations></json:item><json:item><name>SOTTOFATTORI Enzo</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>University of Genoa.</json:string></affiliations></json:item><json:item><name>BALBI Alessandro</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>University of Genoa.</json:string></affiliations></json:item><json:item><name>LITVAK Simon</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</json:string></affiliations></json:item><json:item><name>TARRAGO-LITVAK Laura</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</json:string><json:string>Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</json:string></affiliations></json:item><json:item><name>NEVINSKY Georgy A.</name><affiliations><json:string>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</json:string><json:string>Siberian Division of Russian Academy of Sciences.</json:string><json:string>Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-KP3SDW0G-3</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼1.5 orders of magnitude (ΔG° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the kcat term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.</abstract><qualityIndicators><score>10</score><pdfWordCount>9712</pdfWordCount><pdfCharCount>60113</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>13</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>747</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>277</abstractWordCount><abstractCharCount>1893</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>42</volume><issue>30</issue><pages><first>9235</first><last>9247</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-KP3SDW0G-3</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2003</publicationDate><copyrightDate>2003</copyrightDate><doi><json:string>10.1021/bi0300480</json:string></doi><id>CA832659F002CBA73B2DAFEEEC4EBAAB31AE80B5</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation
of DNA by Human Immunodeficiency Virus Type 1 Integrase<ref type="bib" target="#bi0300480AF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2003 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2003</date><date type="Copyright" when="2003">2003</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation
of DNA by Human Immunodeficiency Virus Type 1 Integrase<ref type="bib" target="#bi0300480AF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Bugreev</surname><forename type="first">Dmitrii V.</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF3"><ref>‡</ref><p>
Siberian Division of Russian Academy of Sciences.</p></note></author><author xml:id="author-0001"><persName><surname>Baranova</surname><forename type="first">Svetlana</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF3"><ref>‡</ref><p>
Siberian Division of Russian Academy of Sciences.</p></note></author><author xml:id="author-0002"><persName><surname>Zakharova</surname><forename type="first">Olga D.</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF3"><ref>‡</ref><p>
Siberian Division of Russian Academy of Sciences.</p></note></author><author xml:id="author-0003"><persName><surname>Parissi</surname><forename type="first">Vincent</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF4"><ref>§</ref><p>
UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</p></note></author><author xml:id="author-0004"><persName><surname>Desjobert</surname><forename type="first">Cécile</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF4"><ref>§</ref><p>
UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</p></note></author><author xml:id="author-0005"><persName><surname>Sottofattori</surname><forename type="first">Enzo</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF5"><ref>‖</ref><p>
University of Genoa.</p></note></author><author xml:id="author-0006"><persName><surname>Balbi</surname><forename type="first">Alessandro</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF5"><ref>‖</ref><p>
University of Genoa.</p></note></author><author xml:id="author-0007"><persName><surname>Litvak</surname><forename type="first">Simon</forename></persName><affiliation><orgName type="institution">Novosibirsk Institute of Bioorganic Chemistry</orgName><orgName type="institution">Siberian Division of Russian Academy of Sciences</orgName><address><addrLine>8 Lavrentieva Avenue</addrLine><addrLine>Novosibirsk 630090</addrLine><country key="RU" xml:lang="en">RUSSIAN FEDERATION</country><addrLine>UMR 5097 CNRS-Université Victor Segalen Bordeaux 2</addrLine><addrLine>146 rue Léo Saignat</addrLine><addrLine>33076 Bordeaux Cedex</addrLine><addrLine>France and IFR 66 Bordeaux</addrLine><country key="FR" xml:lang="en">FRANCE</country><addrLine>and University of Genoa</addrLine><addrLine>Department of Pharmaceutical Sciences</addrLine><addrLine>Viale Benedetto XV</addrLine><addrLine>3</addrLine><addrLine>Genoa-3</addrLine><country key="IT" xml:lang="en">ITALY</country></address></affiliation><note place="foot" n="bi0300480AF4"><ref>§</ref><p>
UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</p></note></author><author xml:id="author-0008" role="corresp"><persName><surname>Tarrago-Litvak</surname><forename type="first">Laura</forename></persName><note place="foot" n="bi0300480AF4"><ref>§</ref><p>
UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</p></note><affiliation role="corresp"> Correspondence should be addressed to the following authors. (G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-5 5757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</affiliation></author><author xml:id="author-0009" role="corresp"><persName><surname>Nevinsky</surname><forename type="first">Georgy A.</forename></persName><note place="foot" n="bi0300480AF3"><ref>‡</ref><p>
Siberian Division of Russian Academy of Sciences.</p></note><affiliation role="corresp"> Correspondence should be addressed to the following authors. (G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-5 5757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</affiliation></author><idno type="istex">CA832659F002CBA73B2DAFEEEC4EBAAB31AE80B5</idno><idno type="ark">ark:/67375/TPS-KP3SDW0G-3</idno><idno type="DOI">10.1021/bi0300480</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2003</date><date type="published">2003</date><biblScope unit="vol">42</biblScope><biblScope unit="issue">30</biblScope><biblScope unit="page" from="9235">9235</biblScope><biblScope unit="page" to="9247">9247</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>Specific interactions between retroviral integrase (IN) and long terminal repeats are required
for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate
specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation
of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The
interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific
oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides
through a superposition of weak contacts with their bases, and more importantly, with the internucleotide
phosphate groups. All these structural components contributed in a combined way to the free energy of
binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the
CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN,
specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands
on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by
incubation with Mn<hi rend="superscript">2+</hi>, the specific oligonucleotides were also able to inhibit the processing reaction. We
found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity
(Δ<hi rend="italic">G</hi>° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds
to ∼1.5 orders of magnitude (Δ<hi rend="italic">G</hi>° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN
and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in
the <hi rend="italic">k</hi><hi rend="subscript">cat</hi> term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific
to specific oligonucleotides.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi0300480</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation
of DNA by Human Immunodeficiency Virus Type 1 Integrase<xref rid="bi0300480AF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Bugreev</surname><given-names>Dmitrii V.</given-names></name><xref rid="bi0300480AF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Baranova</surname><given-names>Svetlana</given-names></name><xref rid="bi0300480AF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Zakharova</surname><given-names>Olga D.</given-names></name><xref rid="bi0300480AF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Parissi</surname><given-names>Vincent</given-names></name><xref rid="bi0300480AF4"><sup>§</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Desjobert</surname><given-names>Cécile</given-names></name><xref rid="bi0300480AF4"><sup>§</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Sottofattori</surname><given-names>Enzo</given-names></name><xref rid="bi0300480AF5"><sup>‖</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Balbi</surname><given-names>Alessandro</given-names></name><xref rid="bi0300480AF5"><sup>‖</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Litvak</surname><given-names>Simon</given-names></name><xref rid="bi0300480AF4"><sup>§</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Tarrago-Litvak</surname><given-names>Laura</given-names></name><xref rid="bi0300480AF1">*</xref><xref rid="bi0300480AF4"><sup>§</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Nevinsky</surname><given-names>Georgy A.</given-names></name><xref rid="bi0300480AF1">*</xref><xref rid="bi0300480AF3"><sup>‡</sup></xref></contrib><aff>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,
Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,
33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of Pharmaceutical
Sciences, Viale Benedetto XV, 3, Genoa-3, Italy
</aff></contrib-group><author-notes><fn id="bi0300480AF3"><label>‡</label><p>
Siberian Division of Russian Academy of Sciences.</p></fn><fn id="bi0300480AF4"><label>§</label><p>
UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</p></fn><fn id="bi0300480AF5"><label>‖</label><p>
University of Genoa.</p></fn><corresp id="bi0300480AF1">
Correspondence should be addressed to the following authors.
(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail:
nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-5
5757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</corresp></author-notes><pub-date pub-type="epub"><day>2</day><month>07</month><year>2003</year></pub-date><pub-date pub-type="ppub"><day>05</day><month>08</month><year>2003</year></pub-date><volume>42</volume><issue>30</issue><fpage>9235</fpage><lpage>9247</lpage><history><date date-type="received"><day>24</day><month>02</month><year>2003</year></date><date date-type="rev-recd"><day>07</day><month>05</month><year>2003</year></date><date date-type="asap"><day>2</day><month>07</month><year>2003</year></date><date date-type="issue-pub"><day>05</day><month>08</month><year>2003</year></date></history><permissions><copyright-statement>Copyright © 2003 American Chemical Society</copyright-statement><copyright-year>2003</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Specific interactions between retroviral integrase (IN) and long terminal repeats are required
for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate
specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation
of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The
interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific
oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides
through a superposition of weak contacts with their bases, and more importantly, with the internucleotide
phosphate groups. All these structural components contributed in a combined way to the free energy of
binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the
CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN,
specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands
on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by
incubation with Mn<sup>2+</sup>, the specific oligonucleotides were also able to inhibit the processing reaction. We
found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity
(Δ<italic toggle="yes">G</italic>° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds
to ∼1.5 orders of magnitude (Δ<italic toggle="yes">G</italic>° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN
and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in
the <italic toggle="yes">k</italic><sub>cat</sub> term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific
to specific oligonucleotides.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi0300480</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi0300480AF2"><label>†</label><p>
This work was supported in part by grants from the Russian
Foundation for Basic Research (01-04-49058), the Siberian Division
of the Russian Academy of Sciences, the French Agence Nationale de
Recherches sur le SIDA (ANRS), the Centre National de la Recherche
Scientifique (CNRS), and the University Victor Segalen Bordeaux 2.
V.P. benefited from a postdoctoral fellowship from SIDACTION.
G.A.N. was supported by a short-fellowship from ANRS.</p></notes></front><body><sec id="d7e251"><title></title><p>Replication of retroviruses depends on the integration of
a double-stranded DNA copy of the retroviral genome into
the host cell nuclear genome (reviewed in ref <italic toggle="yes">1</italic>). The
integration step is catalyzed by the retroviral enzyme
integrase (IN),<xref rid="bi0300480b00001" ref-type="bibr"></xref> whose recognition sequence is located at the
ends of the viral long terminal repeats. This LTR sequence
is critical for site-specific cleavage and integration (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00002" ref-type="bibr"></xref>, <xref rid="bi0300480b00003" ref-type="bibr"></xref></named-content></italic>).
HIV-1 integrase catalyses two reactions to insert both ends
of the proviral DNA into the host cell genome: (i) 3‘-processing, in which the two nucleotides (GT) from the 3‘-ends of linear viral DNA are removed, leaving the CA
dinucleotide at each 3‘-end and (ii) the strand transfer or
joining reaction in which the processed viral DNA ends are
inserted into the host DNA (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00004" ref-type="bibr"></xref>, <xref rid="bi0300480b00005" ref-type="bibr"></xref></named-content></italic>).
</p><p>Substrate recognition by IN is critical for retroviral
integration (reviewed in ref <italic toggle="yes">6</italic> and references therein). To
catalyze cleavage and strand transfer reactions, integrase must
recognize viral DNA ends in a sequence-specific manner and
host target DNA in a sequence-independent manner. The
viral DNA sites for attachment to host DNA are at the termini
and internal edges of the LTRs, yet only the terminal sites
are used as attachment sites for integration. Studies on the
interaction of IN with the DNA substrates have established
that the viral enzyme can distinguish between viral DNA
ends and other oligodeoxynucleotides. The most important
sequence feature in specifying the viral attachment site is a
CA/TG dinucleotide pair, invariably found precisely at the
site of joining to host DNA. The presence of CA immediately
upstream of the cleavage site in the LTR is a highly
conserved feature of all retroviruses. These conserved bases
are crucial for recognition of viral DNA ends by IN. The
site of 3‘-end processing and DNA joining always corresponds to the 3‘-OH of the conserved A. Positions just
internal to the conserved CA also play a major role in
susceptibility to integrase.
</p><p>Viral DNA substrates must be double-stranded to be
processed or DNA joined by integrase. In vitro, IN binds to
substrate DNA with affinities similar to those of nonsubstrate
DNA. The specificity of the reaction may involve the
nonspecific binding of viral DNA first, followed by catalysis
that is achieved in a complex way by the contribution of
several nucleotides, both distal and proximal to the scissile
bond, thus cleaving the CA in a site-specific fashion. Binding
interactions extend inward at least 14−21 base pairs from
the viral DNA end. There is no evidence that sequences
further than 15 base pairs from the ends have any role in
substrate specificity of IN. Therefore, most of the specificity
appears to reside in the terminal base pairs.
</p><p>In contrast to the specificity of integration for viral DNA
ends, no obvious sequences have been defined for the large
number of sites in host chromosomal DNA that can serve
as the target for integration. A model has been proposed for
the functional organization of IN in which viral DNA initially
binds nonspecifically to the C-terminal portion of IN, and
the catalytic central domain has an important role both in
specific recognition of viral DNA ends and in positioning
host DNA for nucleophilic attack (<italic toggle="yes"><xref rid="bi0300480b00007" ref-type="bibr"></xref></italic>). Target DNA recognition in vivo is probably influenced by structural features of
the host DNA, as well as by the viral preintegration complex
and cellular factors.
</p><p>As protein−DNA interactions are involved in many of the
fundamental processes that occur inside cells, it is extremely
important to understand their nature. Many nucleic acid-binding proteins have low or no sequence specificity, whereas
others have extremely high specificity for their special target
sites. It is therefore particularly important to determine the
molecular basis of specificity, which requires characterization
of the conformational properties of the protein (and protein−ligand complex), the DNA target size, and the changes that
ensue as a consequence of the interaction. Any differences
observed in the specific complex need to be compared with
any changes that occur in nonspecific complexes to be able
to determine what constitutes specific binding (refs <italic toggle="yes">8−10</italic>
and references therein).
</p><p>In recent years, significant progress has been made in the
detailed analysis of specific protein−DNA interaction using
X-ray crystallographic techniques. However, X-ray structural
analysis of protein−nucleic acid interactions does not provide
quantitative estimates of the relative importance of molecular
contacts, or of the relative contributions of strong and weak,
or of specific and nonspecific contacts to the total affinity
of an enzyme for DNA. X-ray analyses of sequence-specific
enzymes with DNA have led to the somewhat erroneous
concept that specific contacts, such as pseudo-Watson−Crick
interactions, provide high affinity for specific DNA sequences and that such interactions lead to high specificity
and high efficiency in catalysis. In addition, it is commonly
supposed that enzymes recognizing specific double-stranded
(ds) sequences cannot bind mononucleotides or short single-stranded (ss) oligonucleotides with high efficiency. Even
though in vitro studies of specific binding use short DNA
oligonucleotides, nonspecific binding can significantly contribute to binding equilibria for proteins whose specificity
is not very large. Understanding the role DNA plays in
facilitating the association of DNA-binding proteins is
necessary for understanding how sequence specificity is
accomplished. Therefore, the analysis concerning the quantitative evaluation of the relative individual contributions of
the thermodynamic (complex formation) and kinetic (reaction
rate constant) steps of a catalytic process to the DNA affinity
or to the specificity of DNA-binding proteins is very
important.
</p><p>Different studies have been performed to address the
energetic basis of specificity in DNA−protein complexes.
Experiments using restriction endonucleases showed that
<italic toggle="yes">Bam</italic>HI, <italic toggle="yes">Eco</italic>RI, and <italic toggle="yes">Eco</italic>RV have different context preferences, suggesting that the context affects binding by influencing the free energy levels of the complexes rather than
that of free DNA (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00011" ref-type="bibr"></xref>−<xref rid="bi0300480b00012" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0300480b00013" ref-type="bibr"></xref></named-content></italic>). Jen-Jacobson described how
enzymes that catalyze reactions at specific DNA sites have
overcome the problem of inhibition by excess nonspecific
binding sites on DNA (<italic toggle="yes"><xref rid="bi0300480b00014" ref-type="bibr"></xref></italic>). It has been proposed that the
specific protein−DNA recognition complex bears a close
resemblance to the transition state complex, such that very
tight binding to the recognition site on the DNA substrate
does not inhibit catalysis but instead provides energy that is
efficiently utilized along the path to the transition state. More
recently, the potential contribution of nonspecific binding
to the thermodynamics of specific binding has been analyzed
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00015" ref-type="bibr"></xref>, <xref rid="bi0300480b00016" ref-type="bibr"></xref></named-content></italic>). Studies of the DNA-binding domains of several
nuclear receptors reveal differences in structure and dynamics
upon binding to DNA (<italic toggle="yes"><xref rid="bi0300480b00017" ref-type="bibr"></xref></italic>). As demonstrated recently, the
<italic toggle="yes">lac</italic> repressor interacts with its natural operon through
alternative conformations of its DNA-binding domain, thus
showing a high degree of plasticity in DNA−protein
recognition (<italic toggle="yes"><xref rid="bi0300480b00018" ref-type="bibr"></xref></italic>).
</p><p>In general, site-specific protein−DNA complexes vary
greatly in structural properties and in the thermodynamic
strategy to achieve appropriate binding free energy. Overall,
the primary and spatial structures of proteins, DNA, and
RNA, as well as their conformational changes, possible
plasticity, and additional protein−protein, RNA−RNA, and
DNA−DNA interactions upon complex formation play an
important role in the recognition and transformation of
specific DNA and RNA sequences by enzymes and proteins
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00009" ref-type="bibr"></xref>, <xref rid="bi0300480b00010" ref-type="bibr"></xref></named-content></italic>). However, the factors governing the recognition and
transformation of specific DNA may be different for each
enzyme, thus making it difficult to find a relationship
between them.
</p><p>We have developed new approaches (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00009" ref-type="bibr"></xref>, <xref rid="bi0300480b00010" ref-type="bibr"></xref></named-content></italic>) to evaluate
the relative contributions of individual nucleotides, including
their structural elements, to enzyme affinity for DNA. The
analysis of molecular interactions between enzymes and long
nucleic acids by stepwise increase in ligand complexity
(SILC) has shown that complex formation, including contacts
between an enzyme and specific sequences, cannot alone
provide the basis of either specificity or high affinity for
DNA. Virtually all nucleotides within the DNA-binding cleft
interact with the enzyme. High affinity (5−8 orders of
magnitude) is mainly provided by numerous weak, additive
(or close to additive) interactions between the enzyme and
the various structural elements of nonspecific nucleotides.
For specific DNA, the interaction of its nucleotides with
enzymes may be additive or not and can include different
specific cooperative, anticooperative, or other interactions.
However, in contrast to nonspecific DNA, the relative
contribution of specific interactions to the total affinity is
rather small, not exceeding 1−2 orders of magnitude. Thus,
complex formation cannot alone explain the specificity of
enzyme action. Specificity is provided by the enzyme-dependent DNA adaptation to the optimal conformation and
by catalysis (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00009" ref-type="bibr"></xref>, <xref rid="bi0300480b00010" ref-type="bibr"></xref></named-content></italic>).
</p><p>To better understand the basis for viral DNA-binding
specificity of HIV-1 integrase, we used the SILC approach
here. This method allowed us to probe interactions between
integrase and a series of model substrates (single- or double-stranded, specific or nonspecific oligonucleotides) to characterize quantitatively the structural determinants of substrate
specificity and the mechanism of action of integrase. Results
were then analyzed using a thermodynamic model of specific
DNA recognition.
</p></sec><sec id="d7e343"><title>Experimental Procedures</title><p><italic toggle="yes">Materials.</italic> Reagents were purchased from Merck and
Sigma. Electrophoretically homogeneous HIV-1 integrase
was purified from the JSC 310 protease-deficient yeast
strain transformed with the integrase expression plasmid
pHIV1SF2IN as previously described (<italic toggle="yes"><xref rid="bi0300480b00019" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Oligonucleotides.</italic> Synthesis, purification, and characterization of homo- and hetero-oligonucleotides were performed
as described before (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00020" ref-type="bibr"></xref>, <xref rid="bi0300480b00021" ref-type="bibr"></xref></named-content></italic>), and their concentration was
determined as described by Fasman (<italic toggle="yes"><xref rid="bi0300480b00022" ref-type="bibr"></xref></italic>)<italic toggle="yes">.</italic> The specific ds
DNA substrate (termed ds GT-ODN<sub>21</sub>) used for 3‘-end
processing was prepared by annealing the 21-mer ODN (5‘-GTGTGGAAAATCTCTAGCAGT-3‘) with the 21-mer
complementary strand (5‘-ACTGCTAGAGATTTTCCACAC-3‘): both ODNs were heated for 2 min at 90 °C, followed by slow cooling. The ds ODN was then labeled at
the 3‘-end with [α-<sup>32</sup>P]dGTP and [α-<sup>32</sup>P]TTP in the presence
of the exonuclease-free Klenow fragment of<italic toggle="yes"> Escherichia coli</italic>
DNA polymerase I (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00019" ref-type="bibr"></xref>, <xref rid="bi0300480b00023" ref-type="bibr"></xref></named-content></italic>).
</p><p><italic toggle="yes">Enzyme Activity Assay. </italic>Integrase activity was determined
by measuring the 3‘-end processing reaction at 30 °C. The
standard reaction mixture (20−100 μL) contained 20 mM
HEPES/NaOH (pH 7.5), 10 mM DTT, 0.1 mM EDTA,
4 mM NaCl, 7.5 mM MnCl<sub>2</sub>, 0.05% Nonidet P-40, and 1.5−10 nM ds [<sup>32</sup>P]GT-ODN<sub>21</sub> substrate. The reaction mixture
was incubated for different times (2−60 min) in the presence
of 5−40 nM IN. Products were quantified using two
methods, both giving essentially identical results (<italic toggle="yes"><xref rid="bi0300480b00019" ref-type="bibr"></xref></italic>): (i)
in the first method, the [<sup>32</sup>P]GT product was separated by
15% PAGE in the presence of 7 M urea. Gels were autoradiographed, and gel pieces corresponding to the (<sup>32</sup>P)
cleavage products were measured by Cherenkov counting
and (ii) in the second method, the reactions were stopped
by transferring 10−50 μL aliquots to 40 μL of ice-cold
solution of DNA (2 mg/mL) in 20 mM Tris-HCl containing
100 mM NaCl, and after thorough mixing, 1 mL of cold
8% trichloroacetic acid was added. Solutions were kept on
ice for 3−4 h to allow precipitate formation. The precipitates
were pelleted by centrifugation at 2 °C for 20 min (15 000
rpm), and 1 mL of the supernatants was used to count
radioactivity. The content of [<sup>32</sup>P]GT dinucleotides in the
acid-soluble fraction was the same in both cases and
contained <5% of the radioactivity typically observed after
substrate incubation with IN. The following different controls
were used: (i) the reaction mixture incubated with ds[<sup>32</sup>P]GT-ODN<sub>21</sub> in the absence of IN and (ii) the complete reaction
mixture at zero time of incubation. All measurements were
taken within the linear regions of time courses and enzyme
concentration curves.
</p><p><italic toggle="yes">Effect of Oligonucleotides on the Rate of the 3‘-Processing
Reaction. </italic>To analyze the effect of oligonucleotides on IN
activity, the enzyme was preincubated at 30 °C for 5−60
min in different conditions<italic toggle="yes">.</italic> The standard preincubation
mixture (20−100 μL) contained 20 mM HEPES/NaOH (pH
7.5), 10 mM DTT, 0.1 mM EDTA, 50−100 mM NaCl, 2
mM CHAPS, 3% glycerol, 0.05% Nonidet P-40, 40−50 mM
MnCl<sub>2</sub>, 10 nM to 30 μM integrase, and different concentrations of oligonucleotides. At different time intervals, aliquots
(5−10 μL) of the preincubated mixture were diluted with
buffer (20 mM HEPES/NaOH, pH 7.5, 10 mM DTT, 0.1
mM EDTA, 4 mM NaCl) and added to the 3‘-processing
reaction mixture (final concentration of IN 5−10 nM), and
the reaction was performed as described above.
</p><p><italic toggle="yes">Kinetic Parameters.</italic> Initial rates were measured in kinetic
experiments. The <italic toggle="yes">K</italic><sub>M</sub> and <italic toggle="yes">V</italic><sub>max</sub> values were estimated using
nonlinear regression analysis (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00024" ref-type="bibr"></xref>, <xref rid="bi0300480b00025" ref-type="bibr"></xref></named-content></italic>). The type of inhibition
and the <italic toggle="yes">K</italic><sub>i</sub> values were determined by nonlinear regression
analysis and presented as linear transformations using the
Lineweaver−Burk plot. Values of IC<sub>50</sub> were determined at
a substrate concentration equal to 2<italic toggle="yes">K</italic><sub>M</sub>. For competitive
inhibition (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00024" ref-type="bibr"></xref>, <xref rid="bi0300480b00025" ref-type="bibr"></xref></named-content></italic>), IC<sub>50</sub> = 3<italic toggle="yes">K</italic><sub>i</sub> when [S]= 2<italic toggle="yes">K</italic><sub>M</sub>. Errors in
IC<sub>50</sub> and <italic toggle="yes">K</italic><sub>i</sub><italic toggle="yes"></italic>were within 10−30%.
</p><p><italic toggle="yes">Small-Angle X-ray Scattering.</italic> The effect of oligonucleotides on the oligomerization of integrase was analyzed by
preincubating the enzyme in 40 μL of a mixture containing
20 mM HEPES/NaOH pH 7.5, 10 mM DTT, 0.1 mM EDTA,
100 mM NaCl, 2 mM CHAPS, 3% glycerol, 0.05% Nonidet
P-40, and 100 μM specific or nonspecific pentanucleotides.
SAXS roentgenograms were obtained using a Siemens
diffractometer (Germany) by the method of step-by-step
scanning with a goniometer and X-ray scintillation detector
as described earlier (<italic toggle="yes"><xref rid="bi0300480b00026" ref-type="bibr"></xref></italic>). The determination of the fraction
composition using small-angle roentgenograms was performed as described previously (<italic toggle="yes"><xref rid="bi0300480b00026" ref-type="bibr"></xref></italic>).
</p></sec><sec id="d7e500"><title>Results and Discussion</title><p><italic toggle="yes">Complex</italic> <italic toggle="yes">Formation</italic> <italic toggle="yes">of</italic> <italic toggle="yes">HIV</italic>-<italic toggle="yes">1</italic> <italic toggle="yes">Integrase</italic> <italic toggle="yes">with</italic> <italic toggle="yes">Oligonucleotides</italic>. The formation of the IN·DNA complex was analyzed
using the SILC approach, according to the following scheme:
orthophosphate or mononucleotide (as minimal ligands of
IN) → ss nonspecific homo-d(N)<italic toggle="yes"><sub>n</sub></italic> → ss specific hetero-d(N)<italic toggle="yes"><sub>n</sub></italic>
→ ds nonspecific homo-d(N)<italic toggle="yes"><sub>n</sub></italic> → ds specific hetero-d(N)<italic toggle="yes"><sub>n</sub></italic><italic toggle="yes">.</italic></p><p>We have previously shown that IN can bind different
oligonucleotides: single- or double-stranded molecules of
different lengths and with sequences, either related (specific)
or unrelated (nonspecific) to the specific 21-mer substrate
(<italic toggle="yes"><xref rid="bi0300480b00019" ref-type="bibr"></xref></italic>). Here, we extended these results to other oligonucleotides.
</p><p>The 3‘-end processing catalyzed by HIV-1 integrase was
assayed with double-stranded GT-ODN<sub>21</sub>, the specific DNA
substrate derived from the HIV-1 U5 end of the LTR:
<fig id="bi0300480f1" position="float" orientation="portrait"><label></label><graphic xlink:href="bi0300480f1.tif" position="float" orientation="portrait"></graphic></fig></p><p>Kinetic analysis was performed by measuring the sequence-specific removal of [<sup>32</sup>P]GT from the 3‘-end (Figure <xref rid="bi0300480f00001"></xref>A,B).
A<italic toggle="yes"> K</italic><sub>M</sub> of 1.3 ± 0.3 nM was determined for the specific [<sup>32</sup>P]GT-ODN<sub>21</sub>. All nonspecific ONs inhibited the IN reaction
competitively toward the substrate as illustrated for oligo(A)<sub>10</sub> in Figure <xref rid="bi0300480f00001"></xref>C. We showed that even short nonspecific
ribo-ONs or orthophosphate competed with the ds GT-ODN<sub>21</sub>
substrate for the enzyme. The corresponding inhibition values
for these oligonucleotides are reported in Table <xref rid="bi0300480t00001"></xref>. Since
substrate and inhibitors are competitive, the <italic toggle="yes">K</italic><sub>i</sub> value gives
an estimate of the binding affinity (<italic toggle="yes">K</italic><sub>d</sub><italic toggle="yes"> = K</italic><sub>i</sub>) of IN for
oligonucleotides. As most of the short oligonucleotides had
relatively low affinities for IN, <italic toggle="yes">K</italic><sub>i</sub> values were calculated
from their IC<sub>50</sub>. Under the conditions used, in which the
concentration of the ds GT-ODN<sub>21</sub> substrate was equal to
2<italic toggle="yes">K</italic><sub>M</sub>, the IC<sub>50</sub> corresponded to 3<italic toggle="yes">K</italic><sub>i</sub>. This was confirmed
experimentally with various ss and ds oligonucleotides. Thus,
in most cases, the IC<sub>50</sub> values were used to calculate <italic toggle="yes">K</italic><sub>i</sub> values
(Table <xref rid="bi0300480t00001"></xref>).
<fig id="bi0300480f00001" position="float" orientation="portrait"><label>1</label><caption><p>(A) Inhibition of the IN-catalyzed processing reaction by ribo-(A)<sub>10</sub>. Integrase was incubated under the reaction conditions
described in the Experimental Procedures, with the specific 21-mer duplex DNA substrate containing [<sup>32</sup>P]-labeled GT nucleotides
in the 3‘-end. The reaction products were analyzed by 15%
polyacrylamide−7 M urea gel electrophoresis. Complete reaction
mixtures containing the labeled substrate were incubated in the
absence of integrase (lane S) or in the presence of IN (lane IN).
Lanes 1−6: same as lane IN but in the presence of 20, 50, 120,
150, 200, and 300 μM oligo r(A)<sub>10</sub>. Arrows indicate the 21-mer
and the dinucleotide product (2-mer). (B) Integrase activity.
Reaction products were determined by measuring the TCA nonprecipitable radioactivity corresponding to [<sup>32</sup>P]GT remaining after
IN-dependent processing. Results are expressed as relative activity,
100% corresponding to IN activity in the absence of inhibitor. (C)
Double reciprocal plot according to the Lineweaver−Burk representation. The 3‘-processing reaction was performed varying the
GT-specific double-stranded [<sup>32</sup>P]ODN<sub>21</sub> substrate concentrations
as indicated. Different concentrations of ribo-(A)<sub>10</sub> were added: 0
(·), 18 (□), 40 (▪), and 88 μM (○). Values represent the average
of at least two independent determinations. Errors were within 10−30%.
</p></caption><graphic xlink:href="bi0300480f00001.tif" position="float" orientation="portrait"></graphic></fig><table-wrap id="bi0300480t00001" position="float" orientation="portrait"><label>1</label><caption><p>Affinity of HIV-1 Integrase for Nonspecific Oligonucleotides and Its Constituents<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">(A) Nonspecific single-stranded oligonucleotides</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="8"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:colspec colnum="8" colname="8"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">d(T)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">K</italic><sub>i</sub>
(μM)</oasis:entry><oasis:entry namest="3" nameend="3">d(C)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">K</italic><sub>i</sub>
(μM)</oasis:entry><oasis:entry namest="5" nameend="5">r(U)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="6" nameend="6"><italic toggle="yes">K</italic><sub>i</sub>
(μM)</oasis:entry><oasis:entry namest="7" nameend="7">r(C)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="8" nameend="8"><italic toggle="yes">K</italic><sub>i</sub>
(μM)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T-base
</oasis:entry><oasis:entry colname="2">>500000
</oasis:entry><oasis:entry colname="3">C-base
</oasis:entry><oasis:entry colname="4">>500000
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">dTMP
</oasis:entry><oasis:entry colname="2">767
</oasis:entry><oasis:entry colname="3">dCMP
</oasis:entry><oasis:entry colname="4">15000
</oasis:entry><oasis:entry colname="5">UMP
</oasis:entry><oasis:entry colname="6">4000
</oasis:entry><oasis:entry colname="7">CMP
</oasis:entry><oasis:entry colname="8">4000
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>2</sub></oasis:entry><oasis:entry colname="2">330
</oasis:entry><oasis:entry colname="3">C<sub>2</sub></oasis:entry><oasis:entry colname="4">300
</oasis:entry><oasis:entry colname="5">U<sub>2</sub></oasis:entry><oasis:entry colname="6">2600
</oasis:entry><oasis:entry colname="7">C<sub>2</sub></oasis:entry><oasis:entry colname="8">1500
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>3</sub></oasis:entry><oasis:entry colname="2">166
</oasis:entry><oasis:entry colname="3">C<sub>3</sub></oasis:entry><oasis:entry colname="4">100
</oasis:entry><oasis:entry colname="5">U<sub>3</sub></oasis:entry><oasis:entry colname="6">2000
</oasis:entry><oasis:entry colname="7">C<sub>3</sub></oasis:entry><oasis:entry colname="8">650
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>4</sub></oasis:entry><oasis:entry colname="2">73
</oasis:entry><oasis:entry colname="3">C<sub>4</sub></oasis:entry><oasis:entry colname="4">50
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>5</sub></oasis:entry><oasis:entry colname="2">43
</oasis:entry><oasis:entry colname="3">C<sub>5</sub></oasis:entry><oasis:entry colname="4">32
</oasis:entry><oasis:entry colname="5">U<sub>5</sub></oasis:entry><oasis:entry colname="6"><bold>150</bold></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>6</sub></oasis:entry><oasis:entry colname="2">33
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">U<sub>6</sub></oasis:entry><oasis:entry colname="6">100
</oasis:entry><oasis:entry colname="7">C<sub>6</sub></oasis:entry><oasis:entry colname="8">100
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>7</sub></oasis:entry><oasis:entry colname="2">26
</oasis:entry><oasis:entry colname="3">C<sub>7</sub></oasis:entry><oasis:entry colname="4">10
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>10</sub></oasis:entry><oasis:entry colname="2"><bold>8.3</bold></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">U<sub>10</sub></oasis:entry><oasis:entry colname="6">30
</oasis:entry><oasis:entry colname="7">C<sub>10</sub></oasis:entry><oasis:entry colname="8">40
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">C<sub>16</sub></oasis:entry><oasis:entry colname="4">0.100
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">T<sub>21</sub></oasis:entry><oasis:entry colname="2"><bold>1.0</bold></oasis:entry><oasis:entry colname="3">C<sub>21</sub></oasis:entry><oasis:entry colname="4">0.012
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">(B) Nonspecific single-stranded oligonucleotides and Pi<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="8"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:colspec colnum="7" colname="7"></oasis:colspec><oasis:colspec colnum="8" colname="8"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">d(A)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">K</italic><sub>i</sub>
(μM)</oasis:entry><oasis:entry namest="3" nameend="3">r(A)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">K</italic><sub>i</sub>
(μM)</oasis:entry><oasis:entry namest="5" nameend="5">d(pR)<italic toggle="yes"><sub>n</sub></italic>(T)<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry><oasis:entry namest="6" nameend="6"><italic toggle="yes">K</italic><sub>i</sub>
(μM)</oasis:entry><oasis:entry namest="7" nameend="7">compound<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry namest="8" nameend="8"><italic toggle="yes">K</italic><sub>i</sub>
(μM)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A-base
</oasis:entry><oasis:entry colname="2">>500000
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"><sc>d</sc>-ribose
</oasis:entry><oasis:entry colname="6">>500000
</oasis:entry><oasis:entry colname="7">Pi<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="8">33000
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">dAMP
</oasis:entry><oasis:entry colname="2">5000
</oasis:entry><oasis:entry colname="3">AMP
</oasis:entry><oasis:entry colname="4">7100
</oasis:entry><oasis:entry colname="5">d(pR)
</oasis:entry><oasis:entry colname="6">15000
</oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>2</sub></oasis:entry><oasis:entry colname="2">80
</oasis:entry><oasis:entry colname="3">A<sub>2</sub></oasis:entry><oasis:entry colname="4">830
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>3</sub></oasis:entry><oasis:entry colname="2">48
</oasis:entry><oasis:entry colname="3">A<sub>3</sub></oasis:entry><oasis:entry colname="4">310
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>4</sub></oasis:entry><oasis:entry colname="2">3.5
</oasis:entry><oasis:entry colname="3">A<sub>4</sub></oasis:entry><oasis:entry colname="4">70
</oasis:entry><oasis:entry colname="5">d(pR)<sub>3</sub>(T)
</oasis:entry><oasis:entry colname="6">100
</oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>6</sub></oasis:entry><oasis:entry colname="2">1.2
</oasis:entry><oasis:entry colname="3">A<sub>6</sub></oasis:entry><oasis:entry colname="4">55
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>8</sub></oasis:entry><oasis:entry colname="2">1.0
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">d(pR)<sub>7</sub>(T)
</oasis:entry><oasis:entry colname="6">60
</oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>10</sub></oasis:entry><oasis:entry colname="2"><bold>0.52</bold></oasis:entry><oasis:entry colname="3">A<sub>10</sub></oasis:entry><oasis:entry colname="4"><bold>40</bold></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>12</sub></oasis:entry><oasis:entry colname="2">0.40
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>14</sub></oasis:entry><oasis:entry colname="2">0.30
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5">d(pR)<sub>13</sub>(T)
</oasis:entry><oasis:entry colname="6">15
</oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">A<sub>18</sub></oasis:entry><oasis:entry colname="4">4
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">A<sub>21</sub></oasis:entry><oasis:entry colname="2"><bold>0.016</bold></oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">A<sub>24</sub></oasis:entry><oasis:entry colname="4">2
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7"></oasis:entry><oasis:entry colname="8"></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">(C) Nonspecific double-stranded deoxy-oligonucleotides</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">no of units
(<italic toggle="yes">n</italic>)</oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">K</italic><sub>i</sub>: d(T)<italic toggle="yes"><sub>n</sub></italic>·d(A)<italic toggle="yes"><sub>n</sub></italic>
(μM)</oasis:entry><oasis:entry namest="3" nameend="3">ratio: <italic toggle="yes">K</italic><sub>i </sub>(ss)/<italic toggle="yes">K</italic><sub>i</sub>(ds)
ss: d(T)<italic toggle="yes"><sub>n</sub></italic>
ds: d(T)<italic toggle="yes"><sub>n</sub></italic>·d(A)<italic toggle="yes"><sub>n</sub></italic></oasis:entry><oasis:entry namest="4" nameend="4">ratio: <italic toggle="yes">K</italic><sub>i</sub>(ss)/<italic toggle="yes">K</italic><sub>i</sub> (ds)
ss: d(A)<italic toggle="yes"><sub>n</sub></italic>
ds: d(T)<italic toggle="yes"><sub>n</sub></italic>·d(A)<italic toggle="yes"><sub>n</sub></italic></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">3
</oasis:entry><oasis:entry colname="2">45
</oasis:entry><oasis:entry colname="3">3.6
</oasis:entry><oasis:entry colname="4">1.06
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">4
</oasis:entry><oasis:entry colname="2">3.2
</oasis:entry><oasis:entry colname="3">23
</oasis:entry><oasis:entry colname="4">1.1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">6
</oasis:entry><oasis:entry colname="2">0.600
</oasis:entry><oasis:entry colname="3">55
</oasis:entry><oasis:entry colname="4">2.0
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">8
</oasis:entry><oasis:entry colname="2">0.280
</oasis:entry><oasis:entry colname="3">57
</oasis:entry><oasis:entry colname="4">3.6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">10
</oasis:entry><oasis:entry colname="2"><bold>0.150</bold></oasis:entry><oasis:entry colname="3">55
</oasis:entry><oasis:entry colname="4">3.4
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">12
</oasis:entry><oasis:entry colname="2">0.078
</oasis:entry><oasis:entry colname="3">70
</oasis:entry><oasis:entry colname="4">5.1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">14
</oasis:entry><oasis:entry colname="2">0.054
</oasis:entry><oasis:entry colname="3">83
</oasis:entry><oasis:entry colname="4">5.6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">21
</oasis:entry><oasis:entry colname="2"><bold>0.040</bold></oasis:entry><oasis:entry colname="3">25
</oasis:entry><oasis:entry colname="4">4.0</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1">(D) Nonspecific double-stranded deoxy- and ribo-oligonucleotides</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup><oasis:tgroup cols="5"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">ss ON</oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">K</italic><sub>i </sub>(ss)
(μM)</oasis:entry><oasis:entry namest="3" nameend="3">ds ON</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">K</italic><sub>i </sub>(ds)
(μM)</oasis:entry><oasis:entry namest="5" nameend="5">ratio: <italic toggle="yes">K</italic><sub>i </sub>(ss)/<italic toggle="yes">K</italic><sub>i</sub> (ds)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">dT<sub>10</sub></oasis:entry><oasis:entry colname="2"><bold>8.3</bold></oasis:entry><oasis:entry colname="3">d(T)<sub>10</sub>·r(A)<sub>10</sub></oasis:entry><oasis:entry colname="4">1
</oasis:entry><oasis:entry colname="5">8.3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">rU<sub>10</sub></oasis:entry><oasis:entry colname="2">30
</oasis:entry><oasis:entry colname="3">(rU)<sub>10</sub>·d(A)<sub>10</sub></oasis:entry><oasis:entry colname="4">0.26
</oasis:entry><oasis:entry colname="5">115
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">rU<sub>10</sub></oasis:entry><oasis:entry colname="2">30
</oasis:entry><oasis:entry colname="3">(rU)<sub>10</sub>·r(A)<sub>10</sub></oasis:entry><oasis:entry colname="4">5
</oasis:entry><oasis:entry colname="5">6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">rA<sub>10</sub></oasis:entry><oasis:entry colname="2"><bold>40</bold></oasis:entry><oasis:entry colname="3">r(A)<sub>10</sub>·d(T)<sub>10</sub></oasis:entry><oasis:entry colname="4">1
</oasis:entry><oasis:entry colname="5">40
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">dA<sub>10</sub></oasis:entry><oasis:entry colname="2"><bold>0.52</bold></oasis:entry><oasis:entry colname="3">d(A)<sub>10</sub>·r(U)<sub>10</sub></oasis:entry><oasis:entry colname="4">0.26
</oasis:entry><oasis:entry colname="5">2
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">rA<sub>10</sub></oasis:entry><oasis:entry colname="2"><bold>40</bold></oasis:entry><oasis:entry colname="3">r(A)<sub>10</sub>·r(U)<sub>10</sub></oasis:entry><oasis:entry colname="4">5
</oasis:entry><oasis:entry colname="5">8</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Values are the average results of two to three independent
experiments. Errors in <italic toggle="yes">K</italic><sub>i</sub> and IC<sub>50</sub> values were within 10−20%. <italic toggle="yes">K</italic><sub>i</sub>
values (shown in bold) were determined by nonlinear regression analysis
or calculated using the IC<sub>50</sub> values (lightface) as described in the
Experimental Procedures.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Pi corresponds to orthophosphate.<italic toggle="yes"><sup>c</sup></italic><sup></sup> The
d(pR)<italic toggle="yes"><sub>n</sub></italic> series are deoxyribose phosphates in which R is a chemically
stable analogue of deoxyribose (a tetrahydrofuran derivative).</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Interaction of HIV-1 Integrase with Nonspecific ss Oligonucleotide.</italic> Results in Table <xref rid="bi0300480t00001"></xref>A,B show that while <italic toggle="yes">K</italic><sub>i</sub><italic toggle="yes"></italic>values for Pi and dNMPs were comparable, the affinity for
deoxyribose and for all bases was very low (<italic toggle="yes">K</italic><sub>i</sub><italic toggle="yes"></italic>> 500 mM).
We can thus conclude that even if IN recognizes free dNMPs
through interactions with all its structural elements (base,
sugar, and phosphate), the phosphate group makes the major
contribution.
</p><p>The Gibbs' free energy characterizing enzyme−ligand
complex formation can be presented as the sum of Δ<italic toggle="yes">G</italic>°
values for the individual contacts:<xref rid="bi0300480e00001"></xref><disp-formula content-type="pre-labeled" id="bi0300480e00001"><!--%@md;sys;6q@%&Dgr;%@ital@%G%@rsf@%° = &Dgr;%@ital@%G%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth$-$x55)@%%@mh;$-$x63@%%@sb@%1%@sbx@% + &Dgr;%@ital@%G%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth$-$x55)@%%@mh;$-$x63@%%@sb@%2%@sbx@%+ ... + &Dgr;%@ital@%G%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth$-$x55)@%%@mh;$-$x63@%%@ital@%%@sb@%n%@rsf@%%@sbx@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi0300480e00001.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
with<xref rid="bi0300480e00002"></xref><disp-formula content-type="pre-labeled" id="bi0300480e00002"><!--%@md;sys;6q@%&Dgr;%@ital@%G%@rsf@%%@/xs;55;%lnwidth@%°%@/xs;63;(%lnwidth$-$x55)@%%@mh;$-$x63@%%@sb@%i%@sbx@%%@ex@% %@exx@%= −%@ital@%RT%@rsf@% ln %@ital@%K%@rsf@%%@sx@%d%@be@%i%@sxx@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi0300480e00002.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
where<inline-formula><!--%@mt;sys@%%@ital@%K%@rsf@%%@sx@%d%@be@%i%@sxx@%%@mx@%
--><inline-graphic xlink:href="bi0300480e10001.gif"></inline-graphic></inline-formula>
indicates the contribution of the individual contact (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00024" ref-type="bibr"></xref>, <xref rid="bi0300480b00025" ref-type="bibr"></xref></named-content></italic>). Hence, the overall <italic toggle="yes">K</italic><sub>d</sub> value characterizing
complex formation is the product of the <italic toggle="yes">K</italic><sub>d</sub> values for
individual contacts:<xref rid="bi0300480e00003"></xref><disp-formula content-type="pre-labeled" id="bi0300480e00003"><!--%@md;sys;6q@%&Dgr;%@ital@%G%@rsf@%° = −%@ital@%RT%@rsf@% ln %@ital@%K%@rsf@%%@sb@%d%@sbx@% = −%@ital@%RT%@rsf@% ln%@fn;[;vis;full;auto@%%@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;(;vis;full;unlock@%1%@fnx;);vis;full@%%@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;(;vis;full;unlock@%2%@fnx;);vis;full@% ... %@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;(;vis;full;unlock@%%@ital@%n%@rsf@%%@fnx;);vis;full@%%@fnx;];vis;full@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi0300480e00003.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
and<xref rid="bi0300480e00004"></xref><disp-formula content-type="pre-labeled" id="bi0300480e00004"><!--%@md;sys;6q@%%@ital@%K%@rsf@%%@sb@%d%@ital@% %@rsf@%%@sbx@%= %@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;(;vis;full;auto@%1%@fnx;);vis;full@%%@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;(;vis;full;auto@%2%@fnx;);vis;full@% ... %@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;(;vis;full;auto@%%@ital@%n%@rsf@%%@fnx;);vis;full@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi0300480e00004.gif" position="anchor" orientation="portrait"></graphic></disp-formula><fig id="bi0300480f00002" position="float" orientation="portrait"><label>2</label><caption><p>Dependencies of the logarithms of<italic toggle="yes">K</italic><sub>d</sub> (<italic toggle="yes">K</italic><sub>i</sub>) on the length of ss and ds oligonucleotides. Single-stranded deoxy-oligonucleotides
(A); ss ribo- and deoxy-oligonucleotides (B); and ss and ds oligonucleotides (C). Symbols are shown in panels A−C. The −log <italic toggle="yes">K</italic><sub>d</sub> for
orthophosphate corresponds to <italic toggle="yes">n</italic> = 0. Values represent the average of at least two independent determinations. Errors were within 10−30%.</p></caption><graphic xlink:href="bi0300480f00002.tif" position="float" orientation="portrait"></graphic></fig></p><p>To assess the possible additivity in the interactions of
nonspecific oligonucleotides with IN, data from Table <xref rid="bi0300480t00001"></xref>A,B
were analyzed as the logarithmic dependencies of <italic toggle="yes">K</italic><sub>i</sub> (<italic toggle="yes">K</italic><sub>d</sub>)
for d(N)<italic toggle="yes"><sub>n</sub></italic> versus the number (<italic toggle="yes">n</italic>) of mononucleotide units
(0 ≤ <italic toggle="yes">n</italic> ≤ 21, where <italic toggle="yes">n</italic> = 0 corresponds to Pi). As shown in
Figure <xref rid="bi0300480f00002"></xref>A, all log-dependencies for nonspecific ss d(N)<italic toggle="yes"><sub>n</sub></italic>
appear biphasic. The transition from dTMP, dCMP, or dAMP
to the corresponding d(N)<sub>2</sub> leads to an increase in the affinity
by a factor of 2.5, 50, and 62, respectively. Values of <italic toggle="yes">f</italic>,
defined as the increase in affinity of IN for various d(N)<italic toggle="yes"><sub>n</sub></italic>
per unit increase in length, were evaluated from the slopes
of the linear parts of these curves. Integrase showed two
regions close to linear at <italic toggle="yes">n</italic> = 0−2 and <italic toggle="yes">n </italic>= 4−21,
respectively, while the region corresponding to <italic toggle="yes">n</italic> = 2−4 has
a transitional character. Monotonic increases in <italic toggle="yes">K</italic><sub>d</sub> (<italic toggle="yes">n</italic> > 4),
reflecting interaction between IN and each new nucleotide
unit, are equal to the reciprocals of <italic toggle="yes">f</italic> (<italic toggle="yes">K</italic><sub>i</sub> <italic toggle="yes">= </italic>1<italic toggle="yes">/f </italic>= 0.79−0.33
M). The <italic toggle="yes">K</italic><sub>i</sub> values characterizing the affinity of IN for
dNMPs were higher (∼1000 times) than the <italic toggle="yes">K</italic><sub>i</sub> characterizing
the enzyme interaction with any other nucleotide of longer
oligonucleotides (<italic toggle="yes">n</italic> > 4). The linear log-dependencies for
ss oligonucleotides provide evidence of Δ<italic toggle="yes">G</italic>° values close
to additive for the interaction of some of the 21 individual
units of d(N)<sub>21</sub> with integrase.
</p><p>We have previously shown that the interaction of different
sequence-specific DNA enzymes (repair, topoisomerization,
restriction enzymes) with each nucleotide unit of nonspecific
ss or ds oligonucleotides is usually a superposition of weak
electrostatic, hydrophobic, or van der Waals interactions with
the individual structural elements (<italic toggle="yes"><xref rid="bi0300480b00010" ref-type="bibr"></xref></italic>). The interaction can
be described by the descending geometrical progression:<xref rid="bi0300480e00005"></xref><disp-formula content-type="pre-labeled" id="bi0300480e00005"><!--%@md;sys;6q@%%@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;[;vis;full;auto@%%@fn;(;vis;full;unlock@%N%@fnx;);vis;full@%%@ital@%%@sb@%n%@rsf@%%@sbx@%%@fnx;];vis;full@% = %@ital@%K%@rsf@%%@sb@%d%@sbx@%%@fn;[;vis;full;auto@%%@fn;(;vis;full;unlock@%P%@sb@%i%@sbx@%%@fnx;);vis;full@%%@fnx;];vis;full@%%@fn;(;vis;full;auto@%%@ital@%e%@rsf@%%@fnx;);vis;full@%%@ex@%$-$%@ital@%n%@rsf@%%@exx@%%@fn;(;vis;full;auto@%%@ital@%h%@rsf@%%@sb@%C%@sbx@%%@fnx;);vis;full@%%@ex@%$-$%@ital@%l%@rsf@%%@exx@%%@fn;(;vis;full;auto@%%@ital@%h%@rsf@%%@sb@%T%@sbx@%%@fnx;);vis;full@%%@ex@%$-$%@ital@%m%@rsf@%%@exx@%%@fn;(;vis;full;auto@%%@ital@%h%@rsf@%%@sb@%G%@sbx@%%@fnx;);vis;full@%%@ital@%%@ex@%k%@rsf@%%@exx@%%@fn;(;vis;full;auto@%%@ital@%h%@rsf@%%@sb@%A%@sbx@%%@fnx;);vis;full@%%@ex@%$-$%@ital@%g%@rsf@%%@exx@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi0300480e00005.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
where <italic toggle="yes">K</italic><sub>d</sub>[(P<sub>i</sub>)] is the <italic toggle="yes">K</italic><sub>d</sub> for the minimal orthophosphate
ligand; <italic toggle="yes">e</italic> is a factor reflecting an increase of affinity because
of one internucleotide phosphate group; and <italic toggle="yes">h</italic><sub>N</sub> are coefficients of increase in affinity because of hydrophobic and/or van der Waals interactions of the enzyme with one of the
bases: C, T, G, and A, the numbers of which in (N)<italic toggle="yes"><sub>n</sub></italic> are
equal to <italic toggle="yes">l</italic>, <italic toggle="yes">m</italic>, <italic toggle="yes">k</italic>, and <italic toggle="yes">g</italic>, respectively<italic toggle="yes">.</italic> In addition, factor <italic toggle="yes">f</italic> is
equal to (<italic toggle="yes">he</italic>). Only the values of <italic toggle="yes">e</italic> and <italic toggle="yes">h</italic><sub>N</sub> usually change
from one enzyme to another. The affinity of some DNA-interacting enzymes for nonspecific (N)<italic toggle="yes"><sub>n</sub></italic> does not always
depend on the relative hydrophobicity of the bases. However,
if the enzyme interacts with the bases, the increase in affinity
for such oligonucleotides usually follows the same order as
the increase in the relative hydrophobicity of the bases:
C < T < G < A (9, <italic toggle="yes">10</italic>).
</p><p>HIV-1 integrase was the first enzyme we found in which
the affinity for nonspecific d(C)<italic toggle="yes"><sub>n</sub></italic>, d(T)<italic toggle="yes"><sub>n</sub></italic>, and d(A)<italic toggle="yes"><sub>n</sub></italic> did not
follow the relative hydrophobicity of the bases. Thus, the
affinity of d(A)<sub>21</sub> and d(C)<sub>21</sub> (having minimal and maximal
relative hydrophobicities) is similar, while the affinity of
d(T)<sub>21</sub> is significantly lower (Table <xref rid="bi0300480t00001"></xref>A,B). Interestingly, a
faster increase in the affinity for d(C)<italic toggle="yes"><sub>n</sub></italic> as compared with
d(T)<italic toggle="yes"><sub>n</sub></italic> resulted in a difference of ∼2 orders of magnitude for
the 21-mers (Figure <xref rid="bi0300480f00002"></xref>A). As mentioned above, the interaction
of some enzymes recognizing specific DNA can highly
depend on the conformational flexibility and dynamic
behavior of both enzyme and DNA and their capability for
specific adaptation to each other. The higher affinity of d(C)<italic toggle="yes"><sub>n</sub></italic>
can also be explained by a better adaptation of d(C)<italic toggle="yes"><sub>n</sub></italic> to a
specific conformation in the viral DNA·IN complex and
correlates well with a very flexible structure of d(C)<italic toggle="yes"><sub>n</sub></italic>, while
d(T)<italic toggle="yes"><sub>n</sub></italic> adopts a rigid nonflexible structure (<italic toggle="yes"><xref rid="bi0300480b00027" ref-type="bibr"></xref></italic>). To confirm
the importance of the flexibility of d(C)<italic toggle="yes"><sub>n</sub></italic> in its effective
interaction with integrase, we compared the affinity of the
enzyme for d(T)<italic toggle="yes"><sub>n</sub></italic> and oligothymidylates containing several
dC links in different positions (Table <xref rid="bi0300480t00002"></xref>). The affinity for
C/T-ODN<sub>21a</sub> was comparable with that of d(C)<italic toggle="yes"><sub>n</sub></italic> but not d(T)<italic toggle="yes"><sub>n</sub></italic>
of the same length (Table <xref rid="bi0300480t00001"></xref>A). C/T-ODN<sub>21b</sub> and C/A/T-ODN<sub>9</sub>, having 4−5 T nucleotides in the 3‘-end, presented
an affinity intermediate between that for d(T)<italic toggle="yes"><sub>n</sub></italic> and d(C)<italic toggle="yes"><sub>n</sub></italic> of
the same length. Therefore, the flexible structure of d(C)<italic toggle="yes"><sub>n</sub></italic> is
very important for a productive change during mutual
adjustment of IN and DNA conformations.
<table-wrap id="bi0300480t00002" position="float" orientation="portrait"><label>2</label><caption><p>Affinity of Integrase for Nonspecific ss Oligonucleotides<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="4"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">oligonucleotide</oasis:entry><oasis:entry namest="2" nameend="2">given name</oasis:entry><oasis:entry namest="3" nameend="3">No of
units</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">K</italic><sub>i</sub>
(μM)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">CAT
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">3
</oasis:entry><oasis:entry colname="4">20
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">AATT
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">4
</oasis:entry><oasis:entry colname="4">140
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">GGAA
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">4
</oasis:entry><oasis:entry colname="4">33
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(GA)<sub>3</sub></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">6
</oasis:entry><oasis:entry colname="4">1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(AC)<sub>3</sub></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">6
</oasis:entry><oasis:entry colname="4">33
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">CCAACTTTT
</oasis:entry><oasis:entry colname="2">C/A/T-ODN<sub>9</sub></oasis:entry><oasis:entry colname="3">9
</oasis:entry><oasis:entry colname="4">8.3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(T<sub>8</sub>)GT
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">8.3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">AC(A)<sub>8</sub></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">TCACCTCCTT
</oasis:entry><oasis:entry colname="2">C/T-ODN<sub>10</sub></oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4"><bold>6.6</bold></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">CTGCGTCTATCAGCG
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">15
</oasis:entry><oasis:entry colname="4">0.270
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">TTTTCCTCTCTCCCTCT
</oasis:entry><oasis:entry colname="2">T/C-ODN<sub>17</sub></oasis:entry><oasis:entry colname="3">17
</oasis:entry><oasis:entry colname="4">0.200
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">GGAAAATCTCTAGCAGT
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">17
</oasis:entry><oasis:entry colname="4">0.033
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">GTGTGGAAAATCTCTAGCAGT
</oasis:entry><oasis:entry colname="2">GT- specific
ODN<sub>21</sub></oasis:entry><oasis:entry colname="3">21
</oasis:entry><oasis:entry colname="4">0.010
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">CCCTCCTCCTTCTCTCTCCTT
</oasis:entry><oasis:entry colname="2">C/T-ODN<sub>21a</sub></oasis:entry><oasis:entry colname="3">21
</oasis:entry><oasis:entry colname="4">0.013
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">CCCTCCTCCTTCTCTCTTTTT
</oasis:entry><oasis:entry colname="2">C/T-ODN<sub>21b</sub></oasis:entry><oasis:entry colname="3">21
</oasis:entry><oasis:entry colname="4">0.100
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">CTAGCA<bold>p</bold></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">6
</oasis:entry><oasis:entry colname="4">2.6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">[Tp(Et)]<sub>9</sub>T<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">8000
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(T<sub>9</sub>)p(ddT)
</oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">15
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">d(GAGATCGTC)<bold>rA</bold></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4"><bold>0.500</bold></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Results are the average of two to three measurements. <italic toggle="yes">K</italic><sub>i</sub> values
(shown in bold) were determined by nonlinear regression analysis or
calculated using the IC<sub>50</sub> values (lightface) as described in the
Experimental Procedures. Errors in <italic toggle="yes">K</italic><sub>i</sub> and IC<sub>50</sub> values were within 10−20%.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Ethylated analogue.</p></table-wrap-foot></table-wrap></p><p>The contribution of the sugar moieties to binding was
estimated by comparing the affinities for ribo- and deoxyribo-oligonucleotides. The structural characteristics of RNA and
DNA differ markedly in solution: RNA usually exists in A
form and DNA in B form. In addition, all r(N)<italic toggle="yes"><sub>n</sub></italic> and d(T)<italic toggle="yes"><sub>n</sub></italic>
adopt very rigid nonflexible structures (<italic toggle="yes"><xref rid="bi0300480b00027" ref-type="bibr"></xref></italic>). The log-dependencies for all r(N)<italic toggle="yes"><sub>n</sub></italic> were practically the same as those
for d(T)<italic toggle="yes"><sub>n</sub></italic> (Figure <xref rid="bi0300480f00002"></xref>B). Therefore, for IN the superposition
of enzyme interactions with each mononucleotide unit of ss
r(N)<italic toggle="yes"><sub>n</sub></italic> is essentially the same as for d(T)<italic toggle="yes"><sub>n</sub></italic>. Thus, since the
affinity of different d(T)<italic toggle="yes"><sub>n</sub></italic> and r(N)<italic toggle="yes"><sub>n</sub></italic> does not in practice
depend on their bases, it seems reasonable to suggest that
IN interacts mostly with the sugar−phosphate backbone.
</p><p>To probe the contribution of the sugar−phosphate backbone in the formation of many weak contacts between IN
and oligonucleotides, we synthesized two different series of
analogues: (i) the abasic oligomers, d[(pR)<italic toggle="yes"><sub>n</sub></italic>(T)], in which
R is a chemically stable analogue of deoxyribose with the
base in C-1‘ replaced by a hydrogen atom and (ii) the
ethylated analogues. Table <xref rid="bi0300480t00001"></xref>B shows that the affinity of the
abasic analogues was slightly lower than the corresponding
d(T)<italic toggle="yes"><sub>n</sub></italic> (Figure <xref rid="bi0300480f00002"></xref>B). The situation was different for the other
analogues. The ethylation of the negatively charged internucleotide phosphate groups led to a decrease in affinity.
For example, Table <xref rid="bi0300480t00002"></xref> shows that the [Tp(Et)]<sub>9</sub>T analogue
had an affinity that is 3 orders of magnitude lower than that
of d(T)<sub>10</sub> (Table <xref rid="bi0300480t00001"></xref>). These results further confirmed that
integrase interacts through electrostatic interactions mainly
with the phosphodiester backbone of oligonucleotides, a
finding correlating well with data concerning the crystal
structure of the HIV-1 integrase catalytic core and C-terminal
domains (<italic toggle="yes"><xref rid="bi0300480b00028" ref-type="bibr"></xref></italic>). The structure resolved to 2.8 Å presents a
Y-shaped dimer, in which the DNA binding site can be
formed by the interaction of two monomers of the enzyme.
The electrostatic potential map identifies a contiguous strip
of positive charge along the outer face of the IN dimer,
beginning at the active site of one monomer and expanding
along the linker helix of the other monomer. This strip of
positive potential may provide a base for IN interaction with
internucleotide phosphate groups of specific and nonspecific
DNA.
</p><p>A change in affinity of 1.2−2.0-fold when elongating
oligonucleotides (<italic toggle="yes">n</italic> > 4) by one nucleotide unit is lower
(change in Δ<italic toggle="yes">G</italic>° of approximately −0.11 to −0.4 kcal/mol)
than would be expected for strong electrostatic contacts (up
to −1.0 kcal/mol) or hydrogen bonds (−2 to −6 kcal/mol)
but comparable to values for weak ion−dipole and dipole−dipole interactions (<italic toggle="yes"><xref rid="bi0300480b00024" ref-type="bibr"></xref></italic>). Thus, the interaction of negatively
charged internucleotide groups of nonspecific oligonucleotides with the DNA-binding channel of IN might rely on
dipolar electrostatic interactions rather than on electrostatic
interactions of immediate contacting groups. Since the
structures of ribo-ONs, d(T)<italic toggle="yes"><sub>n</sub></italic>, and d[(pR)<italic toggle="yes"><sub>n</sub></italic>(T)] cannot easily
be adjusted by the enzyme, a decreased affinity of IN for
the 5‘-flanks of these ligands may result from the longer
distance between the positively charged surface of IN and
the sugar−phosphate backbone of these oligonucleotides. The
increased efficiency of the interaction between IN and the
5‘-flanks of ss d(C)<italic toggle="yes"><sub>n</sub></italic> or specific ODNs could be due to
bringing the oppositely charged surfaces of IN and oligonucleotides closer together as a result of easier conformational changes in DNA and the enzyme structure.
</p><p><italic toggle="yes">Affinity of IN for Nonspecific DNA Duplexes.</italic> Usually
enzymes interacting with ds oligonucleotides contact both
chains of DNA after partially melting them, thus maintaining
the base-pairing between both strands. However, the contribution of the second strand to the affinity is usually much
lower than that of the first strand (<italic toggle="yes"><xref rid="bi0300480b00010" ref-type="bibr"></xref></italic>).
</p><p>To assess the contribution of the second strand, we
analyzed the interaction of IN with nonspecific d(T)<italic toggle="yes"><sub>n</sub></italic>·d(A)<italic toggle="yes"><sub>n</sub></italic>
duplexes. The affinity increased with the length of the duplex
to a maximal value at <italic toggle="yes">n</italic> = 12−21 (Table <xref rid="bi0300480t00001"></xref>C). The minimal
ligand exhibiting duplex properties toward IN was the
mixture of complementary hexanucleotides, d(T)<sub>6</sub> and d(A)<sub>6</sub>,
for which the <italic toggle="yes">T</italic><sub>m</sub> in solution (<italic toggle="yes"><xref rid="bi0300480b00029" ref-type="bibr"></xref></italic>) is significantly lower (<5
°C) than the reaction temperature (30 °C). Thus, for short
duplexes, some stabilization is obtained by interaction with
IN.
</p><p>Next, we assayed ribo-oligonucleotides (Table <xref rid="bi0300480t00001"></xref>D). Changing from d(T)<sub>10</sub>·d(A)<sub>10</sub> to r(U)<sub>10</sub>·r(A)<sub>10</sub> led to a decrease in
affinity by a factor of ∼30, while in the case of mixed ribo-deoxy duplexes, r(U)<sub>10</sub>·d(A)<sub>10</sub> and r(A)<sub>10</sub>·d(T)<sub>10</sub>, the changes
in affinity were much lower. It is very likely that IN cannot
efficiently force a productive conformation on the structure
of an RNA−RNA. The replacement of one ribo- by a
deoxyribo-strand led to an easier change in conformation,
which was dependent on IN. Ratios between the <italic toggle="yes">K</italic><sub>i</sub> values
of ss and ds oligonucleotides were highly dependent on the
length and structural characteristics of these compounds
(Table <xref rid="bi0300480t00001"></xref>C,D). All these results show that integrase interacts
with both strands of the DNA substrate but that the
contribution of the second strand is significantly lower than
that of the first. Thus, the rules governing the recognition of
nonspecific ss and ds oligonucleotides by IN are very similar.
</p><p><italic toggle="yes">Activation of IN by Specific ODNs.</italic> As previously shown,
the GT dinucleotide produces an inhibitory effect on IN
activity similar to that observed with nonspecific oligonucleotides (<italic toggle="yes"><xref rid="bi0300480b00019" ref-type="bibr"></xref></italic>). However, an unexpected result was obtained
with longer ODNs: the activity of the enzyme was stimulated
after preincubation of IN with low concentrations of specific
ODNs (Figure <xref rid="bi0300480f00003"></xref>A). The level of activation increased with
the length of ODNs (lanes 2−12). Specific ODNs containing
CA in the 3‘-end were better activators (lanes 7−12) than
GT containing ODNs of the same length (lanes 2−6). The
stimulation was even higher in the presence of ds ODNs
corresponding to the 3‘-terminal sequence of ODN<sub>21</sub> (compare lanes 15 with 3 and 16 with 5). On the contrary, a
nonspecific GT-containing compound such as (T)<sub>8</sub>(GT) did
not activate the enzyme, whereas preincubation with CA or
(T)<sub>8</sub>(CA) led to a small but detectable increase in the activity
(lanes 7 and 8). The effect of ODNs corresponding to the
noncleavable strand of DNA was notably lower (lanes 13
and 14). Lane 17 shows the activation of the enzyme after
preincubation with Mn<sup>2+</sup> ions, an effect previously described
by us and others (<italic toggle="yes">19</italic>, <italic toggle="yes">30</italic>, <italic toggle="yes">31</italic>).
<fig id="bi0300480f00003" position="float" orientation="portrait"><label>3</label><caption><p>(A) Relative activity of IN in the processing reaction. The activity was measured using 10 nM GT-specific ds [<sup>32</sup>P]ODN<sub>21</sub>
as substrate. Integrase (2 μM) was previously preincubated for 1 h
at 30 °C under different conditions: 1, in the absence of ODNs or
MnCl<sub>2</sub> (this relative activity was taken as 1); 2−16, in the absence
of MnCl<sub>2</sub> but after addition of 2.5 μM ODNs as follows: 2, AGT;
3, ss GT-specific hexamer (5‘ AGCAGT 3‘); 4, GT-specific ss
octamer (5‘ CTAGCAGT 3‘); 5, GT-specific ss decamer (5‘
CTCTAGCAGT 3‘); 6, GT-specific ss ODN<sub>21</sub> (5‘ GTGTGGAAAATCTCTAGCAGT 3‘); 7, CA dinucleotide; 8, ss (T)<sub>8</sub>(CA);
9, ss CA tetramer (5‘ AGCA 3‘); 10, ss CA hexamer (5‘ CTAGCA
3‘); 11, ss CA decamer (5‘ ATCTCTAGCA 3‘); 12, ss CA-ODN<sub>19</sub>
(5‘ GTGTGGAAAATCTCTAGCA 3‘); 13, a noncleavable decamer
(5‘ AAAAGGTGTG 3‘); 14, a noncleavable 19-mer (5‘ ACGATCTCTAAAAGGTGTG 3‘); 15, double-stranded GT-specific
hexamer (5‘ AGCAGT 3‘(N)<sub>6</sub>); 16, double-stranded GT-specific
decamer (5‘CTCTAGCAGT 3‘(N)<sub>10</sub>); and 17, 40 mM MnCl<sub>2</sub> in
the absence of ODN. (B) Effect of specific ODNs on the rate of
3‘-processing. The reaction mixture was incubated for 30 min at
30 °C in the presence of 2 nM GT-specific ds [<sup>32</sup>P]ODN<sub>21</sub> substrate
and different concentrations of CTCTAGCA (lines 1 and 3) or
CTAGCAGT (lines 2 and 4). The reaction was started by the
addition of 10 nM integrase before (lines 1 and 2) or after
preincubation with 40 mM MnCl<sub>2</sub> (lines 3 and 4). The relative
amount of [<sup>32</sup>P]GT in the acid-soluble fraction for reaction mixtures
in the absence of oligonucleotides was taken as 100%. Values
represent the average of at least two independent determinations.
Errors were within 10−30%.</p></caption><graphic xlink:href="bi0300480f00003.tif" position="float" orientation="portrait"></graphic></fig></p><p>We then analyzed the relative activity of IN in the 3‘-processing reaction as a function of the substrate concentration. This reaction usually requires a long incubation time
(0.5−1 h), thus suggesting that even without preincubation,
specific ODNs can to some extent activate IN during the
course of the reaction. To check this, we analyzed the activity
of IN as a function of the substrate using a fixed concentration (2 nM) of ds [<sup>32</sup>P]GT-ODN<sub>21</sub> and a stepwise increase
in its total concentration (up to 62 nM) because of addition
of nonradioactive ds ODN<sub>21</sub> (Figure <xref rid="bi0300480f00004"></xref>). Such isotopic dilution
of a radioactive substrate should normally cause a decrease
in the accumulation of the labeled product. However, the
increase in total concentration of ODN<sub>21</sub> led to a significant
enhancement of [<sup>32</sup>P]GT dinucleotide removal. The maximal
level of apparent substrate-dependent activation of IN was
about 6-fold. This was significantly lower than the real level
of IN activation since adding 60 nM nonradioactive ODN<sub>21</sub>
to 2 nM [<sup>32</sup>P]ODN<sub>21</sub> diluted the specific activity of the
substrate by a factor of 31. Thus, the level of IN activation
was so high that the activated enzyme molecules were able
to remove GT efficiently, not only from nonlabeled ODN<sub>21</sub>
but also from [<sup>32</sup>P]ODN<sub>21</sub>.
<fig id="bi0300480f00004" position="float" orientation="portrait"><label>4</label><caption><p>Processing activity as a function of oligonucleotide and IN concentrations. The activity was determined as a function of the concentration of nonradioactive ds GT-ODN<sub>21</sub> at a constant
concentration of ds 3‘-[<sup>32</sup>P]GT-ODN<sub>21</sub> (2 nM) specific substrate.
Integrase was used at different concentrations: 50 (1), 128 (2), 320
(3), and 700 nM (4). The relative accumulation of [<sup>32</sup>P]GT was
compared after 1 h of reaction. Before adding IN to the reaction
mixture, the enzyme (2 μM) was preactivated by incubation with
40 mM MnCl<sub>2</sub> at 30 °C for 1 h. Values represent the average of at
least two independent determinations. Errors were within 10−30%.
</p></caption><graphic xlink:href="bi0300480f00004.tif" position="float" orientation="portrait"></graphic></fig></p><p>The concentration of the ODN<sub>21</sub> substrate (1−2 nM) in
the reaction mixture is usually ∼10−100 times lower than
that of the enzyme (10−100 nM). In general, the catalytic
activity of HIV-1 IN is so low that most activity-based assays
require more than stoichiometric amounts of the enzyme as
compared to the concentration of the substrate (<italic toggle="yes"><xref rid="bi0300480b00031" ref-type="bibr"></xref></italic>)<italic toggle="yes">.</italic> At fixed
low concentrations of the ODN<sub>21</sub> substrate, an increase in
enzyme concentration up to ∼1 μM leads to a linear increase
in the 3‘ processing reaction (data not shown). This means
that <1−5% of IN molecules are active in the reaction. It
has been reported that IN can exist in a dynamic equilibrium
of different oligomers. In the micromolar range, IN form
high-order multimers such as tetramers, octamers, or
aggregates (<italic toggle="yes"><xref rid="bi0300480b00032" ref-type="bibr"></xref></italic>). However, at catalytically active concentrations
(10−100 nM), integrase mostly exists as monomers that are
catalytically inactive. Therefore, the activation of IN after
preincubation with specific ODNs might result from a change
in the equilibrium between monomeric and oligomeric forms
in favor of the formation of catalytically active oligomers.
The low affinity of IN subunits for each other and the
increase in the activation effect by specific ss and ds ODNs
with increasing concentrations of IN strongly suggest that
the preincubation of IN with such ODNs leads to the
formation of catalytically active oligomeric forms of the
enzyme.
</p><p>The relative amounts of different enzyme oligomers can
be estimated using small-angle X-ray scattering (SAXS) (26,
<italic toggle="yes">33</italic>)<italic toggle="yes">.</italic> According to our preliminary data on IN analysis by
SAXS, more than 95% of IN (100 nM) exists in monomeric
form, while preincubation (for 0.5−1 h at 30 °C) with a
specific GCAGT oligonucleotide leads to the formation of
enzyme dimers. As was shown above, nonspecific ODNs
compete with the substrate for IN, but under the preincubation conditions, nonspecific d(T)<sub>5</sub> or d(A)<sub>5</sub> did not lead to
formation of IN dimeric forms. This indicates that nonspecific oligonucleotides interact predominantly with the preformed catalytically active oligomeric forms of the enzyme.
</p><p>Besides several factors governing specificity, the viral
DNA sequence-dependent formation of catalytically active
oligomers of IN can be an important way to increase enzyme
specificity. The propensity of IN for self-assembly increases
with the length of specific GT-ODNs, but CA-containing
ODNs corresponding to the cleaved strand are better activators. Therefore, it cannot be excluded that, in addition to
their effect on IN self-assembly, these GT- and CA-containing ODNs can change the oligomer conformation by
alternative routes. Our results on sequence-dependent activation indicate that IN is an extremely dynamic enzyme in
conformational terms. According to the crystal structure,
some residues in the active site region and between the α5
and α6 helices of catalytic core dimers form very flexible
loops (<italic toggle="yes"><xref rid="bi0300480b00028" ref-type="bibr"></xref></italic>). It is possible that easily achievable changes in
the structure of IN are necessary to provide different types
of interactions with nonspecific and specific DNAs before
and after the removal of the GT dinucleotide and the
following reaction of integration. These results probably
indicate sequence-dependent formation of catalytically active
oligomers of IN.
</p><p><italic toggle="yes">Affinity of Integrase for Specific Oligonucleotides.</italic> The
addition of low concentrations of specific ODNs to the
reaction mixture led to activation of IN, whereas increasing
their concentration inhibited the reaction (Figure <xref rid="bi0300480f00003"></xref>B). Thus,
the activation of IN made it difficult to estimate the affinity
of specific ODNs for the enzyme when using the method of
inhibitory analysis.
</p><p>To evaluate inhibition, it was important to eliminate the
activation produced by specific ODNs on IN (Figure <xref rid="bi0300480f00003"></xref>B,
lines 1 and 2). As shown above, preincubation of IN with
Mn<sup>2+</sup> increased the specific activity of the enzyme. We
therefore preincubated IN in the presence of Mn<sup>2+</sup> to obtain
the activated form of the enzyme, and the processing reaction
was then performed in the presence of specific ODNs. Figure
<xref rid="bi0300480f00003"></xref>B presents the results with two specific ODNs. When
starting with a Mn<sup>2+</sup>-activated form of IN, an inhibition
similar to that obtained with nonspecific ODNs was obtained
(Figure <xref rid="bi0300480f00003"></xref>B, lines 3 and 4). In these conditions, specific ODNs
were also found to be competitive inhibitors toward the
substrate. The <italic toggle="yes">K</italic><sub>i</sub><italic toggle="yes"></italic>values determined for specific ODNs are
summarized in Table <xref rid="bi0300480t00003"></xref>.
<table-wrap id="bi0300480t00003" position="float" orientation="portrait"><label>3</label><caption><p>Affinity of Integrase for Specific ODNs<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="1"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"><graphic xlink:href="bi0300480u00003a.tif" position="float" orientation="portrait"></graphic></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> (A) Single-stranded ODNs. *<italic toggle="yes">N</italic> corresponds to the number of nucleotide units. (B) Double-stranded ODNs. d(N)<italic toggle="yes"><sub>n</sub></italic> corresponds in each case to
the complementary strand. Results are the average of three to four measurements. The <italic toggle="yes">K</italic><sub>i</sub> values were determined by nonlinear regression analysis
(in bold) or calculated using the IC<sub>50</sub> values. The ratio <italic toggle="yes">K</italic><sub>i</sub> (ss)/<italic toggle="yes">K</italic><sub>i</sub> (ds) was calculated with the <italic toggle="yes">K</italic><sub>i</sub> values (ss) given in part A.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Complex Formation of IN with Specific ODNs: Interactions with the Nucleotide Units.</italic> As shown in Figure <xref rid="bi0300480f00002"></xref>, all
log-dependencies for specific and nonspecific ss oligonucleotides are very similar and appear nearly biphasic. The
transition from dTMP, dGMP, or dCMP to the corresponding
d(N)<sub>2</sub> or d(GT) increased their affinity by a factor of 2−115,
while further lengthening by one nucleotide results in a nearly
monotonic increase by a factor of ∼1.26−1.64. In contrast,
the transition from dAMP to d(AA) or d(CA) and then to
the corresponding d(N)<sub>4</sub> led to a significantly higher increase
in affinity (4−330-fold). The increase became slow and
nearly monotonic (<italic toggle="yes">f</italic> = ∼1.2) only at <italic toggle="yes">n</italic> = 5−21. Thus, IN
shows two different modes of interaction with specific short
ODNs containing either the 3‘-terminal GT (and related
nonspecific d(T)<italic toggle="yes"><sub>n</sub></italic> and d(C)<italic toggle="yes"><sub>n</sub></italic> oligonucleotides) or the 3‘-terminal CA (and related d(A)<italic toggle="yes"><sub>n</sub></italic>).
</p><p>Before the processing reaction, the contribution of GT
terminal nucleotides of ODN<sub>21</sub> to the total affinity was
significantly higher than that of any other nucleotide.
However, the high affinity of CA-containing ODNs and their
great ability to activate the enzyme suggest that after IN-dependent removal of the GT-terminal nucleotide, a change
in the enzyme−DNA complex occurs. IN forms new strong
contacts with the four AGCA-terminal nucleotides and
probably retains to some extent the weak contacts previously
formed with the 5‘-flank of ODN<sub>21</sub>.
</p><p>In Table <xref rid="bi0300480t00003"></xref> it is shown that the <italic toggle="yes">K</italic><sub>i</sub> of AGCA is 1 μM. On
the other hand, the contribution of the AGCA sequence
within ss GT-ODN<sub>21</sub> to the affinity of IN could be approximately estimated from the ratio of <italic toggle="yes">K</italic><sub>i</sub> values for
AGCAGT (6.1 μM) and GT (130 μM) (Table <xref rid="bi0300480t00003"></xref>). This
calculation gave a <italic toggle="yes">K</italic><sub>i</sub> of 0.047 M, showing that the contribution of the AGCA tetranucleotide to the enzyme affinity for
CA-ODN<sub>19</sub> was 4.7 × 10<sup>4</sup> times greater that that for GT-ODN<sub>21</sub>. Moreover, from the ratio of <italic toggle="yes">K</italic><sub>i</sub> values for CA (15
μM) and AGCA (1 μM), it can be calculated that the
contribution of the CA-terminal dinucleotide to the affinity
of CA-ODN<sub>19</sub> is about 5.5 × 10<sup>4</sup> times higher than that for
the two additional AG nucleotides (0.067 M) of the AGCA
terminal tetranucleotide.
</p><p>All these data indicate that the 3‘-terminal GT dinucleotide
forms contacts with a specific subsite of IN recognizing the
GT terminus of viral ds DNA. At the same time, specific
ODNs containing CA or even only A at their 3‘-termini
probably are not in contact with the GT-recognizing subsite
of the enzyme but interact with the second CA-recognizing
subsite of IN. Data with nonspecific oligonucleotides also
support this conclusion (Table <xref rid="bi0300480t00002"></xref>). The affinity of short ODNs
such as GGAA or (GA)<sub>3</sub> is comparable to that of specific
CA-ODNs and d(A)<italic toggle="yes"><sub>n</sub></italic>, while the efficiency of interaction of
AATT and d(AC)<sub>3</sub> correlates well with that of GT-ODNs
and d(T)<italic toggle="yes"><sub>n</sub></italic> of the same length. The same conclusion can be
drawn from the change in the affinity of ds ODN<sub>21</sub> (1.3 nM)
after being modified; the removal of T from the 3‘-end
(ODN<sub>20</sub>) or its replacement with rU (rU-ODN<sub>21</sub>) decreased
the affinity ∼30−40-fold (Table <xref rid="bi0300480t00003"></xref>B). The affinity of ds
ODN<sub>20</sub> (60 nM) or rU-ODN<sub>21</sub> (50 nM) is similar to that of
nonspecific ds d(A)<sub>21</sub>·d(T)<sub>21</sub> (40 nM) of the same length,
while ds CA-ODN<sub>19</sub> presents a <italic toggle="yes">K</italic><sub>i</sub> comparable to that of the
specific ds GT-ODN<sub>21</sub> substrate.
</p><p>Thus, these general trends in the interactions of IN with
long and short ss ODNs corresponding to the cleaved strand
of ODN<sub>21</sub> agree very well with those for specific ds ODNs.
The affinities of IN for ss GT-ODN<sub>21</sub> (10 nM) and ss CA-ODN<sub>19</sub> (30 nM) were nearly the same and only 7.7- and 2.5-fold lower, respectively, than those of their duplexes (Table
<xref rid="bi0300480t00003"></xref>). There is no doubt that the 3‘-terminal T forms specific
contacts with IN. In contrast, the interactions of the penultimate G are not so strong. The high affinity for GT-ODN<sub>21</sub>
and CA-ODN<sub>19</sub> was expected since these ODNs have
sequences representing the specific substrates for processing
and strand transfer, respectively. After removal of GT, a
reorganization of the active site of IN probably occurs, which
allows effective interaction with the nascent 3‘-terminal CA
dinucleotide of the cleaved strand. Our results showing a
stronger activation of IN by ODNs containing the 3‘-terminal
CA as compared with GT-containing ODNs also support this
idea. Moreover, in this case, our data point to two different
modes of interaction between IN and these ODNs.
</p><p>The interaction of IN with the invariant 3‘-terminal CAGT
sequence of viral DNA either before or after the removal of
GT contributes significantly to its total affinity for DNA.
This sequence is very important for IN oligomerization into
catalytically active forms and for cooperative interactions
of the enzyme with the 3‘-terminus and the 5‘-flank of the
specific ds ODN<sub>21</sub> substrate. Moreover, the removal of GT
leads to the formation of a free 3‘-OH group on the terminal
dA, which can form additional contacts, such as hydrogen
bonds, with the amino acid residues of IN localized near
this group. Interestingly, a replacement of dA with rA
d(GAGATCGTC)rA (Table <xref rid="bi0300480t00002"></xref>) decreases the affinity of the
specific CA-ODN<sub>10</sub> (Table <xref rid="bi0300480t00003"></xref>) by a factor of ∼5. This might
result from formation of a hydrogen bond between two OH
groups of the rA nucleoside, thus preventing the 3‘-hydroxyl
from having effective contacts with the enzyme. A decrease
in the affinity of CTAGCA<bold>p</bold> by a factor of 6.5 after
phosphorylation of the OH group from the deoxyribose of
the terminal dA also suggests an important role of this OH
group in the interaction with IN.
</p><p><italic toggle="yes">Affinity of IN for Specific DNA Duplexes.</italic> The data reported
in Figure <xref rid="bi0300480f00002"></xref>C show similar log-dependencies of <italic toggle="yes">K</italic><sub>i</sub><italic toggle="yes"></italic>values
versus length for nonspecific or specific double-stranded
ODNs, although the affinity of IN for specific duplexes was
always higher. The maximal contribution to the interaction
between IN and specific DNA can be estimated approximately as <italic toggle="yes">K</italic><sub>d</sub> = 0.033 M (Table <xref rid="bi0300480t00003"></xref>). The apparent relative
contribution of the first strand (corresponding to the cleaved
strand of viral DNA) to the overall affinity may be estimated
as ∼7−8 orders of magnitude, while adding the second
strand increased the affinity by only ∼1 order of magnitude.
The same difference (∼1 order of magnitude) corresponding
to the relative contributions of the two complementary strands
was observed for nonspecific ODNs. This indicates that IN
interacts with both strands of the DNA substrate but that
formally the contribution of the second strand is significantly
lower. However, the increase in affinity because of the
second strand and calculated from the respective affinities
of ss and ds ODNs may be an underestimated value since
the annealing of the second strand might induce changes in
enzyme affinity.
</p><p>Considering our results with HIV-1 integrase and our
previous data (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00009" ref-type="bibr"></xref>, <xref rid="bi0300480b00010" ref-type="bibr"></xref></named-content></italic>) with DNA-specific enzymes (replication, repair, topoisomerization, and restriction enzymes), it
can be concluded that the relative contribution of the second
strand never exceeds 1−2 orders of magnitude. Different
factors are involved, including partial melting of the DNA
duplex in the complex with enzyme leading to a decrease in
complementary interaction between strands. In other cases
(e.g., in the case of a repair enzyme (<italic toggle="yes"><xref rid="bi0300480b00034" ref-type="bibr"></xref></italic>)), the protein forms
more contacts with the first than with the second strand, and
the relative contributions of these strands to the affinity of
the DNA duplex usually correlate with the number of such
contacts.
</p><p>The reduced contribution of the second strand to the
affinity does not mean that the second strand is not important
for recognition and cleavage of specific viral DNA since IN
cannot catalyze 3‘-processing or integration with ss DNA.
In the case of DNA repair, topoisomerization, and restriction
enzymes, the second strand of DNA is usually more
important for adjusting the DNA structure to a conformation
optimal for catalysis than for enzyme binding. However,
some enzymes cannot fit ss DNA to form direct contacts
with the catalytic amino acid residues of the active site<italic toggle="yes">.</italic> The
same might be true for HIV-1 IN.
</p><p><italic toggle="yes">Thermodynamic Models of IN Interactions with Specific
DNA.</italic> The estimation of the relative contributions of different
structural elements of specific and nonspecific DNA to the
total affinity for any enzyme including IN seems difficult
because of the dynamic and cooperative character of DNA−enzyme interactions.
</p><p>In this study, we used the SILC approach to quantitate
the determinants of substrate specificity. Within the limitations of the SILC method, the analysis of the data concerning
the nucleotides from both strands of specific DNA to the
affinity of IN (Figure <xref rid="bi0300480f00002"></xref>) showed that the relative contribution
of the elements from the 3‘-terminal nucleotides of the
cleavable strand can differ by several orders of magnitude.
In contrast, very small monotonic changes of the dependencies of log <italic toggle="yes">K</italic><sub>d</sub> on the number of nucleotides (<italic toggle="yes">n</italic> > 4−8)
indicated weak interactions close to additive.
</p><p>The relative contribution of GT to the affinity of GT-ODN<sub>21</sub> for IN (Δ<italic toggle="yes">G</italic>° ≈ −5.4 kcal/mol) was approximately
assessed from the <italic toggle="yes">K</italic><sub>d</sub> value (Table <xref rid="bi0300480t00003"></xref>) for GT (Figure <xref rid="bi0300480f00005"></xref>). The
contributions of the phosphate, deoxyribose phosphate, and
T nucleotide unit to the affinity of the GT-dinucleotide were
obtained from <italic toggle="yes">K</italic><sub>d</sub> values of free Pi (0.033 M, Δ<italic toggle="yes">G</italic>° ≈ −2.0
kcal/mol), free d(pR) (0.015 M, Δ<italic toggle="yes">G</italic>° ≈ −2.5 kcal/mol), and
free dTMP (0.77 mM, Δ<italic toggle="yes">G</italic>° ≈ −4.3 kcal/mol). Thus, the
contributions of the base and the sugar of dTMP were
estimated as <italic toggle="yes">K</italic><sub>d</sub> ≈ 0.051 M (Δ<italic toggle="yes">G</italic>° ≈ −1.8 kcal/mol) and
0.450 M (Δ<italic toggle="yes">G</italic>° ≈ −0.5 kcal/mol), respectively. Since dCMP
and dGMP have the same affinities as deoxyribose phosphate, these free nucleotides probably interact nonspecifically
with a subsite of IN recognizing the T because of their pR
moieties, similar to deoxyribose phosphate. Therefore, the
approximate contribution of the G unit to the affinity of the
GT dinucleotide for IN may be more correctly estimated from
the ratio of <italic toggle="yes">K</italic><sub>d</sub> values for dTMP and the GT dinucleotide:
<italic toggle="yes">K</italic><sub>d</sub> (G) ≈ 0.170 M (Δ<italic toggle="yes">G</italic>° ≈ −1.1 kcal/mol).
<fig id="bi0300480f00005" position="float" orientation="portrait"><label>5</label><caption><p>Thermodynamic model of the interaction of IN with GT-specific ds ODN<sub>21</sub>. Δ<italic toggle="yes">G</italic>° values characterizing different contacts
between the enzyme and the DNA strands are shown. All contacts
of IN with ss GT-ODN<sub>21</sub> provide an overall total Δ<italic toggle="yes">G</italic>° of
approximately −11.1 kcal/mol (<italic toggle="yes">K</italic><sub>d</sub> = 1 × 10<sup>-8</sup> M). The sum of all
types of nonspecific and specific contacts of IN with the phosphate
backbone and bases of the noncleaved strand together with the
interactions between complementary strands, as compared to ss GT-ODN<sub>21</sub>, increases the affinity approximately by a factor of 8 and
decreases Δ<italic toggle="yes">G</italic>° by −1.25 kcal/mol. All types of nonspecific and
specific interactions of IN and ds GT-ODN<sub>21</sub> provide approximately
Δ<italic toggle="yes">G</italic>° = −12.4 kcal/mol.</p></caption><graphic xlink:href="bi0300480f00005.tif" position="float" orientation="portrait"></graphic></fig></p><p>The relative contributions of the next A and C nucleotide
units of the specific CAGT sequence are comparable and
are characterized by the <italic toggle="yes">K</italic><sub>d</sub> values ≈0.41−0.43 M (Δ<italic toggle="yes">G</italic>° ≈
−0.51 to −0.54 kcal/mol). Each of the next 17 nucleotides
of the 5‘-flank increased the affinity by a factor of ∼1.57
(<italic toggle="yes">K</italic><sub>d</sub> ≈ 0.64 M, Δ<italic toggle="yes">G</italic>° ≈ −0.27 kcal/mol). The total contribution of these 17 links was estimated as Δ<italic toggle="yes">G</italic>° ≈ −4.6 kcal/mol. All specific or nonspecific contacts of IN with the
backbone and the bases of the noncleaved strand, together
with the complementary interactions between the strands,
increased the duplex affinity by a factor of 8 (corresponding
to a decrease in Δ<italic toggle="yes">G</italic>° by −1.25 kcal/mol) as compared with
ss ODN<sub>21</sub>. Such a small increase in affinity may be due to
several factors, including formation of weak contacts of IN
only with the second strand of ds DNA or very weak contacts
between the strands because of partial melting of ds DNA.
Although the current data do not allow us to resolve this
issue, the thermodynamic model of Figure <xref rid="bi0300480f00005"></xref> may approximately describe the interaction of IN with specific ds
GT-ODNs.
</p><p>For the processed substrate (CA-ODN)<sub>19</sub>·(N)<sub>21</sub>, the relative
contribution of CA to the total affinity of CA-ODN for the
enzyme (Δ<italic toggle="yes">G</italic>° ≈ −6.7 kcal/mol) was approximately evaluated from the <italic toggle="yes">K</italic><sub>d</sub> value for free CA (15 μM) (Figure <xref rid="bi0300480f00006"></xref>). The
independent estimation of the contributions of C and A units
to the affinity of IN for the CA dinucleotide is rather difficult.
Free dAMP and dCMP can interact with subsites of the
enzyme different from those with which they are in contact
when in the CA dinucleotide. In addition, the recognition of
CA and AA dinucleotides by IN can occur in a cooperative
fashion since the transition from dAMP to AA or CA led to
a very large increase in affinity (by a factor of 63 and 333,
respectively). Therefore, the minimal approximate
contribution of the A unit to the affinity of the dinucleotides can be
estimated from <italic toggle="yes">K</italic><sub>d</sub> for free dAMP (Δ<italic toggle="yes">G</italic>° ≈ −3.2 kcal/mol).
The extrapolation of the initial parts of biphasic curves for
d(A)<italic toggle="yes"><sub>n</sub></italic> and CA-ODN<sub>21</sub> to <italic toggle="yes">n</italic> = 1 gives a <italic toggle="yes">K</italic><sub>i</sub><italic toggle="yes"></italic>for the A unit
≈1.5−2 mM (−3.2 to −3.9 kcal/mol) for the first 3‘-terminal
nucleotide of these dinucleotides. Thus, a very approximate
estimation of the contribution of the A and C units to the
affinity of the CA dinucleotide is −3.2 to −3.9 and −2.7 to
−3.5 kcal/mol, respectively.
<fig id="bi0300480f00006" position="float" orientation="portrait"><label>6</label><caption><p>Thermodynamic model of the interaction of IN with the duplex CA-specific ODN<sub>19</sub> and complementary 21-mer ODN.
Δ<italic toggle="yes">G</italic>° values characterizing different contacts between the enzyme
and the DNA strands are shown. All contacts of IN with the cleaved
strand after removal of GT provide an overall total Δ<italic toggle="yes">G</italic>° of
approximately −10.4 kcal/mol (<italic toggle="yes">K</italic><sub>d</sub> = 3 × 10<sup>-8</sup> M). The sum of all
types of nonspecific and specific contacts of IN with the phosphate
backbone and bases of the noncleaved strand together with the
interactions between complementary strands, as compared to ss
ODN<sub>21</sub>, increases the affinity approximately by a factor of 8 and
decreases Δ<italic toggle="yes">G</italic>° by −1.25 kcal/mol. All types of nonspecific and
specific interactions of IN and ds DNA provide approximately
Δ<italic toggle="yes">G</italic>° = −11.6 kcal/mol.</p></caption><graphic xlink:href="bi0300480f00006.tif" position="float" orientation="portrait"></graphic></fig></p><p>The increase in affinity upon transition from the CA
dinucleotide to longer ODNs occurs nearly additively, and
the relative contributions of the G and A units (3rd and 4th
from the 3‘-end) to the affinity of CA-ODN was estimated
as −0.88 and −0.75 kcal/mol, respectively (Figure <xref rid="bi0300480f00006"></xref>). Each
of the next six nucleotides of CA-ODN contributed approximately −0.23 kcal/mol to the total Δ<italic toggle="yes">G</italic>° of binding, their
sum being −1.38 kcal/mol. Finally, the interaction of each
of the nine 5‘-terminal nucleotides of CA-ODN is characterized by Δ<italic toggle="yes">G</italic>° ≈ −0.08 kcal/mol, thus adding up to Δ<italic toggle="yes">G</italic>° ≈
−0.72 kcal/mol. All types of interactions of (CA-ODN)<sub>19</sub>
with the complementary ODN<sub>21</sub> increase the total Δ<italic toggle="yes">G</italic>° of
binding by −1.2 kcal/mol. The thermodynamic model in
Figure <xref rid="bi0300480f00006"></xref> might approximately describe the interaction of IN
with ds (CA-ODN)<sub>19</sub>·(N)<sub>21</sub>.
</p><p><italic toggle="yes">Kinetic Factors: Reaction Rate and Specificity of Integrase. </italic>The sequence of the DNA substrate strongly influences
the maximal rate of 3‘-processing and integration reactions
catalyzed by IN (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi0300480b00035" ref-type="bibr"></xref>−<xref rid="bi0300480b00036" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0300480b00037" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0300480b00038" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi0300480b00039" ref-type="bibr"></xref></named-content></italic>). We thus studied the substrate
properties of some of the duplexes mentioned above in the
3‘-processing reaction. As compared to the processing
reaction obtained with the specific ds GT-ODN<sub>21</sub>·(N)<sub>21</sub>
substrate, no removal of dinucleotides from any single-stranded or double-stranded 21-mer ODNs was observed
even at high IN concentration (5 μM) and incubation for 24
h (Table <xref rid="bi0300480t00004"></xref>). However, partially specific ds ODNs such as
(T)<sub>17</sub>CAGT·(N)<sub>21</sub> or (A)<sub>16</sub>CAGT·(N)<sub>20</sub>, which contained the
canonical 3‘-terminal CAGT, were used as substrates by IN
although at a reduced rate. In contrast, the <italic toggle="yes">V</italic><sub>max</sub> of the reaction
was increased when the second strand was partially noncomplementary, as in the case of GT-ODN<sub>21</sub>. These results
together with the data mentioned above point to an important
role of duplex melting for productive interaction with IN.
<table-wrap id="bi0300480t00004" position="float" orientation="portrait"><label>4</label><caption><p>Determination of<italic toggle="yes">K</italic><sub>M</sub> and <italic toggle="yes">V</italic><sub>m</sub><sub>ax</sub> in the 3‘-Processing
Reaction Catalyzed by Integrase with Various ds ODNs Used as
Substrates<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">double-stranded ODNs</oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">V</italic><sub>max</sub>
(%)</oasis:entry><oasis:entry namest="3" nameend="3"><italic toggle="yes">K</italic><sub>M </sub>
(nM)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">[GTGTGGAAAATCTCTAGCAGT]·(N)<sub>21</sub></oasis:entry><oasis:entry colname="2">100
</oasis:entry><oasis:entry colname="3">1.3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">[(T)<sub>17</sub>CAGT]·(N)<sub>21</sub></oasis:entry><oasis:entry colname="2">35
</oasis:entry><oasis:entry colname="3">12
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">[(A)<sub>16</sub>CAGT]·(N)<sub>20</sub></oasis:entry><oasis:entry colname="2">50
</oasis:entry><oasis:entry colname="3">4.8
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(-TGTGGAAAATCTCTA GCAGT)·
·(−ACACCTTTTAGAGAT <bold>GAG</bold>CA)
</oasis:entry><oasis:entry colname="2">120
</oasis:entry><oasis:entry colname="3">2.6
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(-ACTGCTAGAGATTTTCCACA)·
·(-TGTGGAAAATCTCTA<bold>CT</bold> CGT)
</oasis:entry><oasis:entry colname="2">not detectable
</oasis:entry><oasis:entry colname="3"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(T)<sub>19</sub>GT·(N)<sub>21</sub></oasis:entry><oasis:entry colname="2">not detectable
</oasis:entry><oasis:entry colname="3"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(T)<sub>21</sub>·(A)<sub>21</sub></oasis:entry><oasis:entry colname="2">not detectable
</oasis:entry><oasis:entry colname="3"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">(T)<sub>17</sub>CCGT·(N)<sub>21</sub></oasis:entry><oasis:entry colname="2">not detectable
</oasis:entry><oasis:entry colname="3"></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Errors of <italic toggle="yes">K</italic><sub>M</sub> and <italic toggle="yes">V</italic><sub>max</sub> values were within 20−30%. Values are
the average results of two to three independent measurements.</p></table-wrap-foot></table-wrap></p><p>Considering the high concentrations of nonspecific ODNs
and IN at which there is no detectable reaction as compared
with the rate of removal of 3‘-terminal dinucleotides from
specific ds ODNs at low concentrations of substrate and IN,
it can be calculated that the reaction rate is increased by more
than 5−6 orders of magnitude by the transition from
nonspecific to specific ODNs. In contrast, the complex
formation step accounts only for a ∼30-fold difference when
changing from specific to nonspecific DNA. Thus, as in other
enzymes, the catalytic step in integrase appears to be
significantly more sensitive to the DNA structure than the
IN·DNA complex formation step. The specificity of the
enzyme action is mainly evidenced by the enzyme-dependent
DNA adjustment to the optimal conformation and by the
catalytic step of the reaction.
</p><p>In conclusion, several steps of the reaction may provide
enzyme specificity. First, in contrast to nonspecific DNA,
specific DNA can direct the assembly of catalytically active
oligomeric forms of IN and then activate the enzyme because
of a change in its structure. Second, formation of complexes
between preformed catalytically active oligomeric forms of
IN and specific DNA occurs with approximately 1 order of
magnitude higher efficiency as compared with nonspecific
DNA. Upon transition from nonspecific to specific DNA,
the <italic toggle="yes">k</italic><sub>cat</sub> of the processing reaction increases by >5−6 orders
of magnitude. Finally, an additional increase in IN specificity
may be provided by the particular stage of the integration
reaction.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We are grateful to Ray Cooke (English Department,
University Bordeaux 2) who edited the manuscript.
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Virol</italic>. <italic toggle="yes"> 75</italic>, 1359−1370.</mixed-citation></ref><ref id="bi0300480n00001"><mixed-citation><comment>Abbreviations: IN, integrase; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; ON, oligonucleotide; ribo-ON, ribo-oligonucleotide; ODN, oligodeoxynucleotide; ds, double-stranded; ss, single-stranded; SILC, stepwise increase in ligand complexity.</comment></mixed-citation></ref></ref-list></back></article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†</title></titleInfo><titleInfo contentType="CDATA"><title>Dynamic, Thermodynamic, and Kinetic Basis for Recognition and Transformation of DNA by Human Immunodeficiency Virus Type 1 Integrase†</title></titleInfo><name type="personal"><namePart type="family">BUGREEV</namePart><namePart type="given">Dmitrii V.</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> Siberian Division of Russian Academy of Sciences.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">BARANOVA</namePart><namePart type="given">Svetlana</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> Siberian Division of Russian Academy of Sciences.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">ZAKHAROVA</namePart><namePart type="given">Olga D.</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> Siberian Division of Russian Academy of Sciences.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">PARISSI</namePart><namePart type="given">Vincent</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">DESJOBERT</namePart><namePart type="given">Cécile</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">SOTTOFATTORI</namePart><namePart type="given">Enzo</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> University of Genoa.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">BALBI</namePart><namePart type="given">Alessandro</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> University of Genoa.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">LITVAK</namePart><namePart type="given">Simon</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">TARRAGO-LITVAK</namePart><namePart type="given">Laura</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> UMR 5097 CNRS-Université Victor Segalen Bordeaux 2.</affiliation><affiliation> Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal" displayLabel="corresp"><namePart type="family">NEVINSKY</namePart><namePart type="given">Georgy A.</namePart><affiliation>Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 8 Lavrentieva Avenue,Novosibirsk 630090, Russia, UMR 5097 CNRS-Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat,33076 Bordeaux Cedex, France and IFR 66 Bordeaux, France, and University of Genoa, Department of PharmaceuticalSciences, Viale Benedetto XV, 3, Genoa-3, Italy</affiliation><affiliation> Siberian Division of Russian Academy of Sciences.</affiliation><affiliation> Correspondence should be addressed to the following authors.(G.A.N.) Tel.: 7-3832 39 62 26. Fax: 7-3832 33 36 77. E-mail: nevinsky@niboch.nsc.ru. (L.T.-L.) Tel.: 33-5 5757 1740. Fax: 33-55757 1766. E-mail: laura.litvak@reger.u-bordeaux2.fr.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2003-07-02</dateCreated><dateIssued encoding="w3cdtf">2003-08-05</dateIssued><copyrightDate encoding="w3cdtf">2003</copyrightDate></originInfo><note type="footnote" ID="bi0300480AF2"> This work was supported in part by grants from the Russian Foundation for Basic Research (01-04-49058), the Siberian Division of the Russian Academy of Sciences, the French Agence Nationale de Recherches sur le SIDA (ANRS), the Centre National de la Recherche Scientifique (CNRS), and the University Victor Segalen Bordeaux 2. V.P. benefited from a postdoctoral fellowship from SIDACTION. G.A.N. was supported by a short-fellowship from ANRS.</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Specific interactions between retroviral integrase (IN) and long terminal repeats are required for insertion of viral DNA into the host genome. To characterize quantitatively the determinants of substrate specificity, we used a method based on a stepwise increase in ligand complexity. This allowed an estimation of the relative contributions of each nucleotide from oligonucleotides to the total affinity for IN. The interaction of HIV-1 integrase with specific (containing sequences from the LTR) or nonspecific oligonucleotides was analyzed using a thermodynamic model. Integrase interacted with oligonucleotides through a superposition of weak contacts with their bases, and more importantly, with the internucleotide phosphate groups. All these structural components contributed in a combined way to the free energy of binding with the major contribution made by the conserved 3‘-terminal GT, and after its removal, by the CA dinucleotide. In contrast to nonspecific oligonucleotides that inhibited the reaction catalyzed by IN, specific oligonucleotides enhanced the activity, probably owing to the effect of sequence-specific ligands on the dynamic equilibrium between the oligomeric forms of IN. However, after preactivation of IN by incubation with Mn2+, the specific oligonucleotides were also able to inhibit the processing reaction. We found that nonspecific interactions of IN with DNA provide ∼8 orders of magnitude in the affinity (ΔG° ≈ −10.3 kcal/mol), while the relative contribution of specific nucleotides of the substrate corresponds to ∼1.5 orders of magnitude (ΔG° ≈ − 2.0 kcal/mol). Formation of the Michaelis complex between IN and specific DNA cannot by itself account for the major contribution of enzyme specificity, which lies in the kcat term; the rate is increased by more than 5 orders of magnitude upon transition from nonspecific to specific oligonucleotides.</abstract><note type="footnote" ID="bi0300480AF2"> This work was supported in part by grants from the Russian Foundation for Basic Research (01-04-49058), the Siberian Division of the Russian Academy of Sciences, the French Agence Nationale de Recherches sur le SIDA (ANRS), the Centre National de la Recherche Scientifique (CNRS), and the University Victor Segalen Bordeaux 2. V.P. benefited from a postdoctoral fellowship from SIDACTION. 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Virol. 75, 1359−1370.</note></relatedItem><relatedItem type="references" ID="bi0300480n00001" displayLabel="bibbi0300480n00001"><titleInfo><title>Abbreviations: IN, integrase; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; ON, oligonucleotide; ribo-ON, ribo-oligonucleotide; ODN, oligodeoxynucleotide; ds, double-stranded; ss, single-stranded; SILC, stepwise increase in ligand complexity.</title></titleInfo><note type="content-in-line">Abbreviations: IN, integrase; HIV-1, human immunodeficiency virus type 1; LTR, long terminal repeat; ON, oligonucleotide; ribo-ON, ribo-oligonucleotide; ODN, oligodeoxynucleotide; ds, double-stranded; ss, single-stranded; SILC, stepwise increase in ligand complexity.</note></relatedItem><identifier type="istex">CA832659F002CBA73B2DAFEEEC4EBAAB31AE80B5</identifier><identifier type="ark">ark:/67375/TPS-KP3SDW0G-3</identifier><identifier type="DOI">10.1021/bi0300480</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2003 American Chemical Society</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-X5HBJWF8-J">acs</recordContentSource><recordOrigin>Converted from (version 1.2.10) to MODS version 3.6.</recordOrigin><recordCreationDate encoding="w3cdtf">2020-04-10</recordCreationDate></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/record.json</uri></json:item></metadata><annexes><json:item><extension>gif</extension><original>true</original><mimetype>image/gif</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-KP3SDW0G-3/annexes.gif</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/CA832659F002CBA73B2DAFEEEC4EBAAB31AE80B5/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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