Abstract
Nijmegen syndrome (NBS) is a radiation-hypersensitive genetic disorder. NBS and AT (Ataxia-Telangiectasia syndrome) show the similar cellular phenotype such as radiation-hypersensitivity chromosomal instability and radiation-resistant DNA synthesis. So far, it has been clarified that the responsible gene product of NBS, NBS1 interacts with ATM (the responsible gene product of ATM) and this interaction is indispensable for the recruitment of ATM to DNA double-strand break (DSB) sites and activation of ATM kinase. Hence, the functional interaction between NBS1 and ATM is important for regulation of cell cycle checkpoint. Previously, we reported that NBS1 forms the complex with phosphorylated histone H2AX (gamma-H2AX) in response to DSB damage and this interaction is essential to the recruitment of NBS1 to DSB sites. Thus, NBS1 might have important roles in extensive DNA damage response through binding with ATM and H2AX. Therefore, we investigate the functional interaction among these factors in DSB repair.
NBS1 has BRCT and FHA domain in N-terminus, ATM-phosphorylated sites in central region and hMRE11 and ATM-binding sites in C-terminus. Hence, we investigated the role of these domains for homologous recombination (HR) repair using DR-GFP assay. The mutation in BRCT, FHA or MRE-binding domains decreased HR activity, but the mutation in ATM-phosphorylated or ATM-binding sites did not influence HR activity. Moreover, AT cells showed the HR activity in normal level, suggesting that ATM might be dispensable for HR repair. As gamma-H2AX interacts with NBS1 through the FHA/BRCT domain, we examined the role of H2AX in HR repair. H2AX-knockout ES cells showed the decrease in HR activity and the mutation into acetylated sites of H2AX influenced the IR-induced foci formation and HR activity. Further, the repression of acetylation at common sites between H2A and H2AX by a specific inhibitor also decreased IR-induced foci formation and HR activity. Taken together, both NBS1 and H2AX might function in HR repair and the acetylation of histone H2A may be important for DNA damage response.
