title: Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination.
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.
authors.affiliation: College of Veterinary Medicine and Department of Veterinary Pathology, Michigan State University, East Lansing, MI 48824, USA.
authors.forename: L J
authors.initials: LJ
authors.lastname: Kienker
authors.forename: E K
authors.initials: EK
authors.lastname: Shin
authors.forename: K
authors.initials: K
authors.lastname: Meek
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004254:DNA Nucleotidyltransferases / Q000187:drug effects / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D015321:Gene Rearrangement; D007133:Immunoglobulin Joining Region / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D011995:Recombination, Genetic / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects*; D012689:Sequence Homology, Nucleic Acid; D045567:VDJ Recombinases; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D004268:DNA-Binding Proteins; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D011838:Radiation-Sensitizing Agents; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D000077191:Wortmannin