Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics
Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein...
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| Veröffentlicht in: | Nucleic acids research 2007-06, Vol.35 (12), p.4124-4140 |
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| creator | Li, Xuan Zhang, Xiao-Ping Solinger, Jachen A Kiianitsa, Konstantin Yu, Xiong Egelman, Edward H Heyer, Wolf-Dietrich |
| description | Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo. |
| doi_str_mv | 10.1093/nar/gkm412 |
| format | Article |
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Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkm412</identifier><identifier>PMID: 17567608</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Adenosine Triphosphatases - metabolism ; Adenosine Triphosphate - metabolism ; Amino Acid Substitution ; DNA - chemistry ; DNA - metabolism ; DNA - ultrastructure ; DNA Helicases ; DNA Repair Enzymes ; DNA, Superhelical - analysis ; Nucleic Acid Enzymes ; Rad51 Recombinase - genetics ; Rad51 Recombinase - isolation & purification ; Rad51 Recombinase - metabolism ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - isolation & purification ; Saccharomyces cerevisiae Proteins - metabolism</subject><ispartof>Nucleic acids research, 2007-06, Vol.35 (12), p.4124-4140</ispartof><rights>2007 The Author(s) 2007</rights><rights>2007 The Author(s)</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c554t-38808a1b0af03df62ea83020c5d806d377ec11d4db564a0b22028ce7bb6797b3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919488/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1919488/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,1598,27903,27904,53769,53771</link.rule.ids><linktorsrc>$$Uhttps://dx.doi.org/10.1093/nar/gkm412$$EView_record_in_Oxford_University_Press$$FView_record_in_$$GOxford_University_Press</linktorsrc><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17567608$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Li, Xuan</creatorcontrib><creatorcontrib>Zhang, Xiao-Ping</creatorcontrib><creatorcontrib>Solinger, Jachen A</creatorcontrib><creatorcontrib>Kiianitsa, Konstantin</creatorcontrib><creatorcontrib>Yu, Xiong</creatorcontrib><creatorcontrib>Egelman, Edward H</creatorcontrib><creatorcontrib>Heyer, Wolf-Dietrich</creatorcontrib><title>Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.</description><subject>Adenosine Triphosphatases - metabolism</subject><subject>Adenosine Triphosphate - metabolism</subject><subject>Amino Acid Substitution</subject><subject>DNA - chemistry</subject><subject>DNA - metabolism</subject><subject>DNA - ultrastructure</subject><subject>DNA Helicases</subject><subject>DNA Repair Enzymes</subject><subject>DNA, Superhelical - analysis</subject><subject>Nucleic Acid Enzymes</subject><subject>Rad51 Recombinase - genetics</subject><subject>Rad51 Recombinase - isolation & purification</subject><subject>Rad51 Recombinase - metabolism</subject><subject>Saccharomyces cerevisiae</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Saccharomyces cerevisiae Proteins - isolation & purification</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0V1r1TAYB_AgijtOb_wAGgR3IdQ9SdMkvRkc5nyBoaLHSwlPm_Qss23Oknawb29mD75d6FUC-eXhn_wJeczgJYO6PB4xHm-_DYLxO2TFSskLUUt-l6yghKpgIPQBeZDSJQATrBL3yQFTlVQS9Ip8_YS2YhRHS293gq43HzE5iu3kr_3kXaIYHW3CdEGju5p9dJZOgQ7Bzj1O7sctVtj06v2adr7HwY0TtTcjDr5ND8m9DvvkHu3XQ7J5fbY5fVucf3jz7nR9XrRVJaai1Bo0sgawg9J2kjvUJXBoK6tB2lIp1zJmhW0qKRAazoHr1qmmkapWTXlITpaxu7kZnG1zhIi92UU_YLwxAb3582T0F2Ybrg2rWS20zgOO9gNiuJpdmszgU-v6HkcX5mQUKAY5xn9hDlZrpSDDZ3_ByzDHMX9CNiA1l5XM6MWC2hhSiq77GZmBua3W5GrNUm3GT35_5C-67zKD5wsI8-7fg54ursNgcBt9Ml8-c2AlgNK14rL8DvnQtH4</recordid><startdate>20070601</startdate><enddate>20070601</enddate><creator>Li, Xuan</creator><creator>Zhang, Xiao-Ping</creator><creator>Solinger, Jachen A</creator><creator>Kiianitsa, Konstantin</creator><creator>Yu, Xiong</creator><creator>Egelman, Edward H</creator><creator>Heyer, Wolf-Dietrich</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>FBQ</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20070601</creationdate><title>Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics</title><author>Li, Xuan ; Zhang, Xiao-Ping ; Solinger, Jachen A ; Kiianitsa, Konstantin ; Yu, Xiong ; Egelman, Edward H ; Heyer, Wolf-Dietrich</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c554t-38808a1b0af03df62ea83020c5d806d377ec11d4db564a0b22028ce7bb6797b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>Adenosine Triphosphatases - metabolism</topic><topic>Adenosine Triphosphate - metabolism</topic><topic>Amino Acid Substitution</topic><topic>DNA - chemistry</topic><topic>DNA - metabolism</topic><topic>DNA - ultrastructure</topic><topic>DNA Helicases</topic><topic>DNA Repair Enzymes</topic><topic>DNA, Superhelical - analysis</topic><topic>Nucleic Acid Enzymes</topic><topic>Rad51 Recombinase - genetics</topic><topic>Rad51 Recombinase - isolation & purification</topic><topic>Rad51 Recombinase - metabolism</topic><topic>Saccharomyces cerevisiae</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Saccharomyces cerevisiae Proteins - isolation & purification</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Li, Xuan</creatorcontrib><creatorcontrib>Zhang, Xiao-Ping</creatorcontrib><creatorcontrib>Solinger, Jachen A</creatorcontrib><creatorcontrib>Kiianitsa, Konstantin</creatorcontrib><creatorcontrib>Yu, Xiong</creatorcontrib><creatorcontrib>Egelman, Edward H</creatorcontrib><creatorcontrib>Heyer, Wolf-Dietrich</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Li, Xuan</au><au>Zhang, Xiao-Ping</au><au>Solinger, Jachen A</au><au>Kiianitsa, Konstantin</au><au>Yu, Xiong</au><au>Egelman, Edward H</au><au>Heyer, Wolf-Dietrich</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2007-06-01</date><risdate>2007</risdate><volume>35</volume><issue>12</issue><spage>4124</spage><epage>4140</epage><pages>4124-4140</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>17567608</pmid><doi>10.1093/nar/gkm412</doi><tpages>17</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Nucleic acids research, 2007-06, Vol.35 (12), p.4124-4140 |
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| subjects | Adenosine Triphosphatases - metabolism Adenosine Triphosphate - metabolism Amino Acid Substitution DNA - chemistry DNA - metabolism DNA - ultrastructure DNA Helicases DNA Repair Enzymes DNA, Superhelical - analysis Nucleic Acid Enzymes Rad51 Recombinase - genetics Rad51 Recombinase - isolation & purification Rad51 Recombinase - metabolism Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - isolation & purification Saccharomyces cerevisiae Proteins - metabolism |
| title | Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics |
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