DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase
Identifieur interne : 003952 ( Main/Exploration ); précédent : 003951; suivant : 003953DNA Contacts by Protein Domains of the Molluscum Contagiosum Virus Type-1B Topoisomerase
Auteurs : Young Hwang ; Minkyu Park ; Wolfgang H. Fischer ; Alex Burgin Jr. ; Frederic BushmanSource :
- Virology [ 0042-6822 ] ; 1999.
English descriptors
- Teeft :
- Academic press, Active site tyrosine, Additional monomers, Aggregated complexes, Amino, Amino acid sequencing, Amino acids, Apparent rate, Assay, Base pair, Binding activity, Catalytic, Catalytic domain, Catalytic domains, Cheng, Cleavage, Coding region, Complex formation, Contagiosum, Control reactions, Correct contacts, Covalent, Covalent catalysis, Covalent complexes, Covalent form, Digestion, Digestion products, Domain, Domain structure, Edman degradation, Enzyme, Escherichia coli, Expression vector, Fragment, Fragment encoding, High concentrations, Human topoisomerase, Hwang, Individual tubes, Initial binding, Initial cleavage step, Input substrate, Lambda integrase, Lambda integrase family, Linker, Linker region, Modest effects, Molluscum, Molluscum contagiosum, Molluscum contagiosum virus topoisomerase, Nacl, Optimal conditions, Other members, Partial proteolysis, Pentamer, Petersen, Phosphotyrosine linkage, Poxvirus, Poxvirus topoisomerase, Previous studies, Protein domains, Reaction mixture, Reaction mixtures, Reaction products, Recognition site, Relative noncovalent binding nucleotide sequence, Relaxation activity, Religation, Religation activity, Religation reaction, Scissile phosphate, Sequencing type, Short duplex extension, Shuman, Stable domains, Stable form, Structural studies, Suicide substrate, Sulfur atom, Topoisomerase, Trypsin, Unpublished data, Vaccinia, Vaccinia topoisomerase, Vaccinia virus, Various times, Wild type.
Abstract
Abstract: All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.
Url:
DOI: 10.1006/viro.1999.9920
Affiliations:
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Fischer</name><affiliation><wicri:noCountry code="subField">92037</wicri:noCountry></affiliation></author><author><name sortKey="Burgin Jr, Alex" sort="Burgin Jr, Alex" uniqKey="Burgin Jr A" first="Alex" last="Burgin Jr.">Alex Burgin Jr.</name><affiliation><wicri:noCountry code="subField">92182-4614</wicri:noCountry></affiliation></author><author><name sortKey="Bushman, Frederic" sort="Bushman, Frederic" uniqKey="Bushman F" first="Frederic" last="Bushman">Frederic Bushman</name><affiliation><wicri:noCountry code="subField">92037</wicri:noCountry></affiliation></author></analytic><monogr></monogr><series><title level="j">Virology</title><title level="j" type="abbrev">YVIRO</title><idno type="ISSN">0042-6822</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999">1999</date><biblScope unit="volume">262</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="479">479</biblScope><biblScope unit="page" to="491">491</biblScope></imprint><idno type="ISSN">0042-6822</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0042-6822</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Academic press</term><term>Active site tyrosine</term><term>Additional monomers</term><term>Aggregated complexes</term><term>Amino</term><term>Amino acid sequencing</term><term>Amino acids</term><term>Apparent rate</term><term>Assay</term><term>Base pair</term><term>Binding activity</term><term>Catalytic</term><term>Catalytic domain</term><term>Catalytic domains</term><term>Cheng</term><term>Cleavage</term><term>Coding region</term><term>Complex formation</term><term>Contagiosum</term><term>Control reactions</term><term>Correct contacts</term><term>Covalent</term><term>Covalent catalysis</term><term>Covalent complexes</term><term>Covalent form</term><term>Digestion</term><term>Digestion products</term><term>Domain</term><term>Domain structure</term><term>Edman degradation</term><term>Enzyme</term><term>Escherichia coli</term><term>Expression vector</term><term>Fragment</term><term>Fragment encoding</term><term>High concentrations</term><term>Human topoisomerase</term><term>Hwang</term><term>Individual tubes</term><term>Initial binding</term><term>Initial cleavage step</term><term>Input substrate</term><term>Lambda integrase</term><term>Lambda integrase family</term><term>Linker</term><term>Linker region</term><term>Modest effects</term><term>Molluscum</term><term>Molluscum contagiosum</term><term>Molluscum contagiosum virus topoisomerase</term><term>Nacl</term><term>Optimal conditions</term><term>Other members</term><term>Partial proteolysis</term><term>Pentamer</term><term>Petersen</term><term>Phosphotyrosine linkage</term><term>Poxvirus</term><term>Poxvirus topoisomerase</term><term>Previous studies</term><term>Protein domains</term><term>Reaction mixture</term><term>Reaction mixtures</term><term>Reaction products</term><term>Recognition site</term><term>Relative noncovalent binding nucleotide sequence</term><term>Relaxation activity</term><term>Religation</term><term>Religation activity</term><term>Religation reaction</term><term>Scissile phosphate</term><term>Sequencing type</term><term>Short duplex extension</term><term>Shuman</term><term>Stable domains</term><term>Stable form</term><term>Structural studies</term><term>Suicide substrate</term><term>Sulfur atom</term><term>Topoisomerase</term><term>Trypsin</term><term>Unpublished data</term><term>Vaccinia</term><term>Vaccinia topoisomerase</term><term>Vaccinia virus</term><term>Various times</term><term>Wild type</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: All poxviruses studied encode a type 1B topoisomerase that introduces transient nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by different domains. Partial proteolysis of the enzyme revealed two stable domains separated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was sensitive to sequences in the favored 5′-(T/C)CCTT-3′ recognition site and also flanking DNA, indicating that some of the sequence-specific contacts are made by residues 82-323. Assays of initial binding and covalent catalysis by MCV-TOP(82-323) identified the contacts flanking the 5′-CCCTT-3′ sequence at +10, +9, −2, and −3 to be important. Tests with substrates containing a 5-bridging phosphorothiolate that trap the cleaved complex revealed that correct contacts to the flanking sequences were important in the initial cleavage step. MCV-TOP(82-323) differed from the full-length protein in showing reduced sensitivity to mutations at a position within the 5′-(T/C)CCTT-3′ recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide insight into the division of labor within the MCV-TOP enzyme.</div></front></TEI><affiliations><list></list><tree><noCountry><name sortKey="Burgin Jr, Alex" sort="Burgin Jr, Alex" uniqKey="Burgin Jr A" first="Alex" last="Burgin Jr.">Alex Burgin Jr.</name><name sortKey="Bushman, Frederic" sort="Bushman, Frederic" uniqKey="Bushman F" first="Frederic" last="Bushman">Frederic Bushman</name><name sortKey="Fischer, Wolfgang H" sort="Fischer, Wolfgang H" uniqKey="Fischer W" first="Wolfgang H." last="Fischer">Wolfgang H. Fischer</name><name sortKey="Hwang, Young" sort="Hwang, Young" uniqKey="Hwang Y" first="Young" last="Hwang">Young Hwang</name><name sortKey="Park, Minkyu" sort="Park, Minkyu" uniqKey="Park M" first="Minkyu" last="Park">Minkyu Park</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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