The DNA-dependent protein kinase (DNA-PK) complex plays a key role in DNA double-strand break (DSB) repair and V(D)J recombination. Using a genetic approach we have isolated cell mutants sensitive to ionizing radiation (IR) in the hope of elucidating the mechanism and components required for these pathways. We describe here, an X-ray-sensitive and DSB repair defective Chinese hamster ovary (CHO) cell line, XR-C2, which was assigned to the X-Ray Cross Complementation (XRCC) group 7. This group of mutants is defective in the XRCC7/SCID/Prkdc gene, which encodes the catalytic subunit of DNA-PK (DNA-PKcs). Despite the fact that XR-C2 cells expressed normal levels of DNA- PKcs protein, no DNA-PK catalytic activity could be observed in XR-C2, confirming the genetic analyses that these cells harbor a dysfunctional gene for DNA-PKcs. In contrast to other IR group 7 mutants, which contain undetectable or low levels of DNA-PKcs protein and which show a severe defect in V(D)J recombination, XR-C2 cells manifested only a mild defect in both coding and signal junction formation. The unique phenotype of the XR-C2 mutant suggests that a normal level of kinase activity is critical for radiation resistance but not for V(D)J recombination, whereas the overall structure of the DNA-PKcs protein appears to be of great importance for this process. (C) 2000 Published by Elsevier Science B.V.
Bibliographical noteFunding Information:
We would like to thank Laura Wetselaar for her skillful technical assistance. We also would like to thank Graeme C.M. Smith and Nicholas D.Lakin for help and advice on performing the Western blot analysis. This work was supported by the European Union Grant F14PCT90010 and a grant from the Cancer Research Campaign (SPJ). EAH is a Leukemia Scholar of America and was supported by a grant from the NIH.
- Ionizing radiation
- V(D)J recombination