Mutational Analysis of Vaccinia DNA Ligase Defines Residues Essential for Covalent Catalysis
Identifieur interne : 000E88 ( Istex/Corpus ); précédent : 000E87; suivant : 000E89Mutational Analysis of Vaccinia DNA Ligase Defines Residues Essential for Covalent Catalysis
Auteurs : Stewart Shuman ; Xiao-Mei RuSource :
- Virology [ 0042-6822 ] ; 1995.
Abstract
Abstract: DNA ligation entails AMP transfer from ATP to the 5′ end of DNA to form a DNA-adenylate structure, A(5′)pp(5′)N. A similar reaction involving GMP transfer occurs during 5′ capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.
Url:
DOI: 10.1006/viro.1995.1380
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A similar reaction involving GMP transfer occurs during 5′ capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Stewart Shuman</name><affiliations><json:string>Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021</json:string></affiliations></json:item><json:item><name>Xiao-Mei Ru</name><affiliations><json:string>Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021</json:string></affiliations></json:item></author><arkIstex>ark:/67375/6H6-DS7BHGD7-Z</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: DNA ligation entails AMP transfer from ATP to the 5′ end of DNA to form a DNA-adenylate structure, A(5′)pp(5′)N. A similar reaction involving GMP transfer occurs during 5′ capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase AMP formation. 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A similar reaction involving GMP transfer occurs during 5′ capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.</p></abstract></profileDesc><revisionDesc><change when="1995">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-DS7BHGD7-Z/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier converted-article found"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YVIRO</jid><aid>71380</aid><ce:pii>S0042-6822(85)71380-3</ce:pii><ce:doi>10.1006/viro.1995.1380</ce:doi><ce:copyright type="full-transfer" year="1995">Academic Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Regular Article</ce:textfn></ce:dochead><ce:title>Mutational Analysis of Vaccinia DNA Ligase Defines Residues Essential for Covalent Catalysis</ce:title><ce:author-group><ce:author><ce:given-name>Stewart</ce:given-name><ce:surname>Shuman</ce:surname></ce:author><ce:author><ce:given-name>Xiao-Mei</ce:given-name><ce:surname>Ru</ce:surname></ce:author><ce:affiliation><ce:textfn>Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021</ce:textfn></ce:affiliation></ce:author-group><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>DNA ligation entails AMP transfer from ATP to the 5′ end of DNA to form a DNA-adenylate structure, A(5′)pp(5′)N. A similar reaction involving GMP transfer occurs during 5′ capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase AMP formation. These results are concordant with mutational studies of yeast RNA capping enzyme and suggest a common structural basis for covalent nucleotidyl transfer.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Mutational Analysis of Vaccinia DNA Ligase Defines Residues Essential for Covalent Catalysis</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Mutational Analysis of Vaccinia DNA Ligase Defines Residues Essential for Covalent Catalysis</title></titleInfo><name type="personal"><namePart type="given">Stewart</namePart><namePart type="family">Shuman</namePart><affiliation>Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Xiao-Mei</namePart><namePart type="family">Ru</namePart><affiliation>Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length 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>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1995</dateIssued><copyrightDate encoding="w3cdtf">1995</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: DNA ligation entails AMP transfer from ATP to the 5′ end of DNA to form a DNA-adenylate structure, A(5′)pp(5′)N. A similar reaction involving GMP transfer occurs during 5′ capping of eukaryotic mRNA. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. There is local sequence conservation among ligases and capping enzymes in the vicinity of the active site lysine (KxDG) and at three other collinear motifs. The role of these motifs in DNA ligation was tested by mutating individual conserved residues in the vaccinia virus DNA ligase. Wild-type and mutated versions of vaccinia ligase were expressed in bacteria as His-tagged fusion proteins and purified by Ni-affinity and phosphocellulose chromatography steps. We found that Ala substitution for Lys-231 (the presumptive active site) abrogated enzyme-adenylate formation and DNA ligation activities. Ala mutations at conserved residues Glu-283, Glu-377, and Lys-397 also resulted in loss of ligation activity, which correlated with a defect in ligase AMP formation. 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