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Nucleic Acids Res. 2000 Jul 15;28(14):2752-61.

Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination.

Author information

1
College of Veterinary Medicine and Department of Veterinary Pathology, Michigan State University, East Lansing, MI 48824, USA.

Abstract

DNA-dependent protein kinase (DNA-PK) is utilized in both DNA double-strand break repair (DSBR) and V(D)J recombination, but the mechanism by which this multiprotein complex participates in these processes is unknown. To evaluate the importance of DNA-PK-mediated protein phosphorylation in DSBR and V(D)J recombination, we assessed the effects of the phosphatidyl inositol 3-kinase inhibitor wortmannin on the repair of ionizing radiation-induced DNA double-strand breaks and V(D)J recombination in the V(D)J recombinase inducible B cell line HDR37. Wortmannin radiosensitized HDR37, but had no affect on V(D)J recombination despite a marked reduction in DNA-PK activity. On the other hand, studies with mammalian expression vectors for wild-type human DNA-PK catalytic subunit (DNA-PKcs) and a kinase domain mutant demonstrated that only the kinase active form of DNA-PKcs can reconstitute DSBR and V(D)J recombination in a DNA-PKcs-deficient cell line (Sf19), implying that DNA-PKcs kinase activity is essential for both DSBR and V(D)J recombination. These apparently contradictory results were reconciled by analyses of cell lines varying in their expression of recombinant wild-type human DNA-PKcs. These studies establish that minimal DNA-PKcs protein levels are sufficient to support V(D)J recombination, but insufficient to confer resistance to ionizing radiation.

PMID:
10908332
PMCID:
PMC102647
DOI:
10.1093/nar/28.14.2752
[Indexed for MEDLINE]
Free PMC Article

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