Nucleic Acids Research, 2000, Vol. 28, No. 14 2752-2761
© 2000 Oxford University Press
Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination
College of Veterinary Medicine and Department of Veterinary Pathology, Michigan State University, East Lansing, MI 48824, USA and 1Harold C. Simmons Arthritis Research Center, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA
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.
* To whom correspondence should be addressed at: Department of Veterinary Pathology, 350 FST, Michigan State University, East Lansing, MI 48824, USA. Tel: +1 517 432 4505; Fax: +1 517 353 9004; Email: kmeek@msu.edu Present address: Euy Kyun Shin, Genescreen, 2600 Stemmons Freeway, #133, Dallas, TX 75207, USA.
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