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

Nucleic Acids Res. 2000 Jul 15;28(14):2752-61. doi: 10.1093/nar/28.14.2752.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Base Sequence
  • Cell Line
  • Cell Survival / drug effects
  • Cell Survival / radiation effects
  • DNA Nucleotidyltransferases / drug effects
  • DNA Nucleotidyltransferases / metabolism
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins*
  • Gene Rearrangement
  • Immunoglobulin Joining Region / genetics*
  • Immunoglobulin Joining Region / metabolism
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / metabolism
  • Mice
  • Mice, SCID
  • Mutation
  • Protein-Serine-Threonine Kinases / deficiency
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Radiation-Sensitizing Agents / pharmacology
  • Recombination, Genetic / drug effects
  • Recombination, Genetic / genetics
  • Recombination, Genetic / radiation effects*
  • Sequence Homology, Nucleic Acid
  • VDJ Recombinases
  • Wortmannin

Substances

  • Androstadienes
  • DNA-Binding Proteins
  • Immunoglobulin Joining Region
  • Immunoglobulin Variable Region
  • Radiation-Sensitizing Agents
  • DNA-Activated Protein Kinase
  • Protein-Serine-Threonine Kinases
  • DNA Nucleotidyltransferases
  • VDJ Recombinases
  • Wortmannin