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 joini ng of nonhomologous DNA ends, and such activities may play a major rol e in double-strand break repair. In order to dissect the biochemical p rocessing of breaks with oxidatively modified ends, vectors containing various site-specific double-strand breaks with 3'-phosphoglycolate t ermini were constructed and treated with Xenopus egg extracts. These v ectors were rejoined by the extracts at rates 30-100 times slower than comparable 3'-hydroxyl vectors. Vectors with blunt or cohesive 3'-pho sphoglycolate ends yielded single repair products corresponding to sim ple 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 n ot only blocked rejoining, but also suppressed phosphoglycolate remova l, implying an early, essential, kinase-dependent restriction point in the pathway. The results suggest that double-strand breaks with oxida tively modified ends are repaired in Xenopus eggs by a highly conserva tive and stringently regulated end-joining pathway, in which all bioch emical processing of the breaks is contingent on both end alignment an d a specific phosphorylation event. Several lines of indirect evidence suggest DNA-dependent protein kinase as a likely candidate for effect ing this phosphorylation.