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 gamma-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.
Siino
Joseph S
JS
Department of Biological Chemistry, UC Davis School of Medicine, Davis, CA 95616, USA.
Nazarov
Igor B
IB
Zalenskaya
Irina A
IA
Yau
Peter M
PM
Bradbury
E Morton
EM
Tomilin
Nikolai V
NV
D000730:Androstadienes / Q000494:pharmacology; D001483:Base Sequence; D002457:Cell Extracts; D002478:Cells, Cultured; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006657:Histones / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D012995:Solubility; D000077191:Wortmannin
D000730:Androstadienes; D002457:Cell Extracts; D002843:Chromatin; D004791:Enzyme Inhibitors; D006657:Histones; D009687:Nuclear Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D000077191:Wortmannin