Joining of nonhomologous DNA double strand breaks in vitro
Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylink...
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| Veröffentlicht in: | Nucleic acids research 1988-02, Vol.16 (3), p.907-924 |
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| creator | PFEIFFER, P VIELMETTER, W |
| description | Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures. |
| doi_str_mv | 10.1093/nar/16.3.907 |
| format | Article |
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This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/16.3.907</identifier><identifier>PMID: 3344222</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>Oxford: Oxford University Press</publisher><subject>Analytical, structural and metabolic biochemistry ; Animals ; Base Composition ; Biological and medical sciences ; Cell-Free System ; DNA - metabolism ; DNA Damage ; DNA Repair ; DNA, Circular - metabolism ; Dna, deoxyribonucleoproteins ; DNA, Recombinant ; Fundamental and applied biological sciences. Psychology ; Models, Genetic ; Nucleic acids ; Oocytes - metabolism ; Plasmids ; Xenopus laevis</subject><ispartof>Nucleic acids research, 1988-02, Vol.16 (3), p.907-924</ispartof><rights>1988 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c346t-bdf16d738e1a098dea00b91f5710b3cf10215f606f97fd4360e358f7f85833323</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/PMC334727/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC334727/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=7552725$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/3344222$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>PFEIFFER, P</creatorcontrib><creatorcontrib>VIELMETTER, W</creatorcontrib><title>Joining of nonhomologous DNA double strand breaks in vitro</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Base Composition</subject><subject>Biological and medical sciences</subject><subject>Cell-Free System</subject><subject>DNA - metabolism</subject><subject>DNA Damage</subject><subject>DNA Repair</subject><subject>DNA, Circular - metabolism</subject><subject>Dna, deoxyribonucleoproteins</subject><subject>DNA, Recombinant</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Models, Genetic</subject><subject>Nucleic acids</subject><subject>Oocytes - metabolism</subject><subject>Plasmids</subject><subject>Xenopus laevis</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkT1PHDEURS0URJYlHW2kKaJUmcXPz18TKQUiJAEhaJLa8szYi8msDfYuEv8eR6xWpKJycc-z7tUh5BjoAmiHJ9HmE5ALXHRU7ZEZoGQt7yR7R2YUqWiBcv2eHJZyRylwEPyAHCByzhibka-XKcQQl03yTUzxNq3SlJZpU5rv16fNmDb95JqyzjaOTZ-d_VuaEJvHsM7piOx7OxX3YfvOyZ8f57_PfrVXNz8vzk6v2gG5XLf96EGOCrUDSzs9Oktp34EXCmiPgwfKQHhJpe-UHzlK6lBor7wWGhEZzsm3l3_vN_3KjYOLtc5k7nNY2fxkkg3m_ySGW7NMj6aOVEzV-8_b-5weNq6szSqUwU2Tja4ONUoDgkJ4E4QOa2mm3wa57qSs-Jx8eQGHnErJzu9aAzX_5Jkqz4A0aKq8in98vXQHb23V_NM2t2Wwk69WhlB2mBKCKSbwGVU7oWE</recordid><startdate>19880211</startdate><enddate>19880211</enddate><creator>PFEIFFER, P</creator><creator>VIELMETTER, W</creator><general>Oxford University Press</general><scope>IQODW</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>7TM</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19880211</creationdate><title>Joining of nonhomologous DNA double strand breaks in vitro</title><author>PFEIFFER, P ; VIELMETTER, W</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c346t-bdf16d738e1a098dea00b91f5710b3cf10215f606f97fd4360e358f7f85833323</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Base Composition</topic><topic>Biological and medical sciences</topic><topic>Cell-Free System</topic><topic>DNA - metabolism</topic><topic>DNA Damage</topic><topic>DNA Repair</topic><topic>DNA, Circular - metabolism</topic><topic>Dna, deoxyribonucleoproteins</topic><topic>DNA, Recombinant</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Models, Genetic</topic><topic>Nucleic acids</topic><topic>Oocytes - metabolism</topic><topic>Plasmids</topic><topic>Xenopus laevis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>PFEIFFER, P</creatorcontrib><creatorcontrib>VIELMETTER, W</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids 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>PFEIFFER, P</au><au>VIELMETTER, W</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Joining of nonhomologous DNA double strand breaks in vitro</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>1988-02-11</date><risdate>1988</risdate><volume>16</volume><issue>3</issue><spage>907</spage><epage>924</epage><pages>907-924</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Extracts of Xenopus laevis eggs can efficiently join ends of duplex DNA that differ in structure and sequence. This was analysed by recircularisation of linear plasmid DNA molecules with dissimilar termini, generated by successive cuts with two different restriction enzymes within the pSP65 polylinker. Use of various enzymes provided blunt ended or 4 nucleotides long 3' and 5' protruding single strand (PSS) termini which were successfully joined in vitro in any tested combination. Sequence analysis of numerous junctions from cloned reaction products of 7 terminus combinations reveal: apart from very rare base exchanges and single nucleotide insertions less than 10% deletions (1 to 18 nucleotides long) were detected. Blunt/PSS or 3'PSS/5'PSS terminus pairs undergo simple "blunt end" joining which preserves PSS ends by fill-in. In contrast, equally polar 3'PSS/3'PSS or 5'PSS/5'PSS terminus pairs are joined by a complex mode: PSS ends overlap by a defined number of nucleotides, set by matching basepairs. Even one basematch suffices to define the setting. This then determines the final mismatch repair and fill-in pattern. We propose that yet unknown terminal DNA-binding proteins stabilize the energetically highly unfavorable configuration of single matching basepairs and help to support defined overlap structures.</abstract><cop>Oxford</cop><pub>Oxford University Press</pub><pmid>3344222</pmid><doi>10.1093/nar/16.3.907</doi><tpages>18</tpages></addata></record> |
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| identifier | ISSN: 0305-1048 |
| ispartof | Nucleic acids research, 1988-02, Vol.16 (3), p.907-924 |
| issn | 0305-1048 1362-4962 |
| language | eng |
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| source | MEDLINE; Oxford University Press Archive; PubMed Central |
| subjects | Analytical, structural and metabolic biochemistry Animals Base Composition Biological and medical sciences Cell-Free System DNA - metabolism DNA Damage DNA Repair DNA, Circular - metabolism Dna, deoxyribonucleoproteins DNA, Recombinant Fundamental and applied biological sciences. Psychology Models, Genetic Nucleic acids Oocytes - metabolism Plasmids Xenopus laevis |
| title | Joining of nonhomologous DNA double strand breaks in vitro |
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