Synapsis and catalysis by activated Tn3 resolvase mutants
The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I o...
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| Veröffentlicht in: | Nucleic acids research 2008-12, Vol.36 (22), p.7181-7191 |
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| creator | Olorunniji, Femi J. He, Jiuya Wenwieser, Sandra V.C.T. Boocock, Martin R. Stark, W. Marshall |
| description | The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase. |
| doi_str_mv | 10.1093/nar/gkn885 |
| format | Article |
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Marshall</creator><creatorcontrib>Olorunniji, Femi J. ; He, Jiuya ; Wenwieser, Sandra V.C.T. ; Boocock, Martin R. ; Stark, W. Marshall</creatorcontrib><description>The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkn885</identifier><identifier>PMID: 19015124</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Catalysis ; DNA - chemistry ; DNA - metabolism ; Kinetics ; Models, Molecular ; Mutation ; Nucleic Acid Enzymes ; Recombination, Genetic ; Transposon Resolvases - chemistry ; Transposon Resolvases - genetics ; Transposon Resolvases - metabolism</subject><ispartof>Nucleic acids research, 2008-12, Vol.36 (22), p.7181-7191</ispartof><rights>2008 The Author(s) 2008</rights><rights>2008 The Author(s)</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c568t-dccf7a93b56af59c5e7f89f0b69b7749f177dfe491b4c19518f3eaaff17549583</citedby><cites>FETCH-LOGICAL-c568t-dccf7a93b56af59c5e7f89f0b69b7749f177dfe491b4c19518f3eaaff17549583</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602789/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2602789/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,1604,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/19015124$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Olorunniji, Femi J.</creatorcontrib><creatorcontrib>He, Jiuya</creatorcontrib><creatorcontrib>Wenwieser, Sandra V.C.T.</creatorcontrib><creatorcontrib>Boocock, Martin R.</creatorcontrib><creatorcontrib>Stark, W. Marshall</creatorcontrib><title>Synapsis and catalysis by activated Tn3 resolvase mutants</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.</description><subject>Catalysis</subject><subject>DNA - chemistry</subject><subject>DNA - metabolism</subject><subject>Kinetics</subject><subject>Models, Molecular</subject><subject>Mutation</subject><subject>Nucleic Acid Enzymes</subject><subject>Recombination, Genetic</subject><subject>Transposon Resolvases - chemistry</subject><subject>Transposon Resolvases - genetics</subject><subject>Transposon Resolvases - metabolism</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2008</creationdate><recordtype>article</recordtype><sourceid>TOX</sourceid><sourceid>EIF</sourceid><recordid>eNqF0U1rFDEYB_Agit1WL34AGYR6EKZNJu8XQRbbCpUqVhEv4ZlMUqedzWyTzOJ-e1NmqS8HPYUkP_7kyR-hZwQfEazpcYB4fHUTlOIP0IJQ0dRMi-YhWmCKeU0wU3toP6VrjAkjnD1Ge0RjwknDFkh_2gZYpz5VELrKQoZhe7drtxXY3G8gu666DLSKLo3DBpKrVlOGkNMT9MjDkNzT3XqAPp-8vVye1ecXp--Wb85ry4XKdWetl6BpywV4ri130ivtcSt0KyXTnkjZecc0aZklmhPlqQPw5ZwzzRU9QK_n3PXUrlxnXcgRBrOO_Qri1ozQmz9vQv_dXI0b0wjcSKVLwMtdQBxvJ5eyWfXJumGA4MYpGaGV1pjS_8Km5BFNWYEv_oLX4xRD-YVisJCi0bKgVzOycUwpOn__ZILNXW-m9Gbm3gp-_vuQv-iuqAIOZzBO638H1bPrU3Y_7iXEGyMkldycff1mBD398PH98ouh9Cddo7Cp</recordid><startdate>20081201</startdate><enddate>20081201</enddate><creator>Olorunniji, Femi J.</creator><creator>He, Jiuya</creator><creator>Wenwieser, Sandra V.C.T.</creator><creator>Boocock, Martin R.</creator><creator>Stark, W. Marshall</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>BSCLL</scope><scope>TOX</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>20081201</creationdate><title>Synapsis and catalysis by activated Tn3 resolvase mutants</title><author>Olorunniji, Femi J. ; He, Jiuya ; Wenwieser, Sandra V.C.T. ; Boocock, Martin R. ; Stark, W. Marshall</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c568t-dccf7a93b56af59c5e7f89f0b69b7749f177dfe491b4c19518f3eaaff17549583</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2008</creationdate><topic>Catalysis</topic><topic>DNA - chemistry</topic><topic>DNA - metabolism</topic><topic>Kinetics</topic><topic>Models, Molecular</topic><topic>Mutation</topic><topic>Nucleic Acid Enzymes</topic><topic>Recombination, Genetic</topic><topic>Transposon Resolvases - chemistry</topic><topic>Transposon Resolvases - genetics</topic><topic>Transposon Resolvases - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Olorunniji, Femi J.</creatorcontrib><creatorcontrib>He, Jiuya</creatorcontrib><creatorcontrib>Wenwieser, Sandra V.C.T.</creatorcontrib><creatorcontrib>Boocock, Martin R.</creatorcontrib><creatorcontrib>Stark, W. Marshall</creatorcontrib><collection>Istex</collection><collection>Oxford Journals Open Access Collection</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</fulltext></delivery><addata><au>Olorunniji, Femi J.</au><au>He, Jiuya</au><au>Wenwieser, Sandra V.C.T.</au><au>Boocock, Martin R.</au><au>Stark, W. Marshall</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Synapsis and catalysis by activated Tn3 resolvase mutants</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2008-12-01</date><risdate>2008</risdate><volume>36</volume><issue>22</issue><spage>7181</spage><epage>7191</epage><pages>7181-7191</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>19015124</pmid><doi>10.1093/nar/gkn885</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Catalysis DNA - chemistry DNA - metabolism Kinetics Models, Molecular Mutation Nucleic Acid Enzymes Recombination, Genetic Transposon Resolvases - chemistry Transposon Resolvases - genetics Transposon Resolvases - metabolism |
| title | Synapsis and catalysis by activated Tn3 resolvase mutants |
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