Abstract
Defect of homologous recombination (HR), which is a major repair pathway for DNA double strand breaks (DSBs), can result in genomic instability and cancer predisposition. Previously, we have demonstrated that NBS1, responsible for cancer predisposition disorder Nijmegen breakage syndrome, is an important factor of HR. However, the mechanism by which NBS1 functions in HR is unknown. To gain insight into the mechanistic roles of NBS1 in HR, we generated the series of NBS1 mutant cDNAs and examined their HR activities using DR-GFP/SCneo system. These systems utilize the rare-cutting I-SceI endonuclease for the introduction of DSB. The HR activity of NBS1 is completely abolished by deletion of MRE11-binding domain, and severely decreased by mutations in FHA or BRCT domains. In contrast, the mutations in the phosphorylation sites of ATM kinase did not affect the HR activity. We also examined AT cells and found that AT cells have normal HR activities. These results suggest that recruitment MRE11/RAD50 to DSB site by NBS1 is crucial for HR, and that NBS1 can function in HR independent of ATM. We are currently investigating the connections between NBS1 and BRCA1/2 in HR using dominant-negative methods. The results of this current study will also be presented.
