Abstract
The proteins responsible for radiation sensitivity disorders, NBS1, ATM and MRE11, interact through the C-terminus of NBS1 in response to the generation of DNA double strand breaks (DSB), and are all implicated in checkpoint regulation and DSB repair. To gain insights into the mechanistic role of NBS1 in homologous recombination (HR) and the possible relevance of ATM and MRE11 to this process, we generated a series of human NBS1 mutant cDNAs and measured their ability to regulate HR repair using the DR-GFP or SCneo systems. The HR-regulating ability of NBS1 is dramatically reduced by deletion of the MRE11-binding domain at the C-terminus of NBS1, and markedly inhibited by mutations in the FHA/BRCT domains at the N-terminus. This impaired capability in HR is consistent with a failure to observe MRE11 foci formation at damaged sites after transfection of NBS cells with these mutants. In contrast, HR frequencies are only slightly affected by mutations in the serine residues phosphorylated by ATM in response to DSB, or by deletion of ATM binding site at the extreme C-terminus. This was confirmed by findings that ATM deficiency did not reduce the HR repair frequency of an induced DSB, although ATM itself and the ATM-binding domain on NBS1 are essential for regulation of intra-S phase checkpoint. These results suggested that the N- and C-terminal domains of NBS1 are the major regulatory domains for HR pathways, very likely through the recruitment and retention of the MRE11 nuclease to DSB sites in an ATM-independent fashion.
