Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase†
Identifieur interne : 002232 ( Istex/Corpus ); précédent : 002231; suivant : 002233Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase†
Auteurs : Yu Lin Jiang ; James T. StiversSource :
- Biochemistry [ 0006-2960 ] ; 2002.
Abstract
The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described as a three-step process involving phosphodiester backbone pinching, base extrusion through active pushing and plugging by a leucine side chain that inserts in the DNA minor groove, and, finally, pulling by hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the “pinch, push, plug, and pull” base-flipping mechanism. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. (1999) Biochemistry 38, 952−963]. A model that accounts for the temporal and functional roles of these side chain interactions along the reaction pathway for base flipping is presented.
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DOI: 10.1021/bi026226r
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase†</title><author><name sortKey="Jiang, Yu Lin" sort="Jiang, Yu Lin" uniqKey="Jiang Y" first="Yu Lin" last="Jiang">Yu Lin Jiang</name><affiliation><mods:affiliation>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, Maryland 21205-2185</mods:affiliation></affiliation></author><author><name sortKey="Stivers, James T" sort="Stivers, James T" uniqKey="Stivers J" first="James T." last="Stivers">James T. Stivers</name><affiliation><mods:affiliation>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, Maryland 21205-2185</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed: Phone: (410) 502-2758. Fax: (410) 955-3023. E-mail: jstivers@jhmi.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:169EFB7AE2312B134544780300F313FC2A4D6E42</idno><date when="2002" year="2002">2002</date><idno type="doi">10.1021/bi026226r</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-1R2NQZG3-D/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002232</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002232</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase<ref type="bib" target="#bi026226rAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Jiang, Yu Lin" sort="Jiang, Yu Lin" uniqKey="Jiang Y" first="Yu Lin" last="Jiang">Yu Lin Jiang</name><affiliation><mods:affiliation>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, Maryland 21205-2185</mods:affiliation></affiliation></author><author><name sortKey="Stivers, James T" sort="Stivers, James T" uniqKey="Stivers J" first="James T." last="Stivers">James T. Stivers</name><affiliation><mods:affiliation>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, Maryland 21205-2185</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed: Phone: (410) 502-2758. Fax: (410) 955-3023. E-mail: jstivers@jhmi.edu.</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">2002</date><date type="published">2002</date><biblScope unit="vol">41</biblScope><biblScope unit="issue">37</biblScope><biblScope unit="page" from="11236">11236</biblScope><biblScope unit="page" to="11247">11247</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">The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described as a three-step process involving phosphodiester backbone pinching, base extrusion through active pushing and plugging by a leucine side chain that inserts in the DNA minor groove, and, finally, pulling by hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the “pinch, push, plug, and pull” base-flipping mechanism. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. (1999) Biochemistry 38, 952−963]. A model that accounts for the temporal and functional roles of these side chain interactions along the reaction pathway for base flipping is presented.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>tryptophan</json:string><json:string>uracil</json:string><json:string>mutant</json:string><json:string>wtudg</json:string><json:string>mutation</json:string><json:string>stivers</json:string><json:string>biochemistry</json:string><json:string>koff</json:string><json:string>extrahelical</json:string><json:string>kmax</json:string><json:string>tryptophan fluorescence</json:string><json:string>kobsd</json:string><json:string>rate constant</json:string><json:string>serine</json:string><json:string>jiang</json:string><json:string>conformational change</json:string><json:string>excitation</json:string><json:string>mutational effect</json:string><json:string>side chain</json:string><json:string>drohat</json:string><json:string>tryptophan fluorescence change</json:string><json:string>datum</json:string><json:string>microscopic rate constant</json:string><json:string>duplex</json:string><json:string>kmaxtrp</json:string><json:string>phosphodiester</json:string><json:string>tryptophan fluorescence decrease</json:string><json:string>mutant enzyme</json:string><json:string>extrahelical state</json:string><json:string>kflip</json:string><json:string>pyrene</json:string><json:string>mutational</json:string><json:string>uracil base</json:string><json:string>late role</json:string><json:string>transition state</json:string><json:string>ground state</json:string><json:string>active site</json:string><json:string>cutoff filter</json:string><json:string>damaging effect</json:string><json:string>conformational</json:string><json:string>kinetic trace</json:string><json:string>fluorescence increase</json:string><json:string>association measurement</json:string><json:string>enzyme</json:string><json:string>kinetic study</json:string><json:string>fluorescence change</json:string><json:string>binding kinetics</json:string><json:string>glycosidic bond cleavage</json:string><json:string>equilibrium constant</json:string><json:string>base pair</json:string><json:string>extrahelical base</json:string><json:string>fluorescence</json:string><json:string>nucleic acid</json:string><json:string>bimolecular association</json:string><json:string>various enzyme</json:string><json:string>late effect</json:string><json:string>hyperbolic concentration dependence</json:string><json:string>apparent rate constant</json:string><json:string>base extrusion</json:string><json:string>computer simulation</json:string><json:string>kinetic measurement</json:string><json:string>functional role</json:string><json:string>overall process</json:string><json:string>kinetic constant</json:string><json:string>kmax value</json:string><json:string>binding measurement</json:string><json:string>postflipping conformational change</json:string><json:string>previous study</json:string><json:string>phosphodiester backbone</json:string><json:string>transient kinetic study</json:string><json:string>fluorescence kinetic study</json:string><json:string>active site pocket</json:string><json:string>rate equation</json:string><json:string>irreversible dissociation</json:string><json:string>dissociation rate constant</json:string><json:string>kinetic</json:string><json:string>simulation</json:string><json:string>nucleotide</json:string><json:string>conformation</json:string><json:string>dissociation</json:string></teeft></keywords><author><json:item><name>JIANG Yu Lin</name><affiliations><json:string>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, Maryland 21205-2185</json:string></affiliations></json:item><json:item><name>STIVERS James T.</name><affiliations><json:string>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,725 North Wolfe Street, Baltimore, Maryland 21205-2185</json:string><json:string>To whom correspondence should be addressed: Phone: (410) 502-2758. 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Here we employ mutagenesis in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the “pinch, push, plug, and pull” base-flipping mechanism. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. (1999) Biochemistry 38, 952−963]. A model that accounts for the temporal and functional roles of these side chain interactions along the reaction pathway for base flipping is presented.</abstract><qualityIndicators><score>9.58</score><pdfWordCount>8350</pdfWordCount><pdfCharCount>49173</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>696</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>215</abstractWordCount><abstractCharCount>1406</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase†</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>41</volume><issue>37</issue><pages><first>11236</first><last>11247</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-1R2NQZG3-D</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>2002</publicationDate><copyrightDate>2002</copyrightDate><doi><json:string>10.1021/bi026226r</json:string></doi><id>169EFB7AE2312B134544780300F313FC2A4D6E42</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-1R2NQZG3-D/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-1R2NQZG3-D/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-1R2NQZG3-D/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-1R2NQZG3-D/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase<ref type="bib" target="#bi026226rAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2002 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2002</date><date type="Copyright" when="2002">2002</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">Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase<ref type="bib" target="#bi026226rAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Jiang</surname><forename type="first">Yu Lin</forename></persName><affiliation><orgName type="division">Department of Pharmacology and Molecular Sciences</orgName><orgName type="institution">The Johns Hopkins University School of Medicine</orgName><address><addrLine>725 North Wolfe Street</addrLine><addrLine>Baltimore</addrLine><addrLine>Maryland 21205-2185</addrLine></address></affiliation></author><author xml:id="author-0001" role="corresp"><persName><surname>Stivers</surname><forename type="first">James T.</forename></persName><affiliation><orgName type="division">Department of Pharmacology and Molecular Sciences</orgName><orgName type="institution">The Johns Hopkins University School of Medicine</orgName><address><addrLine>725 North Wolfe Street</addrLine><addrLine>Baltimore</addrLine><addrLine>Maryland 21205-2185</addrLine></address></affiliation><affiliation role="corresp"> To whom correspondence should be addressed: Phone: (410) 502-2758. Fax: (410) 955-3023. E-mail: jstivers@jhmi.edu.</affiliation></author><idno type="istex">169EFB7AE2312B134544780300F313FC2A4D6E42</idno><idno type="ark">ark:/67375/TPS-1R2NQZG3-D</idno><idno type="DOI">10.1021/bi026226r</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">2002</date><date type="published">2002</date><biblScope unit="vol">41</biblScope><biblScope unit="issue">37</biblScope><biblScope unit="page" from="11236">11236</biblScope><biblScope unit="page" to="11247">11247</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>The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in
genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide
is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described
as a three-step process involving phosphodiester backbone <hi rend="italic">pinching</hi>, base extrusion through active <hi rend="italic">pushing</hi><hi rend="italic">and plugging</hi> by a leucine side chain that inserts in the DNA minor groove, and, finally, <hi rend="italic">pulling</hi> by
hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in
combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic
groups of UDG that have been implicated in the “pinch, push, plug, and pull” base-flipping mechanism.
Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but
that many of these mutations prevent a subsequent induced fit conformational step in which catalytic
groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping
in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J.
T., et al. (1999) <hi rend="italic">Biochemistry</hi>
<hi rend="italic">38</hi>, 952−963]. A model that accounts for the temporal and functional roles
of these side chain interactions along the reaction pathway for base flipping is presented.
</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/bi026226r</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Mutational Analysis of the Base-Flipping Mechanism of Uracil DNA Glycosylase<xref rid="bi026226rAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Jiang</surname><given-names>Yu Lin</given-names></name></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Stivers</surname><given-names>James T.</given-names></name><xref rid="bi026226rAF1">*</xref></contrib><aff>Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine,
725 North Wolfe Street, Baltimore, Maryland 21205-2185
</aff></contrib-group><author-notes><corresp id="bi026226rAF1">
To whom correspondence should be addressed: Phone: (410) 502-2758. Fax: (410) 955-3023. E-mail: jstivers@jhmi.edu.</corresp></author-notes><pub-date pub-type="epub"><day>23</day><month>08</month><year>2002</year></pub-date><pub-date pub-type="ppub"><day>17</day><month>09</month><year>2002</year></pub-date><volume>41</volume><issue>37</issue><fpage>11236</fpage><lpage>11247</lpage><supplementary-material xlink:href="bi026226r_s1.pdf" orientation="portrait" position="float"></supplementary-material><history><date date-type="received"><day>31</day><month>05</month><year>2002</year></date><date date-type="rev-recd"><day>15</day><month>07</month><year>2002</year></date><date date-type="asap"><day>23</day><month>08</month><year>2002</year></date><date date-type="issue-pub"><day>17</day><month>09</month><year>2002</year></date></history><permissions><copyright-statement>Copyright © 2002 American Chemical Society</copyright-statement><copyright-year>2002</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in
genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide
is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described
as a three-step process involving phosphodiester backbone <italic toggle="yes">pinching</italic>, base extrusion through active <italic toggle="yes">pushing</italic><italic toggle="yes">and plugging</italic> by a leucine side chain that inserts in the DNA minor groove, and, finally, <italic toggle="yes">pulling</italic> by
hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in
combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic
groups of UDG that have been implicated in the “pinch, push, plug, and pull” base-flipping mechanism.
Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but
that many of these mutations prevent a subsequent induced fit conformational step in which catalytic
groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping
in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J.
T., et al. (1999) <italic toggle="yes">Biochemistry</italic> <italic toggle="yes">38</italic>, 952−963]. A model that accounts for the temporal and functional roles
of these side chain interactions along the reaction pathway for base flipping is presented.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi026226r</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi026226rAF2"><label>†</label><p>
Supported by NIH Grant RO1GM56834 (to J.T.S.).</p></notes></front><body><sec id="d7e131"><title></title><p>Enzymes that alter the covalent structure of DNA bases
must solve the general problem of gaining access to sites
that are normally buried in the duplex structure of the DNA.
Nearly 10 years ago the crystal structure of cytosine
5-methyltransferase bound to its DNA substrate revealed that
this enzyme solved the problem by a novel base-flipping
mechanism involving rotation of the entire cytidine nucleotide from the DNA and into the active site where the
5-position of the cytosine base was positioned for methylation
(<italic toggle="yes"><xref rid="bi026226rb00001" ref-type="bibr"></xref></italic>). Subsequently, it has become clear that base flipping is
common in nature, and that many enzymes use similar
approaches to act on DNA bases. In particular, base flipping
appears to be absolutely required for damaged base recognition and glycosidic bond cleavage by DNA repair glycosylases (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00002" ref-type="bibr"></xref>−<xref rid="bi026226rb00003" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026226rb00004" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026226rb00005" ref-type="bibr"></xref></named-content></italic>).
</p><p>Uracil DNA glycosylase (UDG)<xref rid="bi026226rb00001" ref-type="bibr"></xref> has taken a paradigm
role in the pursuit to better understand the process of base
flipping by DNA glycosylases. A crystal structure of human
UDG bound to uracil-containing DNA has been reported (<italic toggle="yes"><xref rid="bi026226rb00006" ref-type="bibr"></xref></italic>),
and this structure suggests several structural requirements
that may be required for flipping bases (Figure <xref rid="bi026226rf00001"></xref>A). First,
the DNA in this structure is sharply bent by about 45°. If
such bending also occurs when UDG first encounters DNA
at a nonspecific site, then this distortion may serve to
destabilize the duplex and promote uracil flipping when the
damaged site is located in correct register with the active
site pocket. Bending may be induced by the interaction of
two serine side chains with the phosphodiester groups on
the 3‘ and 5‘ sides of the deoxyuridine residue (Ser88 and
Ser189), leading to compression of the P−P distance (“serine
pinching”). Although induced DNA strain is an attractive
mechanism for initiation of the base-flipping process, no
direct evidence for this mechanism yet exists. The structure
also shows that a completely conserved leucine residue
(Leu191) is found protruding into the DNA minor groove
opposite the expelled uracil, suggesting that this group acts
as a mechanical wedge to “push” the uracil from the duplex
(<italic toggle="yes"><xref rid="bi026226rb00002" ref-type="bibr"></xref></italic>), and additionally as a “plug” to increase its lifetime in
the active site (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>). Recent biochemical studies have provided
support for both roles (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00007" ref-type="bibr"></xref>, <xref rid="bi026226rb00008" ref-type="bibr"></xref></named-content></italic>). Finally, the uracil base is
stabilized in the extrahelical state by a finely tuned hydrogen-bonding network that completely satisfies all of the hydrogen
bond donor and acceptor groups of the base (Figure <xref rid="bi026226rf00001"></xref>A).
These hydrogen-bonding interactions have been ascribed a
“pulling” function in stabilizing the flipped base (His187 and
Asn123) (<italic toggle="yes"><xref rid="bi026226rb00009" ref-type="bibr"></xref></italic>). Collectively, this constellation of interactions
has been termed the “pinch, push, plug, and pull” mechanism
for base flipping (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00007" ref-type="bibr"></xref>, <xref rid="bi026226rb00009" ref-type="bibr"></xref></named-content></italic>).
<fig id="bi026226rf00001" position="float" orientation="portrait"><label>1</label><caption><p>(A) Arrangement of the active site groups of UDG that interact with the extrahelical uracil (<italic toggle="yes"><xref rid="bi026226rb00006" ref-type="bibr"></xref></italic>). The Ser88 and Ser189 phosphodiester
pinching residues, the Leu191 pushing and plugging side chain, and the Asn123 and His187 pulling groups are shown. The catalytic group,
Asp64, that is important in transition-state stabilization but not base flipping is also shown (<italic toggle="yes"><xref rid="bi026226rb00024" ref-type="bibr"></xref></italic>). (B) Three-step mechanism for uracil base
flipping involving nonspecific DNA binding and dissociation (<italic toggle="yes">k</italic><sub>1</sub> = 220 μM<sup>-1</sup> s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> = 600 s<sup>-1</sup>), reversible uracil flipping into an extrahelical
state (<italic toggle="yes">k</italic><sub>flip</sub> = 700 s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>-</sub><sub>flip</sub> = 180 s<sup>-1</sup>), and conformational docking of UDG around the flipped-out base (<italic toggle="yes">k</italic><sub>conf</sub>, = 350 s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>-</sub><sub>conf</sub> = 100 s<sup>-1</sup>).
The microscopic rate constants were obtained from global kinetic simulations of the stopped-flow kinetic traces (see the Supporting Information
and Table <xref rid="bi026226rt00003"></xref>).</p></caption><graphic xlink:href="bi026226rf00001.tif" position="float" orientation="portrait"></graphic></fig></p><p>Transient kinetic studies and spectroscopic measurements
have built upon these structural insights to illuminate the
pathway for attaining the final extrahelical state (Figure <xref rid="bi026226rf00001"></xref>B).
One useful approach to isolate the base-flipping step was to
arrest the chemical step of the reaction using a deoxyuridine
analogue containing an electron-withdrawing 2‘-fluorine
substituent (2‘-FU) (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). The kinetics of the base-flipping
step could then be monitored in real time using stopped-flow fluorescence, taking advantage of fluorescence intensity
changes of a 2-aminopurine fluorescent reporter group that
was strategically positioned adjacent to the flipped uracil
(<italic toggle="yes"><xref rid="bi026226rb00011" ref-type="bibr"></xref></italic>). This work revealed that UDG follows a minimal two-step mechanism for base flipping in which a weak nonspecific encounter complex is formed before the uracil is rapidly
extruded to form the extrahelical state. Raman spectroscopy
measurements indicated that the structure of the nonspecific
complex was perturbed, with a significant decrease in the
intensities of the DNA base Raman bands, indicating a net
increase in base stacking (<italic toggle="yes"><xref rid="bi026226rb00012" ref-type="bibr"></xref></italic>). A similar hypochromic shift
was observed in the specific complex with 2‘-FU DNA,
suggesting that the nonspecific and specific complexes share
common structural features.
</p><p>Kinetic studies also revealed that uracil flipping is followed
closely by a conformational change in UDG that could be
monitored by following a decrease in the UDG tryptophan
fluorescence, and that this induced fit change only occurs
for DNA that contains uracil (Figure <xref rid="bi026226rf00001"></xref>B) (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). This step
likely involves the closing of the UDG active site around
the flipped uracil, because the free enzyme is found in an
open state that differs appreciably from its conformation in
the specific complex (<italic toggle="yes">2</italic>,<italic toggle="yes"> 6</italic>, <italic toggle="yes">13</italic>, <italic toggle="yes">14</italic>). This conformational
change likely plays a significant role in the high specificity
of UDG for cleavage of the uracil base, because closing of
the active site is a prerequisite for formation of the specific
hydrogen bonds that are essential for leaving group activation
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00015" ref-type="bibr"></xref>−<xref rid="bi026226rb00016" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026226rb00017" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi026226rb00018" ref-type="bibr"></xref></named-content></italic>). These mechanistic insights initially derived from
transient kinetic studies using 2‘-FU substrate analogue DNA
have been recently confirmed using natural deoxyuridine-containing DNA (<italic toggle="yes"><xref rid="bi026226rb00019" ref-type="bibr"></xref></italic>).
</p><p>Here we extend our studies of enzymatic base flipping by
selectively deleting the enzymatic side chains that are
involved in the pinch, push, plug, and pull mechanism
(Figure <xref rid="bi026226rf00001"></xref>A). We interrogate the resultant effects on the
multistep process of base flipping using both thermodynamic
and transient kinetic measurements. We have found that a
single deletion of either serine pinching side chain does not
prevent UDG from flipping the uracil base or undergoing
the conformational clamping step. However, removal of <italic toggle="yes">both</italic>
serine pinching groups or, alternatively, deletion of the
Leu191 or Asn123 side chains prevents the UDG active site
from clamping around the extrahelical base. These mutations
perturb the base-flipping process at discrete points along the
reaction pathway, thereby allowing the temporal mapping
of these interactions. In the following paper in this issue (<italic toggle="yes"><xref rid="bi026226rb00023" ref-type="bibr"></xref></italic>),
we extend our recent “substrate rescue” approach to demonstrate how these base-flipping phenotypes can be partially
or fully rescued by preorganizing the uracil in an extrahelical
conformation using an unnatural pyrene (Y) nucleotide
wedge (i.e., a U/Y base pair) (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>).
</p></sec><sec id="d7e310"><title>Experimental Procedures</title><p><italic toggle="yes">Nucleoside Phosphoramidite and Oligonucleotide Synthesis.</italic> The nucleoside phosphoramidites were purchased from
Applied Biosystems or Glen Research (Sterling, VA), except
for the 2‘-β-fluoro-2‘-deoxyuridine phosphoramidite, which
was synthesized as described (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). The oligonucleotides were
synthesized using standard phosphoramidite chemistry with
an Applied Biosystems 390 synthesizer. After synthesis and
deprotection, the oligonucleotides were purified by anion
exchange HPLC and desalted by C-18 reversed-phase HPLC
(Phenomenex Aqua column). The size, purity, and nucleotide
composition of the DNA were assessed by analytical
reversed-phase HPLC, MALDI mass spectrometry, and
denaturing polyacrylamide gel electrophoresis. The DNA
strands were hybridized as previously described to form the
duplexes used in the binding and kinetic studies as shown
in Table <xref rid="bi026226rt00001"></xref>. In these sequences, U<sup>F</sup> = 2‘-β-fluoro-2‘-deoxyuridine nucleotide. The concentrations of the
oligonucleotides were determined by UV absorption measurements at 260 nm, using the pairwise extinction coefficients
for the constituent nucleotides.
<table-wrap id="bi026226rt00001" position="float" orientation="portrait"><label>1</label><caption><p>DNA Sequences<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="bi026226ru00001a.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> Thermal melting experiments were performed in TMN buffer using
3 μM concentrations of each duplex (see the Experimental Procedures).
The melting temperatures for AU<sup>F</sup>/A and PU<sup>F</sup>/A were 48.8 and 45.8
°C, respectively.</p></table-wrap-foot></table-wrap></p><p><italic toggle="yes">Purification of UDG and Mutants.</italic> As previously described, the recombinant UDG from <italic toggle="yes">E. coli</italic> strain B was
purified to >99% homogeneity using a T7 polymerase-based
overexpression system (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00013" ref-type="bibr"></xref>, <xref rid="bi026226rb00020" ref-type="bibr"></xref></named-content></italic>). The concentration of the
enzyme was determined using an extinction coefficient of
38.5 mM<sup>-1</sup> cm<sup>-1</sup>. All of the mutations in this work were
generated using the Quick-Change double-stranded mutagenesis kit from Stratagene (La Jolla, CA), and the mutations
were confirmed by sequencing both strands of the DNA. The
6X-His-tagged mutant proteins were purified using nickel
chelate chromatography as previously described (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00014" ref-type="bibr"></xref>, <xref rid="bi026226rb00020" ref-type="bibr"></xref></named-content></italic>). The
His-tag was removed by cleavage using biotinylated thrombin
followed by purification using strepavidin beads and nickel
chelate chromatography. The purity of the mutant enzymes
was greater than 95% as judged by SDS−polyacrylamide
gel electrophoresis with visualization by Coomassie Blue
staining.
</p><p><italic toggle="yes">DNA Binding and Competitive Inhibition Studies</italic>. The
dissociation constants (<italic toggle="yes">K</italic><sub>D</sub>) for binding of the various
enzymes to the DNA molecules listed in Table <xref rid="bi026226rt00001"></xref> were
determined using two orthogonal methods. All measurements
were performed in TMN buffer, 10 mM Tris−HCl (pH 8.0),
2.5 mM MgCl<sub>2</sub>, 25 mM NaCl, at 25 °C. In the first method,
direct binding measurements were made by following the
increase in 2-AP fluorescence upon titrating fixed concentrations of the 2-AP-containing DNA (Figure <xref rid="bi026226rf00002"></xref>) with increasing
amounts of UDG. Excitation was at 320 nm, and emission
spectra from 330 to 450 nm were collected using a Spex
Fluoromax 3 fluorimeter. The 2-AP fluorescence intensity
(<italic toggle="yes">F</italic>) at 370 nm was plotted against [UDG]<sub>tot</sub> to obtain the <italic toggle="yes">K</italic><sub>D</sub>
from eqs 1 and 2.<xref rid="bi026226re00001"></xref><disp-formula content-type="pre-labeled" id="bi026226re00001"><!--%@md;sys;6q@%%@ital@%F%@rsf@% = %@ital@%F%@rsf@%%@sb@%o%@sbx@% − %@fn;{;vis;full;auto@%%@fn;(;vis;full;unlock@%%@ital@%F%@rsf@%%@sb@%o%@sbx@% − %@ital@%F%@rsf@%%@sb@%f%@sbx@%%@fnx;);vis;full@%%@fn;[;vis;full;unlock@%DNA%@fnx;];vis;full@%%@sb@%tot%@sbx@%/2%@fnx;};vis;full@% × %@bf@%[S_EL2;quad]\
%@fn;{;vis;full;auto@%%@ital@%b%@rsf@% − %@fn;(;vis;full;unlock@%%@ital@%b%@rsf@%%@ex@%2%@exx@% − 4%@fn;[;vis;full;unlock@%UDG%@fnx;];vis;full@%%@sb@%tot%@sbx@%%@fn;[;vis;full;unlock@%DNA%@fnx;];vis;full@%%@sb@%tot%@sbx@%%@fnx;);vis;full@%%@ex@%1/2%@exx@%%@fnx;};vis;full@%%@id;reqid;1@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00001.gif" position="anchor" orientation="portrait"></graphic></disp-formula><xref rid="bi026226re00002"></xref><disp-formula content-type="pre-labeled" id="bi026226re00002"><!--%@md;sys;6q@%%@ital@%b%@rsf@% = %@ital@%K%@rsf@%%@sb@%D%@sbx@% + %@fn;[;vis;full;auto@%UDG%@fnx;];vis;full@%%@sb@%tot%@sbx@% + %@fn;[;vis;full;auto@%DNA%@fnx;];vis;full@%%@sb@%tot%@sbx@%%@id;reqid;2@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00002.gif" position="anchor" orientation="portrait"></graphic></disp-formula><fig id="bi026226rf00002" position="float" orientation="portrait"><label>2</label><caption><p>Specific DNA Binding of S88A UDG. (A) PU<sup>F</sup>/A DNA
(1500 nM) was titrated with increasing amounts of S88A, and the
2-AP fluorescence increase at 370 nm is plotted as a function of
[S88A]. The curve is the best fit to eq 1. (B) S88A (1500 nM) was
titrated with increasing amounts of AU<sup>F</sup>/A DNA, and the tryptophan
fluorescence decrease at 335 nm is plotted as a function of [AU<sup>F</sup>/A]. The <italic toggle="yes">K</italic><sub>D</sub> values are reported in Table <xref rid="bi026226rt00002"></xref>.</p></caption><graphic xlink:href="bi026226rf00002.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Tryptophan Fluorescence Measurements. </italic>Dissociation
constants were also determined by following the tryptophan
fluorescence decrease in UDG as DNA binds (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). For these
experiments, excitation was at 290 nm and emission scans
were performed over the range 305−450 nm. Corrections
for dilution and inner filter effects were made using the
equation <italic toggle="yes">F</italic><sub>corr</sub>(335 nm) = <italic toggle="yes">F</italic><sub>obsd</sub> × 10<italic toggle="yes"><sup>A</sup></italic><sup><sup>290</sup>/2</sup>, where <italic toggle="yes">A</italic><sup>290</sup> is the
absorption of the DNA ligand at the excitation wavelength.
<italic toggle="yes">F</italic><sub>corr</sub> was then plotted against [DNA]<sub>tot</sub> to obtain the <italic toggle="yes">K</italic><sub>D</sub> using
eq 1. For the binding measurements using tryptophan
fluorescence, the nonfluroescent AU<sup>F</sup>/A duplex was used
(Table <xref rid="bi026226rt00001"></xref>).
</p><p><italic toggle="yes">Fluorescence Measurements of Association and Dissociation Rate Constants.</italic> The observed rate constants for association of 2-AP-labeled DNA molecules with the various
enzymes were obtained using an Applied Photophysics 720
stopped-flow fluorescence instrument (Surrey, U.K.) using
pseudo-first-order conditions in which the concentration of
the enzyme was always more than 4-fold greater than the
concentration of the labeled DNA. In these experiments a
syringe containing a solution of enzyme was rapidly mixed
with a solution of labeled DNA delivered from a second
syringe. The fluorescence change as a function of time was
recorded using a 360 nm cutoff filter with excitation at 320
nm. For stopped-flow measurements in which changes in
UDG tryptophan fluorescence were followed, excitation was
at 290 nm and emission was monitored at wavelengths
greater than 320 nm. In all experiments in which tryptophan
fluorescence was followed, the AU/A nonfluorescent substrate was used. The kinetic traces were fitted to a first-order
rate expression (eq 3)<xref rid="bi026226re00003"></xref><disp-formula content-type="pre-labeled" id="bi026226re00003"><!--%@md;sys;6q@%%@ital@%F%@rsf@%%@ital@%%@sb@%t%@rsf@%%@sbx@% = &Dgr;%@ital@%F%@rsf@% exp%@fn;(;vis;full;auto@%1 −%@ital@% k%@rsf@%%@sb@%obsd%@sbx@%%@ital@%t%@rsf@%%@fnx;);vis;full@% + %@ital@%F%@rsf@%%@sb@%o%@sbx@%%@id;reqid;3@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00003.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
to obtain the observed rate constants (<italic toggle="yes">k</italic><sub>obsd</sub>) at each concentration of enzyme or DNA. With some mutant enzymes, the
<italic toggle="yes">k</italic><sub>obsd</sub> values showed a linear dependence on [E], indicating a
simple one-step binding mechanism (eq 4) or, alternatively,
a multistep mechanism in which bimolecular association is
fully rate-limiting at all achievable concentrations of enzyme.
Accordingly, the association rate was obtained from the slope
of a plot of <italic toggle="yes">k</italic><sub>obsd</sub> against [E], and <italic toggle="yes">k</italic><sub>on</sub> and <italic toggle="yes">k</italic><sub>off</sub> were obtained
from the slope and intercept of the linear regression best-fit
line to the data (eq 5). With other mutant enzymes plots of
<italic toggle="yes">k</italic><sub>obsd</sub> against [AU<sup>F</sup>/A] showed curvature, indicating a more
complex multistep binding mechanism. In these cases the
data were fitted as described below for wtUDG.<xref rid="bi026226re00004"></xref><disp-formula content-type="pre-labeled" id="bi026226re00004"><!--%@md;sys;6q@%E + U %@mspx;$-$90@%%@aw;@rlhar2;;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%on%@sbx@%%@sbx@%%@au@%%@ex@%%@ital@%k%@rsf@%%@sb@%off%@sbx@%%@exx@%%@awx@% E·DNA%@id;reqid;4@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00004.gif" position="anchor" orientation="portrait"></graphic></disp-formula><xref rid="bi026226re00005"></xref><disp-formula content-type="pre-labeled" id="bi026226re00005"><!--%@md;sys;6q@%%@ital@%k%@rsf@%%@sb@%obsd%@sbx@% = %@ital@%k%@rsf@%%@sb@%on%@sbx@%%@fn;[;vis;full;auto@%DNA%@fnx;];vis;full@% + %@ital@%k%@rsf@%%@sb@%off%@sbx@%%@id;reqid;5@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00005.gif" position="anchor" orientation="portrait"></graphic></disp-formula></p><p>For wild-type UDG, curvature in the plot of <italic toggle="yes">k</italic><sub>obsd</sub> against
[E] or [DNA] was observed, indicating a change in rate-limiting step from bimolecular association to unimolecular
isomerization of the DNA or enzyme (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00010" ref-type="bibr"></xref>, <xref rid="bi026226rb00021" ref-type="bibr"></xref></named-content></italic>). Thus, the plot
of <italic toggle="yes">k</italic><sub>obsd</sub> against [E] was fitted to eq 6, which is the general
analytical solution for the hyperbolic concentration dependence of a minimal two-step base-flipping mechanism (eq
7).<xref rid="bi026226re00006"></xref><disp-formula content-type="pre-labeled" id="bi026226re00006"><!--%@md;sys;6q@%%@ital@%k%@rsf@%%@sb@%obsd%@sbx@% = %@fr@%%@ital@%K%@rsf@%‘%@fn;[;vis;full;auto@%E%@fnx;];vis;full@%%@ital@%k%@rsf@%%@sb@%max%@sbx@% + %@ital@%k%@rsf@%%@sb@%off%@sbx@%%@fd@%%@fn;(;vis;full;auto@%%@ital@%K%@rsf@%‘%@fn;[;vis;full;unlock@%E%@fnx;];vis;full@% + 1%@fnx;);vis;full@%%@frx@%%@id;reqid;6@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00006.gif" position="anchor" orientation="portrait"></graphic></disp-formula><xref rid="bi026226re00007"></xref><disp-formula content-type="pre-labeled" id="bi026226re00007"><!--%@md;sys;6q@%E + S %@mspx;$-$90@%%@aw;@rlhar2;;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%1%@sbx@%%@sbx@%%@au@%%@ex@%%@ital@%k%@rsf@%%@sb@%$-$1%@sbx@%%@exx@%%@awx@% ES %@mspx;$-$90@%%@aw;@rlhar2;;none;none@%%@sb@%%@ital@%k%@rsf@%%@sb@%2%@sbx@%%@sbx@%%@au@%%@ex@%%@ital@%k%@rsf@%%@sb@%$-$2%@sbx@%%@exx@%%@awx@% E*F%@id;reqid;7@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00007.gif" position="anchor" orientation="portrait"></graphic></disp-formula>
[As noted previously (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>), there are no observed kinetic lags
in the binding of UDG to DNA because of the rapid rate
constants <italic toggle="yes">k</italic><sub>-</sub><sub>1</sub>, <italic toggle="yes">k</italic><sub>2</sub>, and <italic toggle="yes">k</italic><sub>-</sub><sub>2</sub> in eq 7. Thus, <italic toggle="yes">k</italic><sub>obsd</sub> is always a
single exponential with a hyperbolic dependence on DNA
or enzyme concentration.] In eq 6, the initial slope of the
hyperbolic concentration dependence provides the apparent
second-order rate constant for association of the enzyme with
the DNA [<italic toggle="yes">k</italic><sub>on</sub> = <italic toggle="yes">K</italic>‘<italic toggle="yes">k</italic><sub>max</sub>, where<italic toggle="yes"> K</italic>‘ = <italic toggle="yes">k</italic><sub>1</sub>/(<italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> + <italic toggle="yes">k</italic><sub>max</sub>)], the
asymptotic value provides the observed rate constant for
reversible base flipping (<italic toggle="yes">k</italic><sub>max</sub> = <italic toggle="yes">k</italic><sub>2</sub> + <italic toggle="yes">k</italic><sub>-</sub><sub>2</sub>), and the <italic toggle="yes">y</italic> intercept provides the dissociation rate constant [<italic toggle="yes">k</italic><sub>off</sub> = <italic toggle="yes">k</italic><sub>-</sub><sub>1</sub><italic toggle="yes">k</italic><sub>-</sub><sub>2</sub>/
(<italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> + <italic toggle="yes">k</italic><sub>max</sub>)]. In this minimal two-step mechanism, the base-flipping step (<italic toggle="yes">k</italic><sub>max</sub> = <italic toggle="yes">k</italic><sub>2</sub> + <italic toggle="yes">k</italic><sub>-</sub><sub>2</sub>) is a composite of the base
extrusion and UDG isomerization steps shown in Figure <xref rid="bi026226rf00001"></xref>B
(<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). Depending on whether 2-AP or tryptophan fluorescence
is followed, <italic toggle="yes">k</italic><sub>max</sub> is designated as <italic toggle="yes">k</italic><sub>max</sub><sup>2-AP</sup> or <italic toggle="yes">k</italic><sub>max</sub><sup>trp</sup>, respectively. An equation of form identical to that of eq 6 can result
if more than two steps are involved, but the microscopic rate
constants that comprise the apparent constants <italic toggle="yes">K</italic>‘, <italic toggle="yes">k</italic><sub>max</sub>, and
<italic toggle="yes">k</italic><sub>off</sub> will differ. A three-step mechanism, such as that shown
in Figure <xref rid="bi026226rf00001"></xref>B, is best analyzed by computer simulation
of the kinetic data (see below). Regardless of whether eq 5,
eq 6, or a more complex three-step binding mechanism is
used to analyze the data, <italic toggle="yes">k</italic><sub>off</sub>/<italic toggle="yes">k</italic><sub>on</sub> = <italic toggle="yes">K</italic><sub>D</sub>, the overall dissociation
constant for the interaction. The apparent rate constants
obtained from these analytical expressions are more accurate
than the microscopic rate constants obtained from computer
simulations, and provide informative parameters for comparison of the kinetic properties of the wild-type and mutant
enzymes.
</p><p>To augment the approach-to-equilibrium kinetic measurements, the dissociation rate constant (<italic toggle="yes">k</italic><sub>off</sub>) of U/A from the
various enzymes was also measured using irreversible
conditions. When 2-AP fluorescence was followed, these
experiments were carried out by rapidly mixing a preformed
enzyme−DNA complex with a large excess of nonfluorescent single-stranded trapping DNA. The sequence of the trap
DNA was the same as that of the AU<sup>F</sup>A strand of the duplex
(Table <xref rid="bi026226rt00001"></xref>). The time-dependent decrease in 2-AP fluorescence
was then followed using the stopped-flow fluorescence
instrument. When tryptophan fluorescence was followed, the
free enzyme was trapped using a high concentration of
nonspecific DNA (50 μM). In all cases, the kinetic traces
were fitted to a single-exponential decay (eq 8). Further
experimental details may be found in the figure captions.<xref rid="bi026226re00008"></xref><disp-formula content-type="pre-labeled" id="bi026226re00008"><!--%@md;sys;6q@%%@ital@%F%@rsf@%%@sb@%t%@sbx@% = &Dgr;%@ital@%F%@rsf@% exp%@fn;(;vis;full;auto@%−%@ital@%kt%@rsf@%%@fnx;);vis;full@% + %@ital@%F%@rsf@%%@sb@%o%@sbx@%%@id;reqid;8@%%@mx@%[S_EL2;quad]
--><graphic xlink:href="bi026226re00008.gif" position="anchor" orientation="portrait"></graphic></disp-formula></p><p><italic toggle="yes">Computer Simulations</italic>. The values for <italic toggle="yes">k</italic><sub>on</sub>, <italic toggle="yes">k</italic><sub>off</sub>, <italic toggle="yes">k</italic><sub>max</sub>, and
<italic toggle="yes">K</italic><sub>D</sub> were used as starting values to constrain simulations in
which the microscopic rate constants for a more complex
three-step mechanism for base flipping were determined
(Figure <xref rid="bi026226rf00001"></xref>B). The simulations were performed by globally
fitting the kinetic traces to a single set of six microscopic
rate constants as defined in the three-step mechanism
depicted in Figure <xref rid="bi026226rf00001"></xref>B (<italic toggle="yes">k</italic><sub>-</sub><sub>1</sub>, <italic toggle="yes">k</italic><sub>1</sub>, <italic toggle="yes">k</italic><sub>flip</sub>, <italic toggle="yes">k</italic><sub>-</sub><sub>flip</sub>, <italic toggle="yes">k</italic><sub>conf</sub>, <italic toggle="yes">k</italic><sub>-</sub><sub>conf</sub>) using
the program Dynafit (<italic toggle="yes"><xref rid="bi026226rb00022" ref-type="bibr"></xref></italic>). The requirement for a three-step
mechanism in which the conformational clamping step of
UDG lags behind the initial extrusion of the uracil was
suggested by the observation that the <italic toggle="yes">k</italic><sub>max</sub><sup>trp</sup> value obtained
from following the tryptophan fluorescence of UDG was
about 40% less than the <italic toggle="yes">k</italic><sub>max</sub><sup>2-AP</sup> value obtained from
following the 2-AP fluorescence of the DNA (see the Results
and Discussion).
</p></sec><sec id="d7e873"><title>Results and Discussion</title><p><italic toggle="yes">Definitions. </italic>To allow facile comparison of the mutational
effects with the pyrene rescue effects reported in the
following paper (<italic toggle="yes"><xref rid="bi026226rb00023" ref-type="bibr"></xref></italic>), we have defined the mutational effect
as the kinetic (or binding) parameter for the wild-type
enzyme divided by that for the mutant enzyme. Thus, effects
greater than unity always indicate a fold <italic toggle="yes">damaging </italic>effect
on a rate constant (i.e., a slower on-rate or faster off-rate) or
a <italic toggle="yes">weakening</italic> of binding affinity as a result of the mutation.
To maintain consistency and simplicity in the description of
the effects, we report the mutational effects on the off-rate
and <italic toggle="yes">K</italic><sub>D</sub> values as (wild-type value)<sup>-1</sup>/(mutant value)<sup>-1</sup> such
that ratios greater than unity still reflect the fold damaging
effect of the mutation.<xref rid="bi026226rb00002" ref-type="bibr"></xref></p><p><italic toggle="yes">Mutational Effects on DNA Binding</italic>. Eight different UDG
mutations are investigated in this study (Figure <xref rid="bi026226rf00001"></xref>A): three
serine mutations that remove the hydroxyl groups that are
proposed to <italic toggle="yes">pinch</italic> the phosphodiester backbone (S88A,
S189A, S88A:S189A), two leucine mutations that lack the
bulky side chain that <italic toggle="yes">pushes</italic> into the minor groove (L191A,
L191G), asparagine and histidine mutations (N123G, H187G)
that remove hydrogen-bonding groups that <italic toggle="yes">pull</italic> on the uracil
by bonding to O2, O4, and N3, and an aspartate mutation
that removes the water activating group (D64N). These
mutations probe the four components of the pinch, push,
plug, and pull mechanism for base flipping (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00002" ref-type="bibr"></xref>, <xref rid="bi026226rb00007" ref-type="bibr"></xref></named-content></italic>). The
characterization of D64N mutation serves as a useful control
for the specificity of the substrate rescue effects that are
reported in the following paper (<italic toggle="yes"><xref rid="bi026226rb00023" ref-type="bibr"></xref></italic>). This aspartate group
has previously been shown to have no detrimental effect on
DNA binding or base flipping, and mainly serves to stabilize
the ionic transition state and intermediate for glycosidic bond
cleavage by an electrostatic mechanism (<italic toggle="yes"><xref rid="bi026226rb00024" ref-type="bibr"></xref></italic>). Thus, the
preorganized substrate with the pyrene wedge would not be
expected to rescue the ∼3000-fold damaging effect of
removing Asp64 (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Binding of Wild-Type and Mutant Enzymes to U<sup>F</sup></italic><sup></sup><italic toggle="yes">/A
Analogues</italic>. To begin the mutational analysis, we performed
DNA binding measurements using the nonreactive substrate
analogue constructs PU<sup>F</sup>/A and AU<sup>F</sup>/A employing the 2-AP
and tryptophan fluorescence assays. The 2-AP fluorescence
is very sensitive to base-stacking interactions and reports on
the expulsion of the uracil from the duplex, while the
tryptophan fluorescence detects the postflipping conformational change in UDG. We have employed 11-mer duplex
substrates in this study because of concerns that longer DNA
sequences, such as the 19-mer used in a previous study (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>),
might lead to a significant amount of nonspecific DNA
binding for the base-flipping mutants. Accordingly, we have
truncated the DNA to a minimal length that maintains
essentially full catalytic activity as observed previously for
longer DNA molecules (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00007" ref-type="bibr"></xref>, <xref rid="bi026226rb00025" ref-type="bibr"></xref></named-content></italic>). Several crystal structures of
human UDG productively bound to DNA duplexes of this
size have been solved, indicating that these 11-mers capture
all of the interactions required for base flipping and catalysis
(<italic toggle="yes">2</italic>,<italic toggle="yes"> 6</italic>, <italic toggle="yes">14</italic>). The <italic toggle="yes">K</italic><sub>D</sub> values for wtUDG are reported in Table
<xref rid="bi026226rt00002"></xref> for comparison with those of the mutants.
<table-wrap id="bi026226rt00002" position="float" orientation="portrait"><label>2</label><caption><p>Equilibrium and Kinetic Constants for Binding of Mutant UDG Enzymes to U<sup>F</sup>/A DNA Analogues<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><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">enzyme</oasis:entry><oasis:entry namest="2" nameend="2">substrate</oasis:entry><oasis:entry namest="3" nameend="3"><italic toggle="yes">k</italic><sub>on</sub> (μM<sup>-1</sup> s<sup>-1</sup>)</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">k</italic><sub>off</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="5" nameend="5"><italic toggle="yes">k</italic><sub>max</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="6" nameend="6"><italic toggle="yes">K</italic>‘ (μM<sup>-1</sup>)</oasis:entry><oasis:entry namest="7" nameend="7"><italic toggle="yes">k</italic><sub>off</sub> /<italic toggle="yes">k</italic><sub>on</sub> (μM)</oasis:entry><oasis:entry namest="8" nameend="8"><italic toggle="yes">K</italic><sub>D</sub> (μM)
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">wtUDG
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">160 ± 6
</oasis:entry><oasis:entry colname="4">21 ± 3
</oasis:entry><oasis:entry colname="5">650 ± 43
</oasis:entry><oasis:entry colname="6">0.40 ± 0.08
</oasis:entry><oasis:entry colname="7">0.13 ± 0.02
</oasis:entry><oasis:entry colname="8">0.13 ± 0.03
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">120 ± 11
</oasis:entry><oasis:entry colname="4">22 ± 5
</oasis:entry><oasis:entry colname="5">400 ± 10
</oasis:entry><oasis:entry colname="6">0.3 ± 0.03
</oasis:entry><oasis:entry colname="7">0.18 ± 0.04
</oasis:entry><oasis:entry colname="8">0.11 ± 0.02
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">S88A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">47 ± 15
</oasis:entry><oasis:entry colname="4">80 ± 15
</oasis:entry><oasis:entry colname="5">≥650<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="6">0.032 ± 0.002
</oasis:entry><oasis:entry colname="7">1.7 ± 0.5
</oasis:entry><oasis:entry colname="8">1.2 ± 0.3
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">34 ± 14
</oasis:entry><oasis:entry colname="4">88 ± 6
</oasis:entry><oasis:entry colname="5">680 ± 110
</oasis:entry><oasis:entry colname="6">0.05 ± 0.016
</oasis:entry><oasis:entry colname="7">2.6 ± 1.2
</oasis:entry><oasis:entry colname="8">1.1 ± 0.2
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">S189A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">46 ± 5
</oasis:entry><oasis:entry colname="4">23 ± 1
</oasis:entry><oasis:entry colname="5">650 ± 170
</oasis:entry><oasis:entry colname="6">0.09 ± 0.03
</oasis:entry><oasis:entry colname="7">0.47 ± 0.05
</oasis:entry><oasis:entry colname="8">0.62 ± 0.06
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">13 ± 5
</oasis:entry><oasis:entry colname="4">8 ± 3
</oasis:entry><oasis:entry colname="5">210 ± 40
</oasis:entry><oasis:entry colname="6">0.06 ± 0.02
</oasis:entry><oasis:entry colname="7">0.6 ± 0.2
</oasis:entry><oasis:entry colname="8">0.33 ± 0.03
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">S88A:S189A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">14 ± 3
</oasis:entry><oasis:entry colname="4">205 ± 30
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7">14 ± 4
</oasis:entry><oasis:entry colname="8">8.4 ± 1.4
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">L191A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">20 ± 6
</oasis:entry><oasis:entry colname="4">56 ± 12
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7">2.8 ± 1.0
</oasis:entry><oasis:entry colname="8">0.95 ± 0.05
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">L191G
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">9.9 ± 1.9
</oasis:entry><oasis:entry colname="4">93 ± 20
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7">9.4 ± 2.7
</oasis:entry><oasis:entry colname="8">5.1 ± 1.5
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N123G
</oasis:entry><oasis:entry colname="2">PU/A
</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">2.1 ± 0.4<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">H187G
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">34 ± 5
</oasis:entry><oasis:entry colname="4">4.6 ± 0.5
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7">0.14 ± 0.03
</oasis:entry><oasis:entry colname="8">0.28 ± 0.04
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D64N
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">216 ± 9
</oasis:entry><oasis:entry colname="4">8.9 ± 0.5
</oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry><oasis:entry colname="7">0.041 ± 0.003
</oasis:entry><oasis:entry colname="8">0.020 ± 0.002</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Some of the equilibrium binding parameters for wtUDG have been previously published (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>). The parameters <italic toggle="yes">k</italic><sub>on</sub>, <italic toggle="yes">k</italic><sub>off</sub>, <italic toggle="yes">k</italic><sub>max</sub>, and <italic toggle="yes">K</italic>‘ are defined
in eqs 6 and 7. The <italic toggle="yes">k</italic><sub>max</sub> values for AU<sup>F</sup>/A were obtained from the rates of tryptophan fluorescence changes in UDG (<italic toggle="yes">k</italic><sub>max</sub><sup>trp</sup>). The <italic toggle="yes">k</italic><sub>max</sub> values for
PU<sup>F</sup>/A were obtained from the rates of 2-AP fluorescence changes of the DNA (<italic toggle="yes">k</italic><sub>max</sub><sup>2-AP</sup>). The parameters for PU<sup>F</sup>/A and AU<sup>F</sup>/A were determined
in stopped-flow experiments by following the 2-AP fluorescence of the DNA or the tryptophan fluorescence of UDG, respectively.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Only a lower
limit value was obtained with the S88A mutant.<italic toggle="yes"><sup>c</sup></italic><sup></sup> This is the <italic toggle="yes">K</italic><sub>m</sub> value of N123G for the corresponding substrate. Binding measurements with
PU<sup>F</sup>/A were complicated by apparent sigmoidicity, which was not observed in the kinetic measurements (data not shown).</p></table-wrap-foot></table-wrap><table-wrap id="bi026226rt00003" position="float" orientation="portrait"><label>3</label><caption><p>Microscopic Rate Constants for a Three-Step Base-Flipping Mechanism by wtUDG Determined from Kinetic Simulations<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="7"><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:tbody><oasis:row><oasis:entry namest="1" nameend="1"><italic toggle="yes">k</italic><sub>1</sub> (μM<sup>-1</sup> s<sup>-1</sup>)</oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">k</italic><sub>-1</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="3" nameend="3"><italic toggle="yes">k</italic><sub>flip</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">k</italic><sub>-flip</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="5" nameend="5"><italic toggle="yes">k</italic><sub>conf</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="6" nameend="6"><italic toggle="yes">k</italic><sub>-conf</sub> (s<sup>-1</sup>)</oasis:entry><oasis:entry namest="7" nameend="7"><italic toggle="yes">K</italic><sub>flip</sub><italic toggle="yes">K</italic><sub>conf</sub><italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">220 ± 50
</oasis:entry><oasis:entry colname="2">600 ± 100
</oasis:entry><oasis:entry colname="3">700 ± 200
</oasis:entry><oasis:entry colname="4">180 ± 50
</oasis:entry><oasis:entry colname="5">350 ± 50
</oasis:entry><oasis:entry colname="6">100 ± 10
</oasis:entry><oasis:entry colname="7">14 ± 4</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> The rate constants were obtained from global simulations of the transient kinetic data shown in Figure <xref rid="bi026226rf00004"></xref>A,B,D,E using the program Dynafit
(<italic toggle="yes"><xref rid="bi026226rb00022" ref-type="bibr"></xref></italic>) and correspond to the steps in Figure <xref rid="bi026226rf00001"></xref>B. The simulated fits to the data and the Dynafit script files are provided in the Supporting Information.
A unique fit to the data required an estimate for the equilibrium constant for the first nonspecific binding step. This estimate (<italic toggle="yes">K</italic><sub>D</sub><sup>ns</sup> = 3.6 ± 0.5 μM)
was obtained for an 11-mer duplex using competition binding measurements as previously described (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>). The off-rate of nonspecific DNA (<italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> =
400−750 s<sup>-1</sup>) has been previously estimated (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). These estimates for <italic toggle="yes">K</italic><sub>D</sub><sup>ns</sup> and <italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> are in good agreement with fluorescence binding measurements
using a nonspecific pyrene-containing 11-mer duplex (<italic toggle="yes">K</italic><sub>D</sub><sup>ns</sup> = 2.7 ± 0.3 μM, <italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> = 440 ± 40 s<sup>-1</sup>). The data were also constrained by the overall
dissociation constant <italic toggle="yes">K</italic><sub>D</sub> = (<italic toggle="yes">K</italic><sub>1</sub><italic toggle="yes">K</italic><sub>flip</sub><italic toggle="yes">K</italic><sub>conf</sub>)<sup>-1</sup> = 0.15 ± 0.04 μM (average value). The estimated errors in the microscopic constants are based on
systematic sweeping of the individual rate constants over a 4-fold range, and then comparing visual fits to all the rate and equilibrium data. The
final optimized fits to all of the kinetic data were obtained by constrained nonlinear regression fitting using the same set of six microscopic rate
constants (a ±10% deviation in the rate constants was allowed during fitting). The reported values are average values obtained from fitting all of
the four data sets.<italic toggle="yes"><sup>b</sup></italic><sup></sup> <italic toggle="yes">K</italic><sub>flip</sub><italic toggle="yes">K</italic><sub>conf</sub> is the overall equilibrium constant for base flipping on the enzyme ([E*F]/[ES]). This product of equilibrium constants
is comparable to the previously reported net equilibrium constant for base flipping based on a two-step mechanism (<italic toggle="yes">K</italic><sub>flip</sub> = 19 ± 8 for a U/A base
pair) (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>).</p></table-wrap-foot></table-wrap></p><p>Representative binding data for the S88A “pinching”
mutant using the 2-AP and tryptophan fluorescence assays
are shown in parts A and B, respectively, of Figure <xref rid="bi026226rf00002"></xref>. The
<italic toggle="yes">K</italic><sub>D</sub> values obtained from these data are 1.2 ± 0.3 and 1.1 ±
0.2 μM (Table <xref rid="bi026226rt00002"></xref>), which indicates that removal of the
hydroxyl group of Ser88 weakens binding by 9-fold as
compared to that of wtUDG (Table <xref rid="bi026226rt00004"></xref>). Analogous measurements were performed for the other mutations, and the <italic toggle="yes">K</italic><sub>D</sub>
values and mutational effects are reported in Tables <xref rid="bi026226rt00002"></xref> and
<xref rid="bi026226rt00004"></xref>, respectively. In general, the binding defects arising from
removal of the Ser88, Ser189, and L191 side chains are in
the range 5−40-fold, with the largest effect being observed
for the L191G “pushing” mutation. The double mutation,
S88A:S189A, shows a 65-fold detrimental effect on binding,
which is only modestly greater than the 44-fold damaging
effect expected from multiplying the individual effects of
each single mutation.
<table-wrap id="bi026226rt00004" position="float" orientation="portrait"><label>4</label><caption><p>Mutational Effects on DNA Binding, Association, and Dissociation<italic toggle="yes"><sup>a</sup></italic><sup></sup></p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><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:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="6">fold mutational effect</oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1">enzyme</oasis:entry><oasis:entry namest="2" nameend="2">DNA</oasis:entry><oasis:entry namest="3" nameend="3">1/<italic toggle="yes">K</italic><sub>D</sub></oasis:entry><oasis:entry namest="4" nameend="4"><italic toggle="yes">k</italic><sub>on</sub></oasis:entry><oasis:entry namest="5" nameend="5">1/<italic toggle="yes">k</italic><sub>off</sub></oasis:entry><oasis:entry namest="6" nameend="6"><italic toggle="yes">k</italic><sub>max</sub></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">S88A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">9.2
</oasis:entry><oasis:entry colname="4">3.4
</oasis:entry><oasis:entry colname="5">3.8
</oasis:entry><oasis:entry colname="6"><italic toggle="yes">b</italic></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">3.5
</oasis:entry><oasis:entry colname="5">4
</oasis:entry><oasis:entry colname="6">0.59
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">S189A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">4.8
</oasis:entry><oasis:entry colname="4">3.5
</oasis:entry><oasis:entry colname="5">1.1
</oasis:entry><oasis:entry colname="6">1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">AU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">3
</oasis:entry><oasis:entry colname="4">9.2
</oasis:entry><oasis:entry colname="5">0.36
</oasis:entry><oasis:entry colname="6">1.9
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">S88A:S189A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">65
</oasis:entry><oasis:entry colname="4">11
</oasis:entry><oasis:entry colname="5">9.8
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">L191A
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">7.3
</oasis:entry><oasis:entry colname="4">8
</oasis:entry><oasis:entry colname="5">2.7
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">L191G
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">39
</oasis:entry><oasis:entry colname="4">16
</oasis:entry><oasis:entry colname="5">4.4
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">N123G
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">16
</oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">H187G
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">2.1
</oasis:entry><oasis:entry colname="4">4.7
</oasis:entry><oasis:entry colname="5">0.22
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">D64N
</oasis:entry><oasis:entry colname="2">PU<sup>F</sup>/A
</oasis:entry><oasis:entry colname="3">0.15
</oasis:entry><oasis:entry colname="4">0.74
</oasis:entry><oasis:entry colname="5">0.42
</oasis:entry><oasis:entry colname="6"></oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Mutational effects are defined as wild-type value/mutant value.
Therefore, effects greater than unity indicate a damaging effect of the
mutation (i.e., a decrease in the on-rate or an increase in the off-rate or
<italic toggle="yes">K</italic><sub>D</sub>). All values are derived from measurements using the PU<sup>F</sup>/A
analogue except for the effects on the S88A and S189A mutants, which
were obtained with both the AU<sup>F</sup>/A and PU<sup>F</sup>/A analogues as indicated.
The definitions of the parameters may be found in eqs 4−7. The average
error for all the mutational effects is ±14%; the largest errors are less
than ±30%.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Only a lower limit value for <italic toggle="yes">k</italic><sub>max</sub><sup>2-AP</sup> of ∼650 s<sup>-1</sup> was
obtained for S88A.</p></table-wrap-foot></table-wrap></p><p>We observed a large difference between the mutational
effects of removing the two side chains that hydrogen bond
with the flipped-out uracil (Asn123 and His187), indicating
distinct roles for these two “pulling” groups (Tables <xref rid="bi026226rt00002"></xref> and
<xref rid="bi026226rt00004"></xref>). The N123G mutation binds 16-fold more weakly than
wtUDG as determined from its <italic toggle="yes">K</italic><sub>m</sub> = 2.1 ± 0.4 μM, which
was measured using the 2-AP steady-state kinetic assay (<italic toggle="yes"><xref rid="bi026226rb00011" ref-type="bibr"></xref></italic>).
In contrast, the H187G mutation has a small 2-fold detrimental effect on DNA binding, even though this mutation
has a ∼4000-fold damaging effect on the activation barrier
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00020" ref-type="bibr"></xref>, <xref rid="bi026226rb00026" ref-type="bibr"></xref></named-content></italic>). Thus, Asn123 interacts strongly with the O4 and
H3 atoms of the uracil in the ground state, while the strong
hydrogen bond between uracil O2 and H<sup>ε</sup> of His187 develops
later during the transition state for glycosidic bond cleavage
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00017" ref-type="bibr"></xref>, <xref rid="bi026226rb00018" ref-type="bibr"></xref></named-content></italic>). As we have previously observed, the D64N
mutation actually enhances binding by about 8-fold, which
we have attributed to ablation of an unfavorable electrostatic
interaction between Asp64 and the anionic phosphodiester
backbone of the DNA (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Nature of the Extrahelical State for Each Mutant. </italic>Several
of the mutant enzymes that showed substantial increases in
2-AP fluorescence did not show any decrease in tryptophan
fluorescence upon binding of AU<sup>F</sup>/A, indicating that these
enzymes were defective in the postflipping conformational
change in UDG (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). Shown in Figure <xref rid="bi026226rf00003"></xref> are the maximal
2-AP and tryptophan fluorescence changes for wtUDG and
each mutant (<italic toggle="yes">F</italic><sub>bound</sub>/<italic toggle="yes">F</italic><sub>free</sub>). The S88A:S189A, L191A, H187G,
and D64N enzymes show almost the same <italic toggle="yes">F</italic><sub>bound</sub>/<italic toggle="yes">F</italic><sub>free</sub> for
binding PU<sup>F</sup>/A as the wild-type enzyme (<italic toggle="yes">F</italic><sub>bound</sub>/<italic toggle="yes">F</italic><sub>free</sub>(wtUDG)
= 3.1 ± 0.2), indicating that for these enzymes the DNA
adopts a conformation similar to that found in the wild-type
enzyme. The ratio <italic toggle="yes">F</italic><sub>bound</sub>/<italic toggle="yes">F</italic><sub>free</sub> for the S88A, S189A, and
L191G mutants is approximately 35% smaller than that for
wtUDG, suggesting modest differences in the extrahelical
state that is detected by 2-AP fluorescence for these enzymes.
<fig id="bi026226rf00003" position="float" orientation="portrait"><label>3</label><caption><p>Maximal changes in DNA 2-AP fluorescence and UDG tryptophan fluorescence. The changes are reported as the ratio<italic toggle="yes">F</italic><sub>bound</sub>/<italic toggle="yes">F</italic><sub>free</sub>, where <italic toggle="yes">F</italic><sub>bound</sub> is the fluorescence of the PU<sup>F</sup>/A DNA or enzyme
at saturation and <italic toggle="yes">F</italic><sub>free</sub> is the fluorescence of the free DNA or
enzyme.</p></caption><graphic xlink:href="bi026226rf00003.tif" position="float" orientation="portrait"></graphic></fig></p><p>Most of the mutants show tryptophan fluorescence decreases upon DNA binding that are similar to those of wtUDG (<italic toggle="yes">F</italic><sub>bound</sub>/<italic toggle="yes">F</italic><sub>free</sub>(Trp) = 0.59 ± 0.05) (Figure <xref rid="bi026226rf00004"></xref>). However,
neither the Leu191 deletion mutants nor the N123G and
serine double mutant shows a significant decrease. These
results strongly indicate that removal of these groups severely
alters the internal equilibrium for the conformational docking step that is required to lock in the flipped-out uracil
(<italic toggle="yes">k</italic><sub>conf</sub>/<italic toggle="yes">k</italic><sub>-</sub><sub>conf</sub>, Figure <xref rid="bi026226rf00001"></xref>B). Given that a significant 2-AP fluorescence increase was detected with these mutants, the absence
of a tryptophan fluorescence decrease suggests that the
reaction has been arrested at an otherwise sparsely populated
intermediate in which the uracil is extrahelical, but the
enzyme has not yet enveloped the base. These results support
our two previous studies of base flipping where we concluded
that uracil expulsion was followed closely by a conformational change in UDG (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>), and that part of the action of
Leu191 <italic toggle="yes">followed</italic> the complete or partial expulsion of the
uracil from the base stack (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00007" ref-type="bibr"></xref>, <xref rid="bi026226rb00008" ref-type="bibr"></xref></named-content></italic>). Further insights into the
roles of Leu191 are suggested from the binding and kinetic
studies described below.
<fig id="bi026226rf00004" position="float" orientation="portrait"><label>4</label><caption><p>Stopped-flow fluorescence kinetic studies of DNA association and dissociation from wtUDG. (A) Approach-to-equilibrium association measurements where 100 nM PU<sup>F</sup>/A DNA was mixed with the indicated concentrations of UDG and the 2-AP fluorescence
increase was monitored with a 360 nm cutoff filter with excitation at 320 nm. The lines are the fits to a first-order rate equation. (B)
Irreversible dissociation of PU<sup>F</sup>/A DNA from UDG. A solution of 500 nM PU<sup>F</sup>/A DNA and 1 μM UDG was mixed with a 10 μM concentration
of an ssAU<sup>F</sup> 11-mer nonfluorescent DNA trap to prevent reassociation of the enzyme to PU<sup>F</sup>/A. The time-dependent decrease in 2-AP
fluorescence was measured. (C) Observed rate constants from (A) (○) and (B) (·) against [wtUDG]. The curve is a best fit to eq 6. (D)
Approach-to-equilibrium association measurements where 100 nM UDG was mixed with the indicated concentrations of AU<sup>F</sup>/A DNA and
the tryptophan fluorescence decrease was monitored with a 320 nm cutoff filter with excitation at 290 nm. The lines are fits to a first-order
decay rate equation. (E) Irreversible dissociation of AU<sup>F</sup>/A DNA from UDG as monitored by UDG tryptophan fluorescence. A solution of
500 nM AU<sup>F</sup>/A DNA and 1 μM UDG was mixed with a 50 μM concentration of a 19-mer nonfluorescent DNA to trap the enzyme as it
dissociated (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). The time-dependent increase in 2-AP fluorescence was measured. (F) Observed rate constants from (D) (○) and (E) (·)
against [AU<sup>F</sup>/A]. The curve is a best fit to eq 6. For comparison, the dashed line is the theoretical curve obtained in (C) from the 2-AP
measurements. The parameters are reported in Table <xref rid="bi026226rt00002"></xref>.</p></caption><graphic xlink:href="bi026226rf00004.tif" position="float" orientation="portrait"></graphic></fig></p><p>The fluorescence studies indicate that the N123G mutation
results in a highly unusual binding mode, leading to an
increased unstacking of the 2-AP base (Figure <xref rid="bi026226rf00003"></xref>). Binding
of the N123G mutant to PU<sup>F</sup>/A gives rise to an anomalous
21-fold increase in 2-AP fluorescence that is 7-fold <italic toggle="yes">larger</italic>
than that of wild-type UDG.
</p><p><italic toggle="yes">Transient Kinetic Studies of Base Flipping by Wild-Type
UDG.</italic> Although we have already investigated the kinetic
process of base flipping by UDG using a 19-mer DNA in
which the uracil was located in an A tract sequence (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>),
the significantly different substrates used here required
further measurements. This was especially important because
of the strong DNA sequence dependence of the UDG activity
(<italic toggle="yes">25</italic>, <italic toggle="yes">27</italic>, <italic toggle="yes">28</italic>). The approach we have taken is based on our
previous findings that base flipping and the postflipping
conformational change in UDG can be followed using the
2-AP fluorescence increase of the DNA, or the tryptophan
fluorescence decrease of UDG, respectively (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>).
</p><p>Several stopped-flow kinetic traces for approach-to-equilibrium binding of the PU<sup>F</sup>/A 11-mer to wtUDG are
shown in Figure <xref rid="bi026226rf00004"></xref>A in which the increase in 2-AP fluorescence is followed. We also measured the off-rate of PU<sup>F</sup>/A
from wtUDG using irreversible conditions in which the
dissociated enzyme was trapped by a large excess of
nonfluorescent 11-mer DNA, and the dissociation of this
complex was well-fitted as a single-exponential kinetic
process (Figure <xref rid="bi026226rf00004"></xref>B). A plot of the observed association rate
constants against [wtUDG] is hyperbolic (Figure <xref rid="bi026226rf00004"></xref>C),
suggesting at least a two-step binding mechanism. Fitting
these data to eq 6 yields <italic toggle="yes">k</italic><sub>on</sub> = 160 ± 6 μM<sup>-1</sup> s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>off</sub> =
21 ± 3 s<sup>-1</sup>, <italic toggle="yes">K</italic>‘ = 0.40 ± 0.08 μM<sup>-1</sup>, and <italic toggle="yes">k</italic><sub>max</sub> = 650 ± 43
s<sup>-1</sup> (Table <xref rid="bi026226rt00002"></xref>). The measured off-rate (closed circle, Figure
<xref rid="bi026226rf00004"></xref>C) agrees very well with the <italic toggle="yes">y</italic> intercept obtained from fitting
the observed association rate constants to eq 6 (Figure <xref rid="bi026226rf00004"></xref>C),
and the ratio <italic toggle="yes">k</italic><sub>off</sub><italic toggle="yes">/k</italic><sub>on</sub> = 0.13 ± 0.02 is in excellent agreement
with the measured <italic toggle="yes">K</italic><sub>D</sub> = 0.13 ± 0.05 (Table <xref rid="bi026226rt00002"></xref>). The single-exponential off-rate of PU<sup>F</sup>/A from wtUDG indicates that
the multistep process of DNA dissociation has<italic toggle="yes"> one </italic>major
rate-limiting transition state. Further studies described below
indicate that this transition state is for the reverse conformational change shown in Figure <xref rid="bi026226rf00001"></xref>B (<italic toggle="yes">k</italic><sub>-</sub><sub>conf</sub>).
</p><p>The hyperbolic concentration dependence of <italic toggle="yes">k</italic><sub>obsd</sub> for
wtUDG binding to PU<sup>F</sup>/A indicates a change in rate-limiting
step from concentration-dependent association of the enzyme
with the DNA to concentration-independent isomerization
of the enzyme−DNA complex. These results using the
PU<sup>F</sup>/A 11-mer are similar to those previously reported using
a U<sup>F</sup>/A 19-mer duplex in which the uracil was located in an
A-rich tract (5‘-AAPU<sup>F</sup>AAAAA-3‘). With the previous 19-mer, <italic toggle="yes">k</italic><sub>on</sub> = 320 ± 50 μM<sup>-1</sup> s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>off</sub> = 28 ± 2 s<sup>-1</sup>, <italic toggle="yes">K</italic><sub>D</sub> =
0.09 ± 0.02, and <italic toggle="yes">k</italic><sub>max</sub> = 1200 ± 200 s<sup>-1</sup>. The 1.8-fold larger
<italic toggle="yes">k</italic><sub>max</sub> value, and the 1.6-fold tighter binding of the 19-mer as
compared to the 11-mer, quantitatively accounts for the
observation that the 19-mer shows a 1.6-fold larger increase
in 2-AP fluorescence upon UDG binding as compared to
the 11-mer used here (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). These results indicate that the
base-flipping rates (<italic toggle="yes">k</italic><sub>flip</sub> and <italic toggle="yes">k</italic><sub>-</sub><sub>flip</sub>), and the equilibrium
constant for base flipping (<italic toggle="yes">K</italic><sub>flip</sub> = <italic toggle="yes">k</italic><sub>flip</sub>/<italic toggle="yes">k</italic><sub>-</sub><sub>flip</sub>) may be modestly
affected by the sequence context in which the uracil is
located. DNA sequence effects on the steady-state <italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub>
for human (<italic toggle="yes"><xref rid="bi026226rb00027" ref-type="bibr"></xref></italic>), <italic toggle="yes">E. coli</italic> (<italic toggle="yes"><xref rid="bi026226rb00025" ref-type="bibr"></xref></italic>), and herpes virus UDG (<italic toggle="yes"><xref rid="bi026226rb00028" ref-type="bibr"></xref></italic>)
have been reported, and the results here suggest that part of
the <italic toggle="yes">k</italic><sub>cat</sub>/<italic toggle="yes">K</italic><sub>m</sub> effects may arise from a DNA sequence dependence of the base-flipping step.
</p><p>We also followed the forward binding kinetics and the
reverse dissociation kinetics by monitoring tryptophan
fluorescence of wtUDG (Figure <xref rid="bi026226rf00004"></xref>D,E). The closing of
the UDG clamp upon AU<sup>F</sup>/A binding results in a 1.7-fold decrease in tryptophan fluorescence, and the reverse
clamp-opening step results in a recovery of tryptophan
fluorescence of the same magnitude. The observed rate
constants for AU<sup>F</sup>/A binding showed a hyperbolic dependence on [AU<sup>F</sup>/A] similar to that seen when the 2-AP
fluorescence of PU<sup>F</sup>/A was monitored (Figure <xref rid="bi026226rf00004"></xref>F). Although
the values <italic toggle="yes">k</italic><sub>on</sub> = 120 ± 11 μM<sup>-1</sup> s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>off</sub> = 22 ± 5 s<sup>-1</sup>, and
<italic toggle="yes">K</italic>‘ = 0.3 ± 0.03 μM<sup>-1</sup> were similar to those measured by
following the 2-AP fluorescence, the <italic toggle="yes">k</italic><sub>max</sub> value was found
to be 40% smaller (<italic toggle="yes">k</italic><sub>max</sub> = 400 ± 10 s<sup>-1</sup>). (For comparison,
the best-fit curve from fitting the <italic toggle="yes">k</italic><sub>obsd</sub> values obtained using
2-AP fluorescence is shown as a dashed line in Figure <xref rid="bi026226rf00004"></xref>F.)
An important observation from these data is that the off-rates measured using tryptophan and 2-AP fluorescence are
<italic toggle="yes">identical</italic> (Figure <xref rid="bi026226rf00004"></xref>B,E). This result indicates that the same
rate-limiting step is detected in both experiments, and
requires that opening of the conformational clamp (<italic toggle="yes">k</italic><sub>-</sub><sub>conf</sub> in
Figure <xref rid="bi026226rf00001"></xref>B) is slow compared to the reverse step of base
flipping (<italic toggle="yes">k</italic><sub>-</sub><sub>flip</sub>) and DNA dissociation (<italic toggle="yes">k</italic><sub>-</sub><sub>1</sub>).
</p><p><italic toggle="yes">A Three-Step Mechanism for Uracil Flipping</italic>. The 40%
higher <italic toggle="yes">k</italic><sub>max</sub> value that was measured using the 2-AP
fluorescence signal strongly indicates that the UDG conformational change slightly lags behind the base-flipping step
with these substrates that contain a U/A base pair. Our
previous study using a 19-mer DNA with a significantly
different sequence surrounding the U<sup>F</sup>/A base pair showed
smaller rate differences for the 2-AP and tryptophan fluorescence changes (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>). These differences were hardly beyond
the error limits in the measurements [<italic toggle="yes">k</italic><sub>max</sub>(2-AP) = 1200 ±
200 s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>max</sub>(trp) = 800 ± 200 s<sup>-1</sup>], but we concluded that
base flipping was “followed closely by a conformational
change in UDG”. Because the binding kinetics and mutational effects reported below clearly reveal that the conformational change lags behind the flipping step, we have
used global kinetic simulation of the data to obtain the microscopic rate constants for a three-step mechanism (Figure <xref rid="bi026226rf00001"></xref>B).
The kinetic constants obtained from these simulations are
<italic toggle="yes">k</italic><sub>1</sub> = 220 ± 50, <italic toggle="yes">k</italic><sub>-</sub><sub>1</sub> = 600 ± 100 s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>flip</sub> = 700 ± 200 s<sup>-1</sup>,
<italic toggle="yes">k</italic><sub>-</sub><sub>flip</sub> = 180 ± 50 s<sup>-1</sup>, <italic toggle="yes">k</italic><sub>conf</sub> = 350 ± 50 s<sup>-1</sup>, and <italic toggle="yes">k</italic><sub>-</sub><sub>conf</sub> =
100 ± 10 s<sup>-1</sup>; <italic toggle="yes">K</italic><sub>flip</sub><italic toggle="yes">K</italic><sub>conf</sub> = 14 ± 4 (Table <xref rid="bi026226rt00003"></xref>). The simulated
fits to the individual kinetic traces are shown in the
Supporting Information.
</p><p><italic toggle="yes">Kinetic Roles of the Pinch, Push, Plug, and Pull Groups</italic>.
Analogous stopped-flow fluorescence experiments were
performed for each mutant to assess whether each side chain
interaction was important for the forward rate of DNA
association or, alternatively, served to stabilize the major
extrahelical state (E*F, Figure <xref rid="bi026226rf00001"></xref>B). Representative kinetic
experiments for binding of L191A to PU<sup>F</sup>/A are shown in
Figure <xref rid="bi026226rf00005"></xref> in which changes in 2-AP fluorescence were
followed. From the linear plot of <italic toggle="yes">k</italic><sub>obsd</sub> against [L191A] in
Figure <xref rid="bi026226rf00005"></xref>C we obtained <italic toggle="yes">k</italic><sub>on</sub> = 20 ± 6 μM<sup>-1</sup> s<sup>-1</sup> and <italic toggle="yes">k</italic><sub>off</sub> = 56
± 12 s<sup>-1</sup>. Thus, for L191A, <italic toggle="yes">k</italic><sub>on</sub> is decreased by 8-fold and
<italic toggle="yes">k</italic><sub>off</sub> is increased by 3-fold as compared to those of wtUDG,
while slightly larger effects of 16-fold and 4.4-fold were
measured for the L191G mutant (Tables <xref rid="bi026226rt00002"></xref> and <xref rid="bi026226rt00004"></xref>, data not
shown). L191A did not show any evidence of curvature in
the plots of <italic toggle="yes">k</italic><sub>obsd</sub> against [enzyme] when the PU<sup>F</sup>/A substrate
was used, indicating that bimolecular association was slower
than any subsequent isomerization events that may be
occurring. It should be reemphasized that L191A (as well
as L191G and S88A:S189A) does not proceed to the
conformational docking step that results in tryptophan
fluorescence quenching of wtUDG (Figure <xref rid="bi026226rf00004"></xref>). Thus, the
kinetic results for L191A binding to PU<sup>F</sup>/A measure the
observed rate for formation of a metastable extrahelical
intermediate that most likely resembles the EF complex in
Figure <xref rid="bi026226rf00001"></xref>B.
<fig id="bi026226rf00005" position="float" orientation="portrait"><label>5</label><caption><p>Stopped-flow fluorescence kinetic studies of DNA association and dissociation from L191A. (A) Approach-to-equilibrium association measurements where 1 μM PU<sup>F</sup>/A DNA was mixed
with the indicated concentrations of L191A and the 2-AP fluorescence increase was monitored with a 360 nm cutoff filter with
excitation at 320 nm. The lines are the fits to a first-order rate
equation. (B) Irreversible dissociation of PU<sup>F</sup>/A DNA from L191A.
A solution of 500 nM PU<sup>F</sup>/A DNA and 1 μM L191A was mixed
with a 10 μM concentration of an ssAU<sup>F</sup> 11-mer nonfluorescent
DNA trap to prevent reassociation of the enzyme to PU<sup>F</sup>/A. The
time-dependent decrease in 2-AP fluorescence was measured.
(C) Observed rate constants from (A) (○) and (B) (·) against
[L191A]. The curve is a best fit to eq 5. The parameters are reported
in Table <xref rid="bi026226rt00002"></xref>.</p></caption><graphic xlink:href="bi026226rf00005.tif" position="float" orientation="portrait"></graphic></fig></p><p>Since the S88A and S189A serine pinching mutants both
showed tryptophan fluorescence decreases upon binding
AU<sup>F</sup>/A (Figure <xref rid="bi026226rf00003"></xref>), it was possible to probe the binding
kinetics by following 2-AP fluorescence and tryptophan
fluorescence. Both of these enzymes bind DNA weakly, and
show a lesser increase in 2-AP fluorescence than the wild-type enzyme, which led to very weak signal changes when
using the 2-AP assay (Figure <xref rid="bi026226rf00006"></xref>A). Nevertheless, the results
were reproducible on different days, with different batches
of enzyme and DNA, and the approach-to-equilibrium kinetic
results were in good or excellent agreement with both the
thermodynamic measurements and the irreversible off-rate
measurements (Table <xref rid="bi026226rt00002"></xref>). In addition, the tryptophan fluorescence measurements were more robust than the 2-AP
measurements (compare parts A and B of Figure <xref rid="bi026226rf00006"></xref>), and
yielded results that were consistent with those obtained using
the 2-AP probe. For both the S88A and S189A mutants
(Figure <xref rid="bi026226rf00006"></xref>C,D), the plots of <italic toggle="yes">k</italic><sub>obsd</sub> against enzyme concentration
were slightly curved when 2-AP fluorescence was followed,
but were distinctly hyperbolic when tryptophan fluorescence
was monitored. In addition, the observed rate constants were
always faster for the 2-AP fluorescence changes as compared
to the tryptophan fluorescence changes. These results are
reminiscent of wtUDG where the formation of the extrahelical base was more rapid than the conformational change
(Figure <xref rid="bi026226rf00004"></xref>F), and indicate that the conformational change
remains the rate-limiting step for the S88A and S189A
mutants. These mutants have 3.5−9-fold slower association
rates as compared to wtUDG, showing that these groups play
a modest role in facilitating the formation of the extrahelical
states (EF and E*F in Figure <xref rid="bi026226rf00001"></xref>B). However, the rate of the
conformational change that is detected by tryptophan
fluorescence with these mutants is not significantly impaired as
compared to the wild-type enzyme (Table <xref rid="bi026226rt00002"></xref>). Thus, removal
of these individual serine side chains does not prevent
attainment of the closed conformation as judged by the
similar tryptophan fluorescence decreases with wtUDG, nor
does it dramatically slow its rate of formation. However, as
noted above, removal of <italic toggle="yes">both</italic> side chains in the S88A:S189A
double mutant abrogates the conformational change entirely,
suggesting cooperative action of these groups in the overall
process of attaining this final state that immediately precedes
glycosidic bond cleavage. Although these data suggest that
the serine mutants follow a three-step mechanism for base
flipping like wtUDG, the data do not constrain the modeling
sufficiently to allow unambiguous assignment of microscopic
rate constants. Therefore, in the analysis of these results
below we merely compare the apparent rate constants for
the 2-AP and tryptophan fluorescence changes with those
of wtUDG.
<fig id="bi026226rf00006" position="float" orientation="portrait"><label>6</label><caption><p>Stopped-flow fluorescence kinetic studies of DNA association and dissociation from S189A and S88A. (A) Approach-to-equilibrium association measurements where 200 nM PU<sup>F</sup>/A DNA was mixed with the indicated concentrations of S189A and the 2-AP
fluorescence increase was monitored with a 360 nm cutoff filter with excitation at 320 nm. The lines are the fits to a first-order rate
equation. (B) Approach-to-equilibrium association measurements where 100 nM S189A was mixed with the indicated concentrations of
AU<sup>F</sup>/A DNA and the tryptophan fluorescence decrease was monitored with a 320 nm cutoff filter with excitation at 290 nm. The lines are
fits to a first-order decay rate equation. (C) Observed rate constants from (A) (▵) and (B) (○) against [S189A]. The curve is a best fit to
eq 6. The kinetic parameters are reported in Table <xref rid="bi026226rt00002"></xref>. (D) Observed rate constants for S88A binding to PU<sup>F</sup>/A (▵) and against [AU<sup>F</sup>/A] (○).
The curve is a best fit to eq 6.
</p></caption><graphic xlink:href="bi026226rf00006.tif" position="float" orientation="portrait"></graphic></fig></p><p>The N123G and H187G pulling mutations and the D64N
control were also investigated using the same kinetic
approaches (Tables <xref rid="bi026226rt00002"></xref> and <xref rid="bi026226rt00004"></xref>). Due to the weak DNA binding
by N123G, and the total lack of a tryptophan fluorescence
change upon DNA binding, we were unable to determine
any kinetic constants for this enzyme. Surprisingly, the
kinetic measurements with H187G revealed compensating
effects on <italic toggle="yes">k</italic><sub>on</sub> and <italic toggle="yes">k</italic><sub>off</sub> (Table <xref rid="bi026226rt00002"></xref>). That is, the 5-fold decrease
in <italic toggle="yes">k</italic><sub>on</sub> is offset by a nearly equal decrease in <italic toggle="yes">k</italic><sub>off</sub>, such that
the ratio <italic toggle="yes">k</italic><sub>off</sub>/<italic toggle="yes">k</italic><sub>on</sub> is unchanged from that of the wild-type
enzyme (Table <xref rid="bi026226rt00002"></xref>). Finally, the kinetic studies of D64N
indicate that the ∼3-fold smaller <italic toggle="yes">k</italic><sub>off</sub>/<italic toggle="yes">k</italic><sub>on</sub> ratio for D64N as
compared to wtUDG arises from a 1.3-fold increase in <italic toggle="yes">k</italic><sub>on</sub>
and a 2-fold smaller <italic toggle="yes">k</italic><sub>off</sub>. These small effects on DNA binding
and base flipping are consistent with the primary function
of Asp64 in transition-state stabilization.
</p></sec><sec id="d7e2638"><title>Conclusions</title><p>These results allow construction of a temporal pathway
for base flipping, and suggest functional roles for the
conserved serine, leucine, and asparagine side chains at
several steps in the overall process of base flipping (Figure
<xref rid="bi026226rf00007"></xref>). In this model, which is depicted as a free energy reaction
coordinate diagram in Figure <xref rid="bi026226rf00007"></xref>, we assign the temporal
formation of these interactions as “early” or “late” in the
overall process of forming the final extrahelical state (E*F).
The early classification includes all ground states and
transition states leading to the metastable state where the
uracil is extrahelical and the enzyme is in the open
conformation (EF). The late classification includes the
transition state and ground state for formation of the closed
conformation, which can be monitored by tryptophan fluorescence (E*F). The functional implications suggested here
by mutagenesis are further supported by the pyrene substrate
rescue studies in the following paper (<italic toggle="yes"><xref rid="bi026226rb00023" ref-type="bibr"></xref></italic>).
<fig id="bi026226rf00007" position="float" orientation="portrait"><label>7</label><caption><p>Free energy reaction coordinate diagram for the temporal action of the serine, leucine, and asparagine groups that are involved in the three-step pinch, push, plug, and pull base-flipping mechanism. The interactions are described loosely as “early” if they are important in the ground states or transition states that precede the formation of the EF intermediate and “late” if they are important in the formation or stabilization of the final docked complex (E*F). The first step involves formation of a weak nonspecific encounter complex (ES), which is rapidly isomerized in the second transition state to the extrahelical state detected by 2-AP fluorescence (EF). Removal of Leu191, Ser88, or Ser189 diminishes the rate of formation of the EF species. The E*F complex is severely destabilized (a late effect) when Leu191 or Asn123 is removed or when<italic toggle="yes">both</italic> Ser88 and Ser189 are removed (the long vertical arrow depicts this large destabilization). In addition, the
S189A mutation destabilizes the transition state for formation of the E*F state, while the S88A mutation increases the energy of the ground
state of the complex. These modest effects are depicted as short vertical arrows. To emphasize the energetic features of the pathway, the
diagram is drawn on a roughly linear scale to correspond with the rate constants reported in Table <xref rid="bi026226rt00003"></xref>.
</p></caption><graphic xlink:href="bi026226rf00007.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Serine Pinching</italic>. Deletion of Ser88 or Ser189, which
interact with the 5‘ and 3‘ phosphodiester groups of the
deoxyuridine nucleotide (Figure <xref rid="bi026226rf00001"></xref>A), results in less than a
+1.8 kcal/mol effect on the overall binding equilibrium
(Table <xref rid="bi026226rt00004"></xref>), reflecting perturbations both at early and late steps
in the base-flipping pathway (Figure <xref rid="bi026226rf00007"></xref>). An early and late
role for these pinching interactions is suggested by the effect
of the S88A:S189A double mutation, which shows an 11-fold decrease in the rate of formation of the metastable
intermediate (an early effect), and a strong destabilization
of the closed state reflected in the absence of a tryptophan
fluorescence change and a large <italic toggle="yes">k</italic><sub>off</sub> (late effects). Serine 88
does not appear to form a strong interaction in the apparent
transition state for formation of the closed conformation,
because its <italic toggle="yes">k</italic><sub>max</sub><sup>trp</sup> is similar to that of wtUDG (Table <xref rid="bi026226rt00002"></xref>).
However, a modest interaction of Ser88 in the ground-state
E*F complex is indicated by the 4-fold faster off-rate of
S88A as compared to wtUDG, suggesting that E*F is
destabilized when the Ser88 side chain is removed (Figure
<xref rid="bi026226rf00007"></xref>). In contrast, the 2-fold slower <italic toggle="yes">k</italic><sub>max</sub><sup>trp</sup> resulting from the
removal of Ser189 is most simply accounted for by a
transition-state effect rather than a ground-state effect. This
conclusion is supported by the observation that the same ∼2-fold damaging effect is seen both in the forward rate of
formation of E*F by Ser189A (i.e., <italic toggle="yes">k</italic><sub>max</sub><sup>trp</sup>) and in the reverse
direction (i.e., <italic toggle="yes">k</italic><sub>off</sub>) (Figure <xref rid="bi026226rf00007"></xref>). Most importantly, for both
S88A and S189A, the off-rates measured using tryptophan
fluorescence are equal to or slower than the values measured
using the 2-AP probe. Thus, as observed for wtUDG, the
reverse conformational change is still the rate-limiting step,
and not the reverse flipping or DNA dissociation steps.
</p><p><italic toggle="yes">Leucine Pushing and Plugging</italic>. An early and late role for
Leu191 is also suggested. Removal of this side chain
destabilizes the closed conformation such that no tryptophan
fluorescence changes can be detected, requiring a late role
for Leu191 in stabilizing the E*F complex and perhaps in
accelerating its formation. Since the major bound DNA form
with the Leu191 mutants is the open EF complex, the 8-
and 16-fold smaller <italic toggle="yes">k</italic><sub>on</sub> values for these enzymes must reflect
the removal of important interactions of this side chain that
occur in the early steps of the base-flipping reaction. An early
and late role is also consistent with our previous pyrene
rescue studies where we suggested that Leu191 pushed the
uracil base in the early stages of the base-flipping process,
and later served a plugging role to increase the lifetime of
the base in the active site pocket (<italic toggle="yes"><xref rid="bi026226rb00007" ref-type="bibr"></xref></italic>). The pyrene rescue
results reported in the following paper (<italic toggle="yes"><xref rid="bi026226rb00023" ref-type="bibr"></xref></italic>) further support
an early and late role for Leu191.
</p><p><italic toggle="yes">Pulling by Asn123 and His187</italic>. Of these two putative
pulling groups, only the removal of Asn123 shows a strong
damaging effect on base flipping. Although we were not able
to measure the binding kinetics with N123G, the weak
binding and the absence of a tryptophan fluorescence change
suggest that this Asn123 interacts late in the base-flipping
process, although an additional early role cannot be excluded.
The enormous 2-AP fluorescence increase upon N123G
binding is so large it suggests that the removal of Asn123
may have carved out a hole in the base binding pocket that
is large enough to allow partial or complete flipping of the
2-AP probe or, alternatively, that the DNA conformation is
perturbed such that the 2-AP base becomes significantly more
unstacked with its neighboring bases in the complex with
N123G. The former explanation is intriguing because it
implies that UDG could flip other bases, if the active site
were sterically compatible. This is clearly possible because
UDG mutants that act on cytidine and thymidine nucleotides
have been reported. However, there is no evidence that
wtUDG can flip any other base into the active site pocket
due to the strict steric constraints and optimized hydrogen-bonding groups that are specific for uracil (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi026226rb00002" ref-type="bibr"></xref>, <xref rid="bi026226rb00010" ref-type="bibr"></xref></named-content></italic>).
</p><p>The 5-fold smaller <italic toggle="yes">k</italic><sub>on</sub> for the H187G mutant may indicate
an early or late role for this group, but removal of His187
has no large effect on the stability of the flipped-out base or
that of the closed conformation. The observed effects of
His187 on base flipping may arise from its interaction with
uracil O2, and/or the short hydrogen bond between its
backbone amide and the DNA phosphodiester backbone.
These small effects on base flipping further highlight the
primary role of His187 in transition-state stabilization (<italic toggle="yes"><xref rid="bi026226rb00029" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Summary</italic>. The mutational studies performed here provide
strong support for the stepwise nature of the base-flipping
process by UDG that involves both an early destabilization
of the DNA duplex, resulting in a metastable extrahelical
state (EF, Figure <xref rid="bi026226rf00007"></xref>), and a late conformational change in
the enzyme that allows positioning of active site groups
around the extrahelical base (E*F). This gating step is not
detected with nonspecific DNA (<italic toggle="yes"><xref rid="bi026226rb00010" ref-type="bibr"></xref></italic>), and is abrogated by
the removal of the Leu191 pushing and plugging group or
the Asn123 pulling residue or when both of the hydroxyl
side chains of the serine pinching groups are deleted (Figure
<xref rid="bi026226rf00007"></xref>). The uncoupling of the early and late steps in base flipping
by mutagenesis has allowed assignment of the interactions
of these side chains in the apparent transition states and
ground states in both steps of the reaction. Remarkably, the
substrate rescue studies that are described in the following
paper show how the early and late effects can be fully
rescued by a substrate that contains a pyrene nucleotide
wedge (<italic toggle="yes"><xref rid="bi026226rb00023" ref-type="bibr"></xref></italic>).
</p></sec></body><back><notes notes-type="si"><sec id="d7e2745"><title><ext-link xlink:href="/doi/suppl/10.1021%2Fbi026226r">Supporting Information Available</ext-link></title><p>Kinetic simulations of the stopped-flow kinetic traces in
Figure <xref rid="bi026226rf00004"></xref>A,B,E,F and the associated Dynafit script files. This
material is available free of charge via the Internet at <uri xlink:href="http://pubs.acs.org">http://pubs.acs.org</uri>.
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Fax: (410) 955-3023. E-mail: jstivers@jhmi.edu.</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">2002-08-23</dateCreated><dateIssued encoding="w3cdtf">2002-09-17</dateIssued><copyrightDate encoding="w3cdtf">2002</copyrightDate></originInfo><note type="footnote" ID="bi026226rAF2"> Supported by NIH Grant RO1GM56834 (to J.T.S.).</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>The DNA repair enzyme uracil DNA glycosylase (UDG) locates unwanted uracil bases in genomic DNA using a remarkable base-flipping mechanism in which the entire deoxyuridine nucleotide is rotated from the DNA base stack into the enzyme active site. Enzymatic base flipping has been described as a three-step process involving phosphodiester backbone pinching, base extrusion through active pushing and plugging by a leucine side chain that inserts in the DNA minor groove, and, finally, pulling by hydrogen-bonding groups that interact with the extrahelical base. Here we employ mutagenesis in combination with transient kinetic approaches to assess the functional roles of six conserved enzymatic groups of UDG that have been implicated in the “pinch, push, plug, and pull” base-flipping mechanism. Our results show that these mutant enzymes are capable of flipping the uracil base from the duplex, but that many of these mutations prevent a subsequent induced fit conformational step in which catalytic groups of UDG dock with the flipped-out base. These studies support our previous model for base flipping in which a conformational gating step closely follows base extrusion from the DNA duplex [Stivers, J. T., et al. 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Biophys. 2001 396 1 9 10.1006/abbi.2001.2605</note><identifier type="doi">10.1006/abbi.2001.2605</identifier><part><date>2001</date><detail type="volume"><caption>vol.</caption><number>396</number></detail><extent unit="pages"><start>1</start><end>9</end></extent></part></relatedItem><relatedItem type="references" ID="bi026226rn00001" displayLabel="bibbi026226rn00001"><titleInfo><title>Abbreviations: wtUDG, wild-type uracil DNA glycosylase; 2‘-FU, 2‘-fluoro-2‘-deoxyuridine; 2-AP, 2-aminopurine;kmaxtrp, the maximal rate constant in eq 6 for the tryptophan fluorescence change of UDG;kmax2-AP, the maximal rate constant in eq 6 for the 2-AP fluorescence change of the DNA.</title></titleInfo><note type="content-in-line">Abbreviations: wtUDG, wild-type uracil DNA glycosylase; 2‘-FU, 2‘-fluoro-2‘-deoxyuridine; 2-AP, 2-aminopurine; kmaxtrp, the maximal rate constant in eq 6 for the tryptophan fluorescence change of UDG; kmax2-AP, the maximal rate constant in eq 6 for the 2-AP fluorescence change of the DNA.</note></relatedItem><relatedItem type="references" ID="bi026226rn00002" displayLabel="bibbi026226rn00002"><titleInfo><title>The effects of these mutations on the steady-state kinetic parameters of UDG have also been measured and will be reported elsewhere (Jiang and Stivers, manuscript in preparation).</title></titleInfo><note type="content-in-line">The effects of these mutations on the steady-state kinetic parameters of UDG have also been measured and will be reported elsewhere (Jiang and Stivers, manuscript in preparation).</note></relatedItem><identifier type="istex">169EFB7AE2312B134544780300F313FC2A4D6E42</identifier><identifier type="ark">ark:/67375/TPS-1R2NQZG3-D</identifier><identifier type="DOI">10.1021/bi026226r</identifier><accessCondition type="use and reproduction" contentType="restricted">Copyright © 2002 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-1R2NQZG3-D/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-1R2NQZG3-D/annexes.gif</uri></json:item><json:item><extension>tiff</extension><original>true</original><mimetype>image/tiff</mimetype><uri>https://api.istex.fr/document/169EFB7AE2312B134544780300F313FC2A4D6E42/annexes/tiff</uri></json:item></annexes><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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