NBS1 Localizes to γ-H2AX Foci through Interaction with the FHA/BRCT Domain

is phosphorylated and forms discrete foci immediately (within 5 min) after irradiation [5]; hence, it may represent an earlier signaling response than formation of the complex within 2 hr of irradiation, as detected by anti-hMRE11 antibody (Figure 2A). In the absence of NBS1, 5 Medical and Biological Laboratories Nagano 396-0002 hMRE11 protein was confined to the cytoplasm and did not form nuclear foci, while ␥-H2AX foci were detected Japan (Figure 2A). Subsequently, we examined potential protein interactions by using immunoprecipitation assays. A small amount of NBS1 was detected in immunoprecip-Summary itates by using anti-␥-H2AX antibody in nonirradiated normal cells, possibly during S phase [6], and this DNA double-strand breaks represent the most poten-amount was significantly increased after irradiation with tially serious damage to a genome; hence, many repair 10 Gy (Figure 2B). Similarly, ␥-H2AX coimmunoprecipi-proteins are recruited to nuclear damage sites by as tated with anti-NBS1 antibody (Figure 2C). hMRE11 was yet poorly characterized sensor mechanisms. Here, also present in this NBS1/␥-H2AX immuno-complex, we show that NBS1, the gene product defective in while the amount of hMRE11 did not parallel that of Nijmegen breakage syndrome (NBS) [1–3], physically NBS1, probably due to the direct binding of hMRE11 to interacts with histone, rather than damaged DNA, by DNA. However, when NBS cells were irradiated, hMRE11 direct binding to ␥-H2AX. We also demonstrate that was not detected in immunoprecipitates with anti-␥-NBS1 binding can occur in the absence of interaction H2AX antibody (Figure 2B), and this indicates that with hMRE11 or BRCA1. Furthermore, this NBS1 physi-hMRE11/hRAD50 cannot interact with ␥-H2AX in the cal interaction was reduced when anti-␥-H2AX anti-absence of NBS1 and that N/M/R complex interactions body was introduced into normal cells and was also with ␥-H2AX are most likely mediated by binding of delayed in AT cells, which lack the kinase activity for NBS1 to ␥-H2AX. phosphorylation of H2AX. NBS1 has no DNA binding These experiments did not allow us to exclude the region but carries a combination of the fork-head as-possibility of unknown intermediate cofactor(s), which sociated (FHA) and the BRCA1 C-terminal domains might bridge NBS1 and ␥-H2AX. Therefore, H2AX-(BRCT) [4]. We show that the FHA/BRCT domain of N/M/R interactions were investigated by in vitro experi-NBS1 is essential for this physical interaction, since ments with recombinant proteins. When a mixture of NBS1 lacking this domain failed to bind to ␥-H2AX in recombinant H2AX and recombinant full-length NBS1 cells, and a recombinant …