Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract
The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable port...
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| Veröffentlicht in: | Journal of cell science 2002-08, Vol.115 (Pt 15), p.3159-3169 |
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| creator | Kobayashi, Takayuki Tada, Shusuke Tsuyama, Takashi Murofushi, Hiromu Seki, Masayuki Enomoto, Takemi |
| description | The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine. |
| doi_str_mv | 10.1242/jcs.115.15.3159 |
| format | Article |
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In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.</description><identifier>ISSN: 0021-9533</identifier><identifier>EISSN: 1477-9137</identifier><identifier>DOI: 10.1242/jcs.115.15.3159</identifier><identifier>PMID: 12118071</identifier><language>eng</language><publisher>England</publisher><subject>Animals ; Ataxia Telangiectasia Mutated Proteins ; Biotin - analogs & derivatives ; Biotin - pharmacology ; Caffeine - pharmacology ; Camptothecin - pharmacology ; Cell Cycle Proteins - antagonists & inhibitors ; Cell Cycle Proteins - metabolism ; Cell Cycle Proteins - pharmacology ; Cell Extracts ; Cell Nucleus - drug effects ; Cell Nucleus - genetics ; Cell Nucleus - metabolism ; Cell-Free System - drug effects ; Cell-Free System - metabolism ; Deoxyuracil Nucleotides - pharmacology ; DNA Damage - drug effects ; DNA Damage - genetics ; DNA Repair - drug effects ; DNA Repair - genetics ; DNA-Binding Proteins - biosynthesis ; DNA-Binding Proteins - drug effects ; Eukaryotic Cells - drug effects ; Eukaryotic Cells - metabolism ; Female ; Freshwater ; Geminin ; Genes, cdc - drug effects ; Genes, cdc - physiology ; Male ; Oocytes ; Protein Serine-Threonine Kinases - antagonists & inhibitors ; Protein Serine-Threonine Kinases - metabolism ; Replication Protein A ; Site-Specific DNA-Methyltransferase (Adenine-Specific) - pharmacology ; Spermatozoa ; Tumor Suppressor Proteins ; Xenopus ; Xenopus laevis ; Xenopus Proteins</subject><ispartof>Journal of cell science, 2002-08, Vol.115 (Pt 15), p.3159-3169</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c512t-7ad69199a1e04b639bea2277b01c6ae572223271ef158656e106b83c30f3d3d13</citedby><cites>FETCH-LOGICAL-c512t-7ad69199a1e04b639bea2277b01c6ae572223271ef158656e106b83c30f3d3d13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,3678,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/12118071$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kobayashi, Takayuki</creatorcontrib><creatorcontrib>Tada, Shusuke</creatorcontrib><creatorcontrib>Tsuyama, Takashi</creatorcontrib><creatorcontrib>Murofushi, Hiromu</creatorcontrib><creatorcontrib>Seki, Masayuki</creatorcontrib><creatorcontrib>Enomoto, Takemi</creatorcontrib><title>Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract</title><title>Journal of cell science</title><addtitle>J Cell Sci</addtitle><description>The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.</description><subject>Animals</subject><subject>Ataxia Telangiectasia Mutated Proteins</subject><subject>Biotin - analogs & derivatives</subject><subject>Biotin - pharmacology</subject><subject>Caffeine - pharmacology</subject><subject>Camptothecin - pharmacology</subject><subject>Cell Cycle Proteins - antagonists & inhibitors</subject><subject>Cell Cycle Proteins - metabolism</subject><subject>Cell Cycle Proteins - pharmacology</subject><subject>Cell Extracts</subject><subject>Cell Nucleus - drug effects</subject><subject>Cell Nucleus - genetics</subject><subject>Cell Nucleus - metabolism</subject><subject>Cell-Free System - drug effects</subject><subject>Cell-Free System - metabolism</subject><subject>Deoxyuracil Nucleotides - pharmacology</subject><subject>DNA Damage - drug effects</subject><subject>DNA Damage - genetics</subject><subject>DNA Repair - drug effects</subject><subject>DNA Repair - genetics</subject><subject>DNA-Binding Proteins - biosynthesis</subject><subject>DNA-Binding Proteins - drug effects</subject><subject>Eukaryotic Cells - drug effects</subject><subject>Eukaryotic Cells - metabolism</subject><subject>Female</subject><subject>Freshwater</subject><subject>Geminin</subject><subject>Genes, cdc - drug effects</subject><subject>Genes, cdc - physiology</subject><subject>Male</subject><subject>Oocytes</subject><subject>Protein Serine-Threonine Kinases - antagonists & inhibitors</subject><subject>Protein Serine-Threonine Kinases - metabolism</subject><subject>Replication Protein A</subject><subject>Site-Specific DNA-Methyltransferase (Adenine-Specific) - pharmacology</subject><subject>Spermatozoa</subject><subject>Tumor Suppressor Proteins</subject><subject>Xenopus</subject><subject>Xenopus laevis</subject><subject>Xenopus Proteins</subject><issn>0021-9533</issn><issn>1477-9137</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpFkcFq3DAQhkVpSLZpzr0FnXqqdzXS2rKOS5JNCyG5pNCbkeXxRoltOZIcug_Td43cXVoYGEbz_QPiI-QLsCXwNV89m7AEyJepBOTqA1nAWspMgZAfyYIxDpnKhTgjn0J4ZoxJruQpOQMOUDIJC_Jn68wUstb5XkfrBupa6nHsrDmMo3cR7UA336g20b79g8wTmpfR2SHSsA8Re6qHhl7fb-a4tj69DvEJgw3UDs1ksKH1_u--cVPdYRainxO1R_0yM_QXDm6cAsXdjuLvtDXxMzlpdRfw4tjPyc_tzePV9-zu4fbH1eYuMznwmEndFAqU0oBsXRdC1ag5l7JmYAqNueScCy4BW8jLIi8QWFGXwgjWikY0IM7J18Pd9N3XCUOsehsMdp0e0E2hglJxXqoigasDaLwLwWNbjd722u8rYNVspEpGqmSkSjUbSYnL4-mp7rH5zx8ViHfQ94ks</recordid><startdate>20020801</startdate><enddate>20020801</enddate><creator>Kobayashi, Takayuki</creator><creator>Tada, Shusuke</creator><creator>Tsuyama, Takashi</creator><creator>Murofushi, Hiromu</creator><creator>Seki, Masayuki</creator><creator>Enomoto, Takemi</creator><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>F1W</scope><scope>H95</scope><scope>L.G</scope></search><sort><creationdate>20020801</creationdate><title>Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract</title><author>Kobayashi, Takayuki ; Tada, Shusuke ; Tsuyama, Takashi ; Murofushi, Hiromu ; Seki, Masayuki ; Enomoto, Takemi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c512t-7ad69199a1e04b639bea2277b01c6ae572223271ef158656e106b83c30f3d3d13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>Animals</topic><topic>Ataxia Telangiectasia Mutated Proteins</topic><topic>Biotin - analogs & derivatives</topic><topic>Biotin - pharmacology</topic><topic>Caffeine - pharmacology</topic><topic>Camptothecin - pharmacology</topic><topic>Cell Cycle Proteins - antagonists & inhibitors</topic><topic>Cell Cycle Proteins - metabolism</topic><topic>Cell Cycle Proteins - pharmacology</topic><topic>Cell Extracts</topic><topic>Cell Nucleus - drug effects</topic><topic>Cell Nucleus - genetics</topic><topic>Cell Nucleus - metabolism</topic><topic>Cell-Free System - drug effects</topic><topic>Cell-Free System - metabolism</topic><topic>Deoxyuracil Nucleotides - pharmacology</topic><topic>DNA Damage - drug effects</topic><topic>DNA Damage - genetics</topic><topic>DNA Repair - drug effects</topic><topic>DNA Repair - genetics</topic><topic>DNA-Binding Proteins - biosynthesis</topic><topic>DNA-Binding Proteins - drug effects</topic><topic>Eukaryotic Cells - drug effects</topic><topic>Eukaryotic Cells - metabolism</topic><topic>Female</topic><topic>Freshwater</topic><topic>Geminin</topic><topic>Genes, cdc - drug effects</topic><topic>Genes, cdc - physiology</topic><topic>Male</topic><topic>Oocytes</topic><topic>Protein Serine-Threonine Kinases - antagonists & inhibitors</topic><topic>Protein Serine-Threonine Kinases - metabolism</topic><topic>Replication Protein A</topic><topic>Site-Specific DNA-Methyltransferase (Adenine-Specific) - pharmacology</topic><topic>Spermatozoa</topic><topic>Tumor Suppressor Proteins</topic><topic>Xenopus</topic><topic>Xenopus laevis</topic><topic>Xenopus Proteins</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kobayashi, Takayuki</creatorcontrib><creatorcontrib>Tada, Shusuke</creatorcontrib><creatorcontrib>Tsuyama, Takashi</creatorcontrib><creatorcontrib>Murofushi, Hiromu</creatorcontrib><creatorcontrib>Seki, Masayuki</creatorcontrib><creatorcontrib>Enomoto, Takemi</creatorcontrib><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>ASFA: Aquatic Sciences and Fisheries Abstracts</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) 1: Biological Sciences & Living Resources</collection><collection>Aquatic Science & Fisheries Abstracts (ASFA) Professional</collection><jtitle>Journal of cell science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kobayashi, Takayuki</au><au>Tada, Shusuke</au><au>Tsuyama, Takashi</au><au>Murofushi, Hiromu</au><au>Seki, Masayuki</au><au>Enomoto, Takemi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract</atitle><jtitle>Journal of cell science</jtitle><addtitle>J Cell Sci</addtitle><date>2002-08-01</date><risdate>2002</risdate><volume>115</volume><issue>Pt 15</issue><spage>3159</spage><epage>3169</epage><pages>3159-3169</pages><issn>0021-9533</issn><eissn>1477-9137</eissn><abstract>The response to DNA damage was analyzed using a cell-free system consisting of Xenopus egg extract and demembranated sperm nuclei. In the absence of DNA-damaging agents, detergent-resistant accumulation of replication protein A appeared in nuclei after a 30 minute incubation, and a considerable portion of the replication protein A signals disappeared during a further 30 minute incubation. Similar replication protein A accumulation was observed in the nuclei after a 30 minute incubation in the extract containing camptothecin, whereas a further 30 minute incubation generated discrete replication protein A foci. The addition of camptothecin also induced formation of gamma-H2AX foci, which have been previously shown to localize at sites of DSBs. Analysis of the time course of DNA replication and results obtained using geminin, an inhibitor of licensing for DNA replication, suggest that the discrete replication protein A foci formed in response to camptothecin-induced DNA damage occur in a DNA-replication-dependent manner. When the nuclei were incubated in the extract containing EcoRI, discrete replication protein A foci were observed at 30 minutes as well as at 60 and 90 minutes after incubation, and the focus-formation of replication protein A was not sensitive to geminin. DNA replication was almost completely inhibited in the presence of EcoRI and the inhibition was sensitive to caffeine, an inhibitor of ataxia telangiectasia mutated protein (ATM) and ATM- and Rad3-related protein (ATR). However, the focus-formation of replication protein A in the presence of EcoRI was not influenced by caffeine treatment. EcoRI-induced incorporation of biotin-dUTP into chromatin was observed following geminin-mediated inhibition of DNA replication, suggesting that the incorporation was the result of DNA repair. The biotin-dUTP signal co-localized with replication protein A foci and was not significantly suppressed or stimulated by the addition of caffeine.</abstract><cop>England</cop><pmid>12118071</pmid><doi>10.1242/jcs.115.15.3159</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Animals Ataxia Telangiectasia Mutated Proteins Biotin - analogs & derivatives Biotin - pharmacology Caffeine - pharmacology Camptothecin - pharmacology Cell Cycle Proteins - antagonists & inhibitors Cell Cycle Proteins - metabolism Cell Cycle Proteins - pharmacology Cell Extracts Cell Nucleus - drug effects Cell Nucleus - genetics Cell Nucleus - metabolism Cell-Free System - drug effects Cell-Free System - metabolism Deoxyuracil Nucleotides - pharmacology DNA Damage - drug effects DNA Damage - genetics DNA Repair - drug effects DNA Repair - genetics DNA-Binding Proteins - biosynthesis DNA-Binding Proteins - drug effects Eukaryotic Cells - drug effects Eukaryotic Cells - metabolism Female Freshwater Geminin Genes, cdc - drug effects Genes, cdc - physiology Male Oocytes Protein Serine-Threonine Kinases - antagonists & inhibitors Protein Serine-Threonine Kinases - metabolism Replication Protein A Site-Specific DNA-Methyltransferase (Adenine-Specific) - pharmacology Spermatozoa Tumor Suppressor Proteins Xenopus Xenopus laevis Xenopus Proteins |
| title | Focus-formation of replication protein A, activation of checkpoint system and DNA repair synthesis induced by DNA double-strand breaks in Xenopus egg extract |
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