Implication of DNA-dependent protein kinase in an early, essential, local phosphorylation event during end-joining of DNA double-strand breaks in vitro.
Previous work with Xenopus egg extracts suggested that a wortmannin-sensitive protein phosphorylation event precedes both the removal of modified termini from DNA double-strand break ends and the joining of unmodified ends. To assess the possible role of DNA-dependent protein kinase in effecting this phosphorylation, both DNA end-joining and DNA-stimulated phosphorylation were examined in the presence of various inhibitors. Linear but not supercoiled DNA stimulated the phosphorylation of several endogenous proteins in the extracts, including species of approximately 48, 87, and 96 kDa. This phosphorylation was selectively suppressed by the kinase inhibitors wortmannin, dimethylaminopurine, and LY294002, with a dose response that in each case paralleled the inhibition of DNA end-joining. If wortmannin was added while the end-joining reaction was in progress, end-joining of DNA already present in the reaction continued for some time, but newly added DNA was not joined or processed at all. Ends with 3'-hydroxyl termini were joined much faster than those with 3'-phosphoglycolate termini, although both were equally effective in stimulating protein phosphorylation. The results support a role for DNA-dependent protein kinase in regulating end-joining in vitro, and suggest that at least one of the necessary phosphorylations involves a protein bound at or near the DNA end to be joined. In contrast, nuclear extracts from human cells joined double-strand breaks with normal but not modified termini, and the joining was unaffected by kinase inhibitors, suggesting that the dominant mechanism of end-joining in these extracts did not involve DNA-PK.
Gu
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Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298, USA.
Weinfeld
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MA
Povirk
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LF
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005260:Female; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010063:Ovum / Q000201:enzymology; D010766:Phosphorylation; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases