End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini.
Both mammalian cells and Xenopus eggs possess activities for the joining of nonhomologous DNA ends, and such activities may play a major role in double-strand break repair. In order to dissect the biochemical processing of breaks with oxidatively modified ends, vectors containing various site-specific double-strand breaks with 3'-phosphoglycolate termini were constructed and treated with Xenopus egg extracts. These vectors were rejoined by the extracts at rates 30-100 times slower than comparable 3'-hydroxyl vectors. Vectors with blunt or cohesive 3'-phosphoglycolate ends yielded single repair products corresponding to simple phosphoglycolate removal followed by ligation, while a vector with mismatched ends was also rejoined but yielded a mixture of products. Addition of the kinase inhibitors wortmannin and dimethylaminopurine not only blocked rejoining, but also suppressed phosphoglycolate removal, implying an early, essential, kinase-dependent restriction point in the pathway. The results suggest that double-strand breaks with oxidatively modified ends are repaired in Xenopus eggs by a highly conservative and stringently regulated end-joining pathway, in which all biochemical processing of the breaks is contingent on both end alignment and a specific phosphorylation event. Several lines of indirect evidence suggest DNA-dependent protein kinase as a likely candidate for effecting this phosphorylation.
Gu
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Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298, USA.
Bennett
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Povirk
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LF
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals; D008969:Molecular Sequence Data; D010063:Ovum / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors; D000077191:Wortmannin; D014982:Xenopus laevis
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005609:Free Radicals; D004247:DNA; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D000077191:Wortmannin