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Proc Natl Acad Sci U S A. 1995 Jan 3;92(1):320-4.

DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair.

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  • 1Wellcome Trust/Cancer Research Campaign Institute, Cambridge, United Kingdom.

Abstract

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.

PMID:
7816841
[PubMed - indexed for MEDLINE]
PMCID:
PMC42870
Free PMC Article
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