Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli
Identifieur interne : 001856 ( Istex/Corpus ); précédent : 001855; suivant : 001857Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli
Auteurs : Hua-Wei Chen ; Dwight E. Randle ; Monica Gabbidon ; Douglas A. JulinSource :
- Journal of Molecular Biology [ 0022-2836 ] ; 1998.
English descriptors
- KwdEn :
- Teeft :
- Assay, Atpase, Atpase activity, Binding sites, Biochemistry, Biol, Boehmer emmerson, Chamberlin, Chamberlin julin, Chem, Cleavage, Coli, Denatured, Dixon kowalczykowski, Dsdna, Duplex, Emmerson, Enzyme, Escherichia, Escherichia coli, Escherichia coli recbcd enzyme, Goldmark, Goldmark linn, Helicase, Holoenzyme, Hrecd, Hsieh, Hsieh julin, Hydrolysis, Hydrolysis activity, Hydrolysis reaction, Hydrolysis subunits, Julin, Korangy, Korangy julin, Kowalczykowski, Linear dsdna, Linearized, Linn, Lysine residue, Masterson, Mgcl2, Muskavitch linn, Mutant, Mutant protein, Mutant recbchd enzymes, Mutation, Nuclease, Nuclease activities, Nuclease activity, Nuclease reaction, Nuclease reactions, Oligomer, Oligomer substrate, Oligomer substrates, Oligomers, Phosphorimager, Plasmid, Reaction mixtures, Recb, Recb protein, Recb subunit, Recbc, Recbc enzyme, Recbcd, Recbcd enzyme, Recbcd holoenzyme, Recbchd, Recc, Recd, Recd subunit, Recombination, Reconstituted, Reconstituted enzymes, Roman kowalczykowski, Ssdna, Subunit, Subunit concentrations, Taylor smith, Unwinding.
Abstract
Abstract: The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min−1. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼103-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.
Url:
DOI: 10.1006/jmbi.1998.1694
Links to Exploration step
ISTEX:D6E548FC392162DD1A17EAFA9CA43D28F87574DALe document en format XML
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Randle</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author><author><name sortKey="Gabbidon, Monica" sort="Gabbidon, Monica" uniqKey="Gabbidon M" first="Monica" last="Gabbidon">Monica Gabbidon</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author><author><name sortKey="Julin, Douglas A" sort="Julin, Douglas A" uniqKey="Julin D" first="Douglas A" last="Julin">Douglas A. Julin</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:D6E548FC392162DD1A17EAFA9CA43D28F87574DA</idno><date when="1998" year="1998">1998</date><idno type="doi">10.1006/jmbi.1998.1694</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-JFHRDHQN-Q/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001856</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001856</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli</title><author><name sortKey="Chen, Hua Wei" sort="Chen, Hua Wei" uniqKey="Chen H" first="Hua-Wei" last="Chen">Hua-Wei Chen</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author><author><name sortKey="Randle, Dwight E" sort="Randle, Dwight E" uniqKey="Randle D" first="Dwight E" last="Randle">Dwight E. Randle</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author><author><name sortKey="Gabbidon, Monica" sort="Gabbidon, Monica" uniqKey="Gabbidon M" first="Monica" last="Gabbidon">Monica Gabbidon</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author><author><name sortKey="Julin, Douglas A" sort="Julin, Douglas A" uniqKey="Julin D" first="Douglas A" last="Julin">Douglas A. Julin</name><affiliation><mods:affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998">1998</date><biblScope unit="volume">278</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="89">89</biblScope><biblScope unit="page" to="104">104</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>ATP hydrolysis</term><term>ATPγS</term><term>Hepes</term><term>RecBCD</term><term>RecB∗ (or B∗)</term><term>bp</term><term>ddATP</term><term>dsDNA</term><term>hRecD (or hD)</term><term>hRecD∗ (or hD∗)</term><term>helicase nuclease</term><term>nt</term><term>pd(T)12</term><term>recombination</term><term>ssDNA</term></keywords><keywords scheme="Teeft" xml:lang="en"><term>Assay</term><term>Atpase</term><term>Atpase activity</term><term>Binding sites</term><term>Biochemistry</term><term>Biol</term><term>Boehmer emmerson</term><term>Chamberlin</term><term>Chamberlin julin</term><term>Chem</term><term>Cleavage</term><term>Coli</term><term>Denatured</term><term>Dixon kowalczykowski</term><term>Dsdna</term><term>Duplex</term><term>Emmerson</term><term>Enzyme</term><term>Escherichia</term><term>Escherichia coli</term><term>Escherichia coli recbcd enzyme</term><term>Goldmark</term><term>Goldmark linn</term><term>Helicase</term><term>Holoenzyme</term><term>Hrecd</term><term>Hsieh</term><term>Hsieh julin</term><term>Hydrolysis</term><term>Hydrolysis activity</term><term>Hydrolysis reaction</term><term>Hydrolysis subunits</term><term>Julin</term><term>Korangy</term><term>Korangy julin</term><term>Kowalczykowski</term><term>Linear dsdna</term><term>Linearized</term><term>Linn</term><term>Lysine residue</term><term>Masterson</term><term>Mgcl2</term><term>Muskavitch linn</term><term>Mutant</term><term>Mutant protein</term><term>Mutant recbchd enzymes</term><term>Mutation</term><term>Nuclease</term><term>Nuclease activities</term><term>Nuclease activity</term><term>Nuclease reaction</term><term>Nuclease reactions</term><term>Oligomer</term><term>Oligomer substrate</term><term>Oligomer substrates</term><term>Oligomers</term><term>Phosphorimager</term><term>Plasmid</term><term>Reaction mixtures</term><term>Recb</term><term>Recb protein</term><term>Recb subunit</term><term>Recbc</term><term>Recbc enzyme</term><term>Recbcd</term><term>Recbcd enzyme</term><term>Recbcd holoenzyme</term><term>Recbchd</term><term>Recc</term><term>Recd</term><term>Recd subunit</term><term>Recombination</term><term>Reconstituted</term><term>Reconstituted enzymes</term><term>Roman kowalczykowski</term><term>Ssdna</term><term>Subunit</term><term>Subunit concentrations</term><term>Taylor smith</term><term>Unwinding</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min−1. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼103-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>recbcd</json:string><json:string>nuclease</json:string><json:string>recb</json:string><json:string>subunit</json:string><json:string>recbchd</json:string><json:string>julin</json:string><json:string>hrecd</json:string><json:string>oligomer</json:string><json:string>dsdna</json:string><json:string>recbc</json:string><json:string>coli</json:string><json:string>reconstituted</json:string><json:string>escherichia</json:string><json:string>recc</json:string><json:string>recd</json:string><json:string>nuclease activity</json:string><json:string>ssdna</json:string><json:string>nuclease 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linn</json:string><json:string>emmerson</json:string><json:string>recombination</json:string><json:string>chamberlin julin</json:string><json:string>oligomer substrate</json:string><json:string>unwinding</json:string><json:string>linearized</json:string><json:string>holoenzyme</json:string><json:string>dixon kowalczykowski</json:string><json:string>biochemistry</json:string><json:string>escherichia coli recbcd enzyme</json:string><json:string>recbc enzyme</json:string><json:string>masterson</json:string><json:string>recbcd holoenzyme</json:string><json:string>taylor smith</json:string><json:string>hydrolysis subunits</json:string><json:string>phosphorimager</json:string><json:string>hydrolysis</json:string><json:string>recb protein</json:string><json:string>subunit concentrations</json:string><json:string>cleavage</json:string><json:string>oligomer substrates</json:string><json:string>recd subunit</json:string><json:string>assay</json:string><json:string>duplex</json:string><json:string>recb subunit</json:string><json:string>muskavitch linn</json:string><json:string>atpase activity</json:string><json:string>linear dsdna</json:string><json:string>mutant protein</json:string><json:string>mutant recbchd enzymes</json:string><json:string>nuclease activities</json:string><json:string>lysine residue</json:string><json:string>boehmer emmerson</json:string><json:string>hydrolysis reaction</json:string><json:string>binding sites</json:string><json:string>roman kowalczykowski</json:string></teeft></keywords><author><json:item><name>Hua-Wei Chen</name><affiliations><json:string>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</json:string></affiliations></json:item><json:item><name>Dwight E Randle</name><affiliations><json:string>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</json:string></affiliations></json:item><json:item><name>Monica Gabbidon</name><affiliations><json:string>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</json:string></affiliations></json:item><json:item><name>Douglas A Julin</name><affiliations><json:string>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular article</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RecBCD</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>helicase nuclease</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ATP hydrolysis</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>recombination</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>RecB∗ (or B∗) : RecB-K29Q protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>hRecD (or hD) : histidine-tagged RecD protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>hRecD∗ (or hD∗) : histidine-tagged RecD-K177Q protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ATPγS : adenosine 5′- O -(3-thiotriphosphate)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ddATP : 2′,3′-dideoxyadenosine 5′-triphosphate</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>bp : base-pair</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Hepes : N -(2-hydroxyethyl)piperazine- N ′-(2-ethanesulfonic acid)</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>nt : nucleotide residue</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>pd(T)12 : 5′-phosphorylated oligodeoxythymidine, 12 nucleotides in length</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>dsDNA : double-stranded DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ssDNA : single-stranded DNA</value></json:item></subject><arkIstex>ark:/67375/6H6-JFHRDHQN-Q</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min−1. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼103-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.</abstract><qualityIndicators><refBibsNative>true</refBibsNative><abstractWordCount>243</abstractWordCount><abstractCharCount>1611</abstractCharCount><keywordCount>16</keywordCount><score>9.916</score><pdfWordCount>10262</pdfWordCount><pdfCharCount>59902</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>16</pdfPageCount><pdfPageSize>595 x 842 pts (A4)</pdfPageSize></qualityIndicators><title>Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli</title><pmid><json:string>9571036</json:string></pmid><pii><json:string>S0022-2836(98)91694-1</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>1998</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0367-1</json:string></pii><volume>278</volume><issue>1</issue><pages><first>89</first><last>104</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1998</json:string><json:string>2152</json:string><json:string>2071</json:string><json:string>3000</json:string></date><geogName></geogName><orgName><json:string>Hamon Center, Dallas</json:string><json:string>Biochemicals Corp.</json:string><json:string>Biochemicals Corp</json:string><json:string>Life Technologies</json:string><json:string>DNA International</json:string><json:string>National Institutes of Health</json:string><json:string>University of Maryland</json:string><json:string>Promega Corp.</json:string><json:string>Molecular Dynamics Corp.</json:string></orgName><orgName_funder></orgName_funder><orgName_provider></orgName_provider><persName><json:string>M. Chamberlin</json:string><json:string>D. E. Randle</json:string><json:string>RecBC</json:string><json:string>Douglas A. Julin</json:string><json:string>E. Randle</json:string><json:string>Monica Gabbidon</json:string><json:string>Harry Hines</json:string></persName><placeName><json:string>MD.</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Ganesan & Smith, 1993</json:string><json:string>Dixon & Kowalczykowski, 1995</json:string><json:string>Williamson & Celander, 1990</json:string><json:string>Muskavitch & Linn, 1982</json:string><json:string>Finch et al., 1986a,b</json:string><json:string>Taylor & Smith 1995</json:string><json:string>Wright et al., 1971</json:string><json:string>Goldmark & Linn, 1972</json:string><json:string>98 Korangy & Julin, 1992a,b</json:string><json:string>Boehmer & Emmerson, 1992</json:string><json:string>$800-fold less than RecBCD; Hsieh & Julin, 1992</json:string><json:string>Taylor & Smith, 1980</json:string><json:string>Figure 8b; Ganesan & Smith, 1993</json:string><json:string>Mead et al., 1986</json:string><json:string>Roman & Kowalczykowski, 1989b</json:string><json:string>Korangy & Julin, 1992b</json:string><json:string>Amundsen et al., 1986</json:string><json:string>Haijema et al., 1996</json:string><json:string>Eggleston & Kowalczykowski, 1993</json:string><json:string>Roman et al., 1992</json:string><json:string>Julin & Lehman, 1987</json:string><json:string>Boehmer & Emmerson, 1991</json:string><json:string>Taylor et al., 1985</json:string><json:string>Karu et al., 1975</json:string><json:string>Chamberlin & Julin, 1996</json:string><json:string>Roman & Kowalczykowski, 1989a</json:string><json:string>Chen et al. (1997)</json:string><json:string>Dixon & Kowalczykowski, 1993</json:string><json:string>Gross & Shuman, 1995</json:string><json:string>Korangy & Julin, 1992a</json:string><json:string>George et al., 1994</json:string><json:string>Dixon et al., 1994</json:string><json:string>Taylor & Smith, 1995</json:string><json:string>Chen et al., 1997</json:string><json:string>Murphy, 1991</json:string><json:string>Farah & Smith, 1997</json:string><json:string>Hsieh & Julin, 1992</json:string><json:string>Cantor et al., 1970</json:string><json:string>Eichler & Lehman, 1977</json:string><json:string>Connolly et al., 1991</json:string><json:string>Korangy & Julin, 1994</json:string><json:string>data not shown; Korangy & Julin, 1993</json:string><json:string>[3H]pDJ01 plasmid DNA (Korangy & Julin, 1992a</json:string><json:string>Dixon & Kowalczykowski, 1991, 1995</json:string><json:string>MacKay & Linn, 1974</json:string><json:string>Woodbury & von Hippel, 1983</json:string><json:string>Smith et al., 1995</json:string><json:string>Korangy & Julin, 1993</json:string><json:string>Ponticelli et al., 1985</json:string><json:string>Palas and Kushner (1990)</json:string><json:string>Phillips et al., 1997</json:string><json:string>lacIq; Boehmer & Emmerson, 1991</json:string><json:string>Dixon & Kowalczykowski, 1993, 1995</json:string><json:string>Korangy & Julin, 1993, 1994</json:string><json:string>Patel et al., 1994</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-JFHRDHQN-Q</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 - Molecular Biology</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><json:string>4 - vertebres: anatomie et physiologie, organisme dans son ensemble ou etude de plusieurs organes ou systemes</json:string></inist></categories><publicationDate>1998</publicationDate><copyrightDate>1998</copyrightDate><doi><json:string>10.1006/jmbi.1998.1694</json:string></doi><id>D6E548FC392162DD1A17EAFA9CA43D28F87574DA</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-JFHRDHQN-Q/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-JFHRDHQN-Q/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-JFHRDHQN-Q/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1998 Academic Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1998</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note>Edited by G. Smith</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Nuclease reactions with the oligomer substrate catalyzed by the RecBCD (a) and RecBChD (b) enzymes in the presence of ATP. a, The reaction mixtures contained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, 0.5 mM ATP, the 26-mer substrate (100 nM oligomer) labeled at the 3′ or 5′-end as described in Materials and Methods, and 0.4 nM RecBCD, at 37°C. Samples were removed at the times indicated above the lanes, quenched, and loaded on a non-denaturing 20% polyacrylamide gel. The gel was dried and the radioactivity was visualized using a Phosphorimager. The lanes labeled GA, G, and T are markers prepared from the 5′-labeled 26-mer (left-hand set of lanes) or 3′-labeled 26-mer (right-hand lanes), as described in Materials and Methods. The sizes of some of the product bands and the expected position of a 5′-labeled 22-mer are indicated. b, Reaction mixtures were as in a, with 100 nM (5′-32P)-labeled 25-mer substrate. The subunit concentrations in the reaction mixture were 6 nM RecB, 30 nM RecC, and 30 nM hRecD. Samples were removed at the indicated times and analyzed as in a. The arrow indicates the 4-mer product discussed in the text.</note><note type="content">Figure 2: Nuclease reactions with the 26-mer substrate catalyzed by the RecBCD enzyme in the absence of ATP. The reaction mixtures contained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, 300 nM (5′-32P) or (3′-32P)-labeled 26-mer, and 30 nM RecBCD, with no ATP. Samples were removed at time=0 (lanes 1), 15 minutes (lanes 2), 30 minutes (lanes 3), 60 minutes (lanes 4), 90 minutes (lanes 5), and 120 minutes (lanes 6) and analyzed as inFigure 1.</note><note type="content">Figure 3: Binding of RecBCD to the 15-mer and 25-mer in the absence of ATP. Binding mixtures contained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, and: (•) 1 nM (5′-32P)-labeled 15-mer; (□) 0.1 nM (5′-32P)-labeled 25-mer. The fraction of the DNA bound at varied enzyme concentrations was determined using a nitrocellulose filter binding assay (Materials and Methods). The error bars show the range of duplicate determinations. The continuous lines are fits toequation (1)(Materials and Methods) withKd=15 nM, ζ=0.044 (15-mer), and Kd=0.09 nM, ζ=0.2 (25-mer).</note><note type="content">Figure 4: Nuclease reactions catalyzed by the reconstituted wild-type and mutant RecBChD enzymes with 5′-labeled substrate in the absence of ATP. Reaction mixtures were as inFigure 2, with 100 nM (5′-32P)-labeled 25-mer-C, at 37°C. Samples were removed at the indicated times (minutes), quenched, and analyzed as in Figure 1a. The enzymes were reconstituted as described in Materials and Methods, and the subunit concentrations in the reaction mixtures were 6 nM RecB or RecB∗, 30 nM RecC, and 30 nM hRecD or hRecD∗.</note><note type="content">Figure 5: Nuclease reactions catalyzed by the reconstituted wild-type and mutant RecBChD enzymes with 5′-labeled 25-mer substrate in the presence of ATP. Reactions were as inFigure 1a, with 100 nM 5′-labeled 25-mer-C substrate (see Materials and Methods). The subunit concentrations in the reaction mixtures were 3 nM RecB, 15 nM RecC, and 15 nM hRecD or hRecD∗, or 6 nM RecB∗, 30 nM RecC, and 30 nM hRecD or hRecD∗. The arrow indicates the 4-mer product discussed in the text.</note><note type="content">Figure 6: Nuclease reactions catalyzed by the reconstituted wild-type and mutant RecBChD enzymes with 3′-labeled 25-mer substrate in the presence of ATP. Reaction mixtures were as in Figure 1a, with 100 nM (3′-32P)-labeled 26-mer substrate, at 37°C. The subunit concentrations in the reaction mixtures were 3 nM RecB or RecB∗, 15 nM RecC, and 15 nM hRecD or hRecD∗, except the double mutant, which was 6 nM RecB∗, 30 nM RecC, and 30 nM hRecD∗. The arrow indicates the 12-mer product discussed in the text.</note><note type="content">Figure 7: Product distributions in nuclease reactions catalyzed by RecBChD, RecBChD∗, and RecB∗ChD. a, The amount of the 5′-labeled 4-mer product (arrows in Figures. 1b and 5) in the reactions with the 5′-labeled 25-mer substrate, as a percentage of all products at each time-point. The amount of radioactivity in each band at every time-point was determined by integration using the Phosphorimager, and the concentrations of the corresponding labeled DNA species were calculated using equation (2)(Materials and Methods). The concentration of the product band in question was then divided by the sum of all products in the same gel lane and converted to a percentage. The data are from three separate experiments. (•) RecBChD reactions. (□) RecBChD∗ reactions. (▴) RecB∗ChD reactions. b, The percentage of the 3′-labeled 12-mer product (arrow in Figure 6) in the reactions with the 3′-labeled 26-mer substrate. Symbols are as in a. The plotted points are the average from two separate experiments.</note><note type="content">Figure 8: Model for the interaction of RecBCD with DNA oligomer substrates (a) and with dsDNA (b). DNA oligomers bound separately in two DNA binding sites stimulate ATP hydrolysis by the RecB and RecD subunits. The small broken-line oval represents the nuclease active site situated near the ends of the bound DNA. Cleavage towards the 3′-end of one oligomer depends on ATP hydrolysis by RecB, while cleavage dependent on RecD occurs towards the 5′-end of the second oligomer.</note><note type="content">Table 1: ATP hydrolysis by the RecB and RecB∗ proteins</note><note type="content">Table 2: ATP hydrolysis by the reconstituted RecBChD enzymes stimulated by poly(dT)</note><note type="content">Table 3: Nuclease activities of the reconstituted enzymes and RecBCD with linearized plasmid substrates</note><note type="content">Table 4: Nuclease rates with the oligomer substrates</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli</title><author xml:id="author-0000"><persName><forename type="first">Hua-Wei</forename><surname>Chen</surname></persName><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Dwight E</forename><surname>Randle</surname></persName><note type="biography">Present address: D. E. Randle, 6000 Harry Hines Blvd., Hamon Center, Dallas, TX 75235-8593, USA.</note><affiliation>Present address: D. E. Randle, 6000 Harry Hines Blvd., Hamon Center, Dallas, TX 75235-8593, USA.</affiliation><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Monica</forename><surname>Gabbidon</surname></persName><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Douglas A</forename><surname>Julin</surname></persName><note type="correspondence"><p>Corresponding author</p></note><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation></author><idno type="istex">D6E548FC392162DD1A17EAFA9CA43D28F87574DA</idno><idno type="ark">ark:/67375/6H6-JFHRDHQN-Q</idno><idno type="DOI">10.1006/jmbi.1998.1694</idno><idno type="PII">S0022-2836(98)91694-1</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0367-1</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1998"></date><biblScope unit="volume">278</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="89">89</biblScope><biblScope unit="page" to="104">104</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1998</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min−1. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼103-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Regular article</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>RecBCD</term></item><item><term>helicase nuclease</term></item><item><term>ATP hydrolysis</term></item><item><term>recombination</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>RecB∗ (or B∗)</term><term>RecB-K29Q protein</term></item><item><term>hRecD (or hD)</term><term>histidine-tagged RecD protein</term></item><item><term>hRecD∗ (or hD∗)</term><term>histidine-tagged RecD-K177Q protein</term></item><item><term>ATPγS</term><term>adenosine 5′- O -(3-thiotriphosphate)</term></item><item><term>ddATP</term><term>2′,3′-dideoxyadenosine 5′-triphosphate</term></item><item><term>bp</term><term>base-pair</term></item><item><term>Hepes</term><term>N -(2-hydroxyethyl)piperazine- N ′-(2-ethanesulfonic acid)</term></item><item><term>nt</term><term>nucleotide residue</term></item><item><term>pd(T)12</term><term>5′-phosphorylated oligodeoxythymidine, 12 nucleotides in length</term></item><item><term>dsDNA</term><term>double-stranded DNA</term></item><item><term>ssDNA</term><term>single-stranded DNA</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1998-01-26">Modified</change><change when="1998">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-JFHRDHQN-Q/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><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:entity SYSTEM="gr1b" NDATA="IMAGE" name="GR1b"></istex:entity><istex:entity SYSTEM="gr1a" NDATA="IMAGE" name="GR1a"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="GR4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="GR5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="GR6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="GR8"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>91694</aid><ce:pii>S0022-2836(98)91694-1</ce:pii><ce:doi>10.1006/jmbi.1998.1694</ce:doi><ce:copyright type="full-transfer" year="1998">Academic Press</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from<ce:italic>Escherichia coli</ce:italic><ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para><ce:bold><ce:italic>Edited by G. Smith</ce:italic></ce:bold></ce:note-para></ce:footnote></ce:title><ce:author-group><ce:author><ce:given-name>Hua-Wei</ce:given-name><ce:surname>Chen</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Dwight E</ce:given-name><ce:surname>Randle</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref><ce:cross-ref refid="FN2"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Monica</ce:given-name><ce:surname>Gabbidon</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Douglas A</ce:given-name><ce:surname>Julin</ce:surname><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:cross-ref refid="AFF1"><ce:sup>a</ce:sup></ce:cross-ref></ce:author><ce:affiliation id="AFF1"><ce:label>a</ce:label><ce:textfn>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author</ce:text></ce:correspondence><ce:footnote id="FN2"><ce:label>2</ce:label><ce:note-para>Present address: D. E. Randle, 6000 Harry Hines Blvd., Hamon Center, Dallas, TX 75235-8593, USA.</ce:note-para></ce:footnote></ce:author-group><ce:date-received day="4" month="3" year="1997"></ce:date-received><ce:date-revised day="26" month="1" year="1998"></ce:date-revised><ce:date-accepted day="27" month="1" year="1998"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The RecBCD enzyme from <ce:italic>Escherichia coli</ce:italic> is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min<ce:sup>−1</ce:sup>. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼10<ce:sup>3</ce:sup>-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>RecBCD</ce:text></ce:keyword><ce:keyword><ce:text>helicase nuclease</ce:text></ce:keyword><ce:keyword><ce:text>ATP hydrolysis</ce:text></ce:keyword><ce:keyword><ce:text>recombination</ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>RecB∗ (or B∗)</ce:text><ce:keyword><ce:text>RecB-K29Q protein</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>hRecD (or hD)</ce:text><ce:keyword><ce:text>histidine-tagged RecD protein</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>hRecD∗ (or hD∗)</ce:text><ce:keyword><ce:text>histidine-tagged RecD-K177Q protein</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ATPγS</ce:text><ce:keyword><ce:text>adenosine 5′-<ce:italic>O</ce:italic>-(3-thiotriphosphate)</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ddATP</ce:text><ce:keyword><ce:text>2′,3′-dideoxyadenosine 5′-triphosphate</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>bp</ce:text><ce:keyword><ce:text>base-pair</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>Hepes</ce:text><ce:keyword><ce:text><ce:italic>N</ce:italic>-(2-hydroxyethyl)piperazine-<ce:italic>N</ce:italic>′-(2-ethanesulfonic acid)</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>nt</ce:text><ce:keyword><ce:text>nucleotide residue</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>pd(T)<ce:inf>12</ce:inf></ce:text><ce:keyword><ce:text>5′-phosphorylated oligodeoxythymidine, 12 nucleotides in length</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>dsDNA</ce:text><ce:keyword><ce:text>double-stranded DNA</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ssDNA</ce:text><ce:keyword><ce:text>single-stranded DNA</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from Escherichia coli</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Functions of the ATP hydrolysis subunits (RecB and RecD) in the nuclease reactions catalyzed by the RecBCD enzyme from</title></titleInfo><name type="personal"><namePart type="given">Hua-Wei</namePart><namePart type="family">Chen</namePart><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Dwight E</namePart><namePart type="family">Randle</namePart><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation><description>Present address: D. E. Randle, 6000 Harry Hines Blvd., Hamon Center, Dallas, TX 75235-8593, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Monica</namePart><namePart type="family">Gabbidon</namePart><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Douglas A</namePart><namePart type="family">Julin</namePart><affiliation>Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, USA</affiliation><description>Corresponding author</description><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">1998</dateIssued><dateModified encoding="w3cdtf">1998-01-26</dateModified><copyrightDate encoding="w3cdtf">1998</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: The RecBCD enzyme from Escherichia coli is an ATP-dependent nuclease and helicase. Two of its subunits, the RecB and RecD proteins, are DNA-dependent ATPases. We have purified RecB and RecD proteins with mutations in their consensus ATP binding sites to study the functions of these subunits in the ATP-dependent nuclease activities of RecBCD. Reconstituted heterotrimeric enzymes were prepared by mixing wild-type RecB or RecB-K29Q mutant protein (RecB∗) with purified RecC protein, and with a histidine-tagged wild-type RecD (hD) or mutant hRecD-K177Q (hD∗) protein. RecBCD and all four reconstituted enzymes (wild-type, two single mutants, and the double mutant) cleave a single-stranded DNA oligomer substrate (25-mer) in the absence of ATP at rates of 0.03 to 0.06 min−1. The nuclease reaction catalyzed by RecB∗ChD∗ is not stimulated significantly by ATP, while the reactions catalyzed by RecBCD, RecBChD, RecBChD∗, and RecB∗ChD are 300 to 3000 fold faster in the presence of 0.5 mM ATP. RecB∗ChD∗ also has very low ATP hydrolysis activity (∼103-fold less than RecBCD), as do the individual mutant RecB∗ and hRecD∗ proteins (∼100-fold less than RecB or hRecD). The products from the ATP-stimulated nuclease reaction with the oligomer substrate suggest a mechanism where two DNA molecules bind to the enzyme in opposite orientations and are cleaved by the nuclease active site. Cleavage towards the 3′-end of one oligomer (observed with RecBChD∗) depends on the wild-type RecB subunit, while RecD-dependent cleavage (observed with RecB∗ChD) occurs towards the 5′-end of the second bound oligomer.</abstract><note type="footnote">Edited by G. Smith</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Nuclease reactions with the oligomer substrate catalyzed by the RecBCD (a) and RecBChD (b) enzymes in the presence of ATP. a, The reaction mixtures contained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, 0.5 mM ATP, the 26-mer substrate (100 nM oligomer) labeled at the 3′ or 5′-end as described in Materials and Methods, and 0.4 nM RecBCD, at 37°C. Samples were removed at the times indicated above the lanes, quenched, and loaded on a non-denaturing 20% polyacrylamide gel. The gel was dried and the radioactivity was visualized using a Phosphorimager. The lanes labeled GA, G, and T are markers prepared from the 5′-labeled 26-mer (left-hand set of lanes) or 3′-labeled 26-mer (right-hand lanes), as described in Materials and Methods. The sizes of some of the product bands and the expected position of a 5′-labeled 22-mer are indicated. b, Reaction mixtures were as in a, with 100 nM (5′-32P)-labeled 25-mer substrate. The subunit concentrations in the reaction mixture were 6 nM RecB, 30 nM RecC, and 30 nM hRecD. Samples were removed at the indicated times and analyzed as in a. The arrow indicates the 4-mer product discussed in the text.</note><note type="content">Figure 2: Nuclease reactions with the 26-mer substrate catalyzed by the RecBCD enzyme in the absence of ATP. The reaction mixtures contained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, 300 nM (5′-32P) or (3′-32P)-labeled 26-mer, and 30 nM RecBCD, with no ATP. Samples were removed at time=0 (lanes 1), 15 minutes (lanes 2), 30 minutes (lanes 3), 60 minutes (lanes 4), 90 minutes (lanes 5), and 120 minutes (lanes 6) and analyzed as inFigure 1.</note><note type="content">Figure 3: Binding of RecBCD to the 15-mer and 25-mer in the absence of ATP. Binding mixtures contained 50 mM Tris HCl (pH 7.5), 10 mM MgCl2, and: (•) 1 nM (5′-32P)-labeled 15-mer; (□) 0.1 nM (5′-32P)-labeled 25-mer. The fraction of the DNA bound at varied enzyme concentrations was determined using a nitrocellulose filter binding assay (Materials and Methods). The error bars show the range of duplicate determinations. The continuous lines are fits toequation (1)(Materials and Methods) withKd=15 nM, ζ=0.044 (15-mer), and Kd=0.09 nM, ζ=0.2 (25-mer).</note><note type="content">Figure 4: Nuclease reactions catalyzed by the reconstituted wild-type and mutant RecBChD enzymes with 5′-labeled substrate in the absence of ATP. Reaction mixtures were as inFigure 2, with 100 nM (5′-32P)-labeled 25-mer-C, at 37°C. Samples were removed at the indicated times (minutes), quenched, and analyzed as in Figure 1a. The enzymes were reconstituted as described in Materials and Methods, and the subunit concentrations in the reaction mixtures were 6 nM RecB or RecB∗, 30 nM RecC, and 30 nM hRecD or hRecD∗.</note><note type="content">Figure 5: Nuclease reactions catalyzed by the reconstituted wild-type and mutant RecBChD enzymes with 5′-labeled 25-mer substrate in the presence of ATP. Reactions were as inFigure 1a, with 100 nM 5′-labeled 25-mer-C substrate (see Materials and Methods). The subunit concentrations in the reaction mixtures were 3 nM RecB, 15 nM RecC, and 15 nM hRecD or hRecD∗, or 6 nM RecB∗, 30 nM RecC, and 30 nM hRecD or hRecD∗. The arrow indicates the 4-mer product discussed in the text.</note><note type="content">Figure 6: Nuclease reactions catalyzed by the reconstituted wild-type and mutant RecBChD enzymes with 3′-labeled 25-mer substrate in the presence of ATP. Reaction mixtures were as in Figure 1a, with 100 nM (3′-32P)-labeled 26-mer substrate, at 37°C. The subunit concentrations in the reaction mixtures were 3 nM RecB or RecB∗, 15 nM RecC, and 15 nM hRecD or hRecD∗, except the double mutant, which was 6 nM RecB∗, 30 nM RecC, and 30 nM hRecD∗. The arrow indicates the 12-mer product discussed in the text.</note><note type="content">Figure 7: Product distributions in nuclease reactions catalyzed by RecBChD, RecBChD∗, and RecB∗ChD. a, The amount of the 5′-labeled 4-mer product (arrows in Figures. 1b and 5) in the reactions with the 5′-labeled 25-mer substrate, as a percentage of all products at each time-point. The amount of radioactivity in each band at every time-point was determined by integration using the Phosphorimager, and the concentrations of the corresponding labeled DNA species were calculated using equation (2)(Materials and Methods). The concentration of the product band in question was then divided by the sum of all products in the same gel lane and converted to a percentage. The data are from three separate experiments. (•) RecBChD reactions. (□) RecBChD∗ reactions. (▴) RecB∗ChD reactions. b, The percentage of the 3′-labeled 12-mer product (arrow in Figure 6) in the reactions with the 3′-labeled 26-mer substrate. Symbols are as in a. The plotted points are the average from two separate experiments.</note><note type="content">Figure 8: Model for the interaction of RecBCD with DNA oligomer substrates (a) and with dsDNA (b). DNA oligomers bound separately in two DNA binding sites stimulate ATP hydrolysis by the RecB and RecD subunits. The small broken-line oval represents the nuclease active site situated near the ends of the bound DNA. Cleavage towards the 3′-end of one oligomer depends on ATP hydrolysis by RecB, while cleavage dependent on RecD occurs towards the 5′-end of the second oligomer.</note><note type="content">Table 1: ATP hydrolysis by the RecB and RecB∗ proteins</note><note type="content">Table 2: ATP hydrolysis by the reconstituted RecBChD enzymes stimulated by poly(dT)</note><note type="content">Table 3: Nuclease activities of the reconstituted enzymes and RecBCD with linearized plasmid substrates</note><note type="content">Table 4: Nuclease rates with the oligomer substrates</note><subject><genre>article-category</genre><topic>Regular article</topic></subject><subject lang="en"><genre>Keywords</genre><topic>RecBCD</topic><topic>helicase nuclease</topic><topic>ATP hydrolysis</topic><topic>recombination</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>RecB∗ (or B∗) : RecB-K29Q protein</topic><topic>hRecD (or hD) : histidine-tagged RecD protein</topic><topic>hRecD∗ (or hD∗) : histidine-tagged RecD-K177Q protein</topic><topic>ATPγS : adenosine 5′- O -(3-thiotriphosphate)</topic><topic>ddATP : 2′,3′-dideoxyadenosine 5′-triphosphate</topic><topic>bp : base-pair</topic><topic>Hepes : N -(2-hydroxyethyl)piperazine- N ′-(2-ethanesulfonic acid)</topic><topic>nt : nucleotide residue</topic><topic>pd(T)12 : 5′-phosphorylated oligodeoxythymidine, 12 nucleotides in length</topic><topic>dsDNA : double-stranded DNA</topic><topic>ssDNA : single-stranded DNA</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1998</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0367-1</identifier><part><date>1998</date><detail type="volume"><number>278</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue-pages"><start>1</start><end>289</end></extent><extent unit="pages"><start>89</start><end>104</end></extent></part></relatedItem><identifier type="istex">D6E548FC392162DD1A17EAFA9CA43D28F87574DA</identifier><identifier type="ark">ark:/67375/6H6-JFHRDHQN-Q</identifier><identifier type="DOI">10.1006/jmbi.1998.1694</identifier><identifier type="PII">S0022-2836(98)91694-1</identifier><accessCondition type="use and reproduction" contentType="copyright">©1998 Academic Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Academic Press, ©1998</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-JFHRDHQN-Q/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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