End‐joining of reconstituted histone H2AX‐containing chromatin in vitro by soluble nuclear proteins from human cells
Non‐homologous end‐joining is an important pathway for the repair of DNA double‐strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nu...
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| Veröffentlicht in: | FEBS letters 2002-09, Vol.527 (1-3), p.105-108 |
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| creator | Siino, Joseph S Nazarov, Igor B Zalenskaya, Irina A Yau, Peter M Bradbury, E.Morton Tomilin, Nikolai V |
| description | Non‐homologous end‐joining is an important pathway for the repair of DNA double‐strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end‐joining is not suppressed by the PI‐3 kinase inhibitor wortmannin. During the end‐joining reaction H2AX is phosphorylated at Ser‐139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end‐joining reaction appears to be independent of H2AX phosphorylation. |
| doi_str_mv | 10.1016/S0014-5793(02)03176-9 |
| format | Article |
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This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end‐joining is not suppressed by the PI‐3 kinase inhibitor wortmannin. During the end‐joining reaction H2AX is phosphorylated at Ser‐139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. 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This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end‐joining is not suppressed by the PI‐3 kinase inhibitor wortmannin. During the end‐joining reaction H2AX is phosphorylated at Ser‐139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end‐joining reaction appears to be independent of H2AX phosphorylation.</description><subject>AFM, atomic force microscopy</subject><subject>Androstadienes - pharmacology</subject><subject>Base Sequence</subject><subject>Cell Extracts</subject><subject>Cells, Cultured</subject><subject>Chromatin - drug effects</subject><subject>Chromatin - metabolism</subject><subject>DNA end-joining</subject><subject>DNA Repair - drug effects</subject><subject>DNA Repair - physiology</subject><subject>DSB, double-strand DNA break</subject><subject>DTT, dithiothreitol</subject><subject>Enzyme Inhibitors - pharmacology</subject><subject>Histone H2AX</subject><subject>Histones - drug effects</subject><subject>Histones - metabolism</subject><subject>Humans</subject><subject>Molecular Biology - methods</subject><subject>Molecular Sequence Data</subject><subject>NCP, nucleosome core particle</subject><subject>NHEJ, non-homologous end-joining</subject><subject>Nuclear Proteins - chemistry</subject><subject>Nuclear Proteins - metabolism</subject><subject>Phosphatidylinositol 3-Kinases - antagonists & inhibitors</subject><subject>Phosphorylation</subject><subject>Reconstituted chromatin</subject><subject>rhH2AX, recombinant human H2AX</subject><subject>Solubility</subject><subject>Wortmannin</subject><issn>0014-5793</issn><issn>1873-3468</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2002</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkMtO3DAYRq2qVWegPEKRVwgWob4ljpd0NNNBQmJBK7GznMTueJTYYDuF2fEIPGOfpJ6LYFvJkmV_57_oAPAVo0uMcPXtDiHMipILeo7IBaKYV4X4AKa45rSgrKo_gukbMgFHMa5RftdYfAYTTAhBFaNT8Dx33d-X17W3zrrf0BsYdOtdTDaNSXdwZWPyTsMlubrPXI6S2qPtKvhBJetgPn9sCh42Gxh9Pza9hm5se60CfAg-aesiNJmGq3FQDra67-MX8MmoPuqTw30Mfi3mP2fL4ub2x_Xs6qZoKUeioB1DJa-MQKShpOkwbQnvWoWpMAgbbAhWWHNlNNaibjjrSlYaxjpas6pqCD0GZ_u-eZPHUcckBxu3Gyin_RglJ4gLwasMlnuwDT7GoI18CHZQYSMxklvlcqdcbn1KROROuRS57vQwYGwG3b1XHRxnYLkHnmyvN__XVS7m38ku2QaI7L4F_Qc0kpP7</recordid><startdate>20020911</startdate><enddate>20020911</enddate><creator>Siino, Joseph S</creator><creator>Nazarov, Igor B</creator><creator>Zalenskaya, Irina A</creator><creator>Yau, Peter M</creator><creator>Bradbury, E.Morton</creator><creator>Tomilin, Nikolai V</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>7X8</scope></search><sort><creationdate>20020911</creationdate><title>End‐joining of reconstituted histone H2AX‐containing chromatin in vitro by soluble nuclear proteins from human cells</title><author>Siino, Joseph S ; Nazarov, Igor B ; Zalenskaya, Irina A ; Yau, Peter M ; Bradbury, E.Morton ; Tomilin, Nikolai V</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3709-3d40576f902b32bd13c27dca139f01f1f21a1e7afe1e98b74d545f44d38466b23</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2002</creationdate><topic>AFM, atomic force microscopy</topic><topic>Androstadienes - pharmacology</topic><topic>Base Sequence</topic><topic>Cell Extracts</topic><topic>Cells, Cultured</topic><topic>Chromatin - drug effects</topic><topic>Chromatin - metabolism</topic><topic>DNA end-joining</topic><topic>DNA Repair - drug effects</topic><topic>DNA Repair - physiology</topic><topic>DSB, double-strand DNA break</topic><topic>DTT, dithiothreitol</topic><topic>Enzyme Inhibitors - pharmacology</topic><topic>Histone H2AX</topic><topic>Histones - drug effects</topic><topic>Histones - metabolism</topic><topic>Humans</topic><topic>Molecular Biology - methods</topic><topic>Molecular Sequence Data</topic><topic>NCP, nucleosome core particle</topic><topic>NHEJ, non-homologous end-joining</topic><topic>Nuclear Proteins - chemistry</topic><topic>Nuclear Proteins - metabolism</topic><topic>Phosphatidylinositol 3-Kinases - antagonists & inhibitors</topic><topic>Phosphorylation</topic><topic>Reconstituted chromatin</topic><topic>rhH2AX, recombinant human H2AX</topic><topic>Solubility</topic><topic>Wortmannin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Siino, Joseph S</creatorcontrib><creatorcontrib>Nazarov, Igor B</creatorcontrib><creatorcontrib>Zalenskaya, Irina A</creatorcontrib><creatorcontrib>Yau, Peter M</creatorcontrib><creatorcontrib>Bradbury, E.Morton</creatorcontrib><creatorcontrib>Tomilin, Nikolai V</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>FEBS letters</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Siino, Joseph S</au><au>Nazarov, Igor B</au><au>Zalenskaya, Irina A</au><au>Yau, Peter M</au><au>Bradbury, E.Morton</au><au>Tomilin, Nikolai V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>End‐joining of reconstituted histone H2AX‐containing chromatin in vitro by soluble nuclear proteins from human cells</atitle><jtitle>FEBS letters</jtitle><addtitle>FEBS Lett</addtitle><date>2002-09-11</date><risdate>2002</risdate><volume>527</volume><issue>1-3</issue><spage>105</spage><epage>108</epage><pages>105-108</pages><issn>0014-5793</issn><eissn>1873-3468</eissn><abstract>Non‐homologous end‐joining is an important pathway for the repair of DNA double‐strand breaks. This type of DNA break is followed by the rapid phosphorylation of Ser‐139 in the histone variant H2AX to form γ‐H2AX. Here we report efficient in vitro end‐joining of reconstituted chromatin containing nucleosomes made with either H2A or H2AX. This reaction is catalyzed by nuclear extracts from human cells and this end‐joining is not suppressed by the PI‐3 kinase inhibitor wortmannin. During the end‐joining reaction H2AX is phosphorylated at Ser‐139 as detected by immunoblot with specific antibodies and this phosphorylation is inhibited by wortmannin. Therefore, in vitro the DNA end‐joining reaction appears to be independent of H2AX phosphorylation.</abstract><cop>England</cop><pmid>12220643</pmid><doi>10.1016/S0014-5793(02)03176-9</doi><tpages>4</tpages></addata></record> |
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| source | Wiley Free Content; MEDLINE; Wiley Online Library Journals Frontfile Complete; Elsevier ScienceDirect Journals; EZB-FREE-00999 freely available EZB journals; Alma/SFX Local Collection |
| subjects | AFM, atomic force microscopy Androstadienes - pharmacology Base Sequence Cell Extracts Cells, Cultured Chromatin - drug effects Chromatin - metabolism DNA end-joining DNA Repair - drug effects DNA Repair - physiology DSB, double-strand DNA break DTT, dithiothreitol Enzyme Inhibitors - pharmacology Histone H2AX Histones - drug effects Histones - metabolism Humans Molecular Biology - methods Molecular Sequence Data NCP, nucleosome core particle NHEJ, non-homologous end-joining Nuclear Proteins - chemistry Nuclear Proteins - metabolism Phosphatidylinositol 3-Kinases - antagonists & inhibitors Phosphorylation Reconstituted chromatin rhH2AX, recombinant human H2AX Solubility Wortmannin |
| title | End‐joining of reconstituted histone H2AX‐containing chromatin in vitro by soluble nuclear proteins from human cells |
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