Chk2 regulation by DNA damage doses in relation to ATM and Mre11-complex integrity

In eukaryotic cells DNA lesions activate a network of pathways (checkpoints) that coordinately delay cell cycle progression and induce repair, preventing genomic instability and cancer. Chk2, a kinase involved in DNA damage-induced checkpoints and apoptosis, is activated by low doses radiation in an ataxia telangiectasia mutated (ATM)-dependent manner and phosphorylates several substrates (p53, Cdc25A, Cdc25C) causing arrest at multiple cell cycle phases. We have found that gamma-radiation induced Chk2 activation requires Nbs1, the protein defective in the Nijmegen Breakage Syndrome (NBS), a disorder which shares similarity with AT. In NBS cells, Chk2 phosphorylation and activation are defective and could be complemented by wild type Nbs1, but not by mutants that impair Mre11 complex formation or fail to correct the RDS defect. Furthermore, the Nbs1-dependence of Chk2 activation is dose-dependent, demonstrating the damage specificity of checkpoints induction. To further investigate the nature of these defects, Chk2 protein complexes were evaluated pre and post irradiation after size-fractionation on gel filtration. We found that the pattern of Chk2 complexes distribution and phosphorylation differs among normal (70-150KDa pre and 70-300KDa post irradiation), NBS and AT cells (70-150KDa pre and post irradiation). Moreover, analysis on ATLD (AT like disease) cells from patients with Mre11 mutations, showed that Chk2 complexes eluted within a MW range similar to NBS and AT. A defective Chk2 activation in AT, NBS and ATLD could explain the checkpoint defects of these cells and emphasizes the role of Nbs1/Mre11/Rad50 complex as a sensor/adaptor in Chk2-dependent checkpoints.