DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair.
DNA-dependent protein kinase (DNA-PK) is a nuclear serine/threonine protein kinase composed of a catalytic subunit called p350 and a DNA binding component termed Ku. Ku consists of two tightly associated polypeptides of approximately 70 kDa and 80 kDa (Ku80). An intriguing feature of DNA-PK is that it binds to DNA ends and other discontinuities in DNA and requires these structures for its activation. This suggests that DNA-PK may function in DNA repair and/or recombination. Consistent with this, Ku DNA binding activity was shown recently to be absent in extracts of hamster xrs-6 cells, which are defective in DNA double-strand (ds) break repair and V(D)J recombination. Furthermore, xrs-6 cells are complemented by expression of the Ku80 cDNA. To date, DNA-PK activity has been demonstrated unequivocally only in extracts of primate cells. Here, we describe an assay that can detect DNA-PK activity in extracts of mouse, hamster, Xenopus, and Drosophila cells. Using this assay, we find that xrs-6 cells completely lack DNA-PK activity. By contrast, xrs-6 derivatives complemented by human chromosome fragments bearing the Ku80 gene have restored both the DNA end binding and kinase activities associated with DNA-PK. Finally, we show that xrs-6 extracts are complemented biochemically by purified Ku. Our findings indicate that the xrs-6 defects are direct consequences of the mutation in Ku80 and implicate DNA-PK in recombination and DNA repair processes.
Finnie
N J
NJ
Wellcome Trust/Cancer Research Campaign Institute, Cambridge, United Kingdom.
Gottlieb
T M
TM
Blunt
T
T
Jeggo
P A
PA
Jackson
S P
SP
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004330:Drosophila; D005816:Genetic Complementation Test; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008322:Mammals; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000032:analysis / Q000172:deficiency* / Q000378:metabolism*; D011995:Recombination, Genetic; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016159:Tumor Suppressor Protein p53; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases