The Forkhead-associated Domain of NBS1 Is Essential for Nuclear Foci Formation after Irradiation but Not Essential for hRAD50·hMRE11·NBS1 Complex DNA Repair Activity

Abstract

NBS1 (p95), the protein responsible for Nijmegen breakage syndrome, shows a weak homology to the yeast Xrs2 protein at the N terminus region, known as the forkhead-associated (FHA) domain and the BRCA1 C terminus domain. The protein interacts with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination. Here, we show in vivo direct evidence that NBS1 recruits the hMRE11 nuclease complex into the cell nucleus and leads to the formation of foci by utilizing different functions from several domains. The amino acid sequence at 665-693 on the C terminus of NBS1, where a novel identical sequence with yeast Xrs2 protein was found, is essential for hMRE11 binding. The hMRE11-binding region is necessary for both nuclear localization of the complex and for cellular radiation resistance. On the other hand, the FHA domain regulates nuclear foci formation of the multiprotein complex in response to DNA damage but is not essential for nuclear transportation of the complex and radiation resistance. Because the FHA/BRCA1 C terminus domain is widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, and DNA repair, the foci formation could be associated with many phenotypes of Nijmegen breakage syndrome other than radiation sensitivity.NBS1 is a responsible gene for Nijmegen breakage syndrome (NBS), 1 a variant of ataxia-telangiectasia. Disruption of NBS1 in NBS patients leads to hypersensitivity to ionizing radiation, chromosomal instability, and a predisposition to cancer (1-3). NBS1 (p95) protein shows a weak (29%) homology to the yeast (Saccharomyces cerevisiae) Xrs2 protein only in the N terminus regions known as forkhead-associated (FHA) domain and BRCA1 C terminus (BRCT) domain (1-3), which are widely conserved in eukaryotic nuclear proteins related to the cell cycle, gene regulation, or DNA repair (4, 5). The protein is known to interact with hMRE11 to form a complex with a nuclease activity for initiation of both nonhomologous end joining and homologous recombination (6, 7). However, the function of most of the NBS1 protein is still not understood, because about 70% of the NBS1 protein on the C terminus end does not show any sequence homology to any known proteins including Xrs2 (1-3). Because the mutations found in NBS patients all occur between codons 220 and 385 of the NBS1 gene (3) and lead to proteins truncated downstream of the FHA/BRCT domain, the C-terminal half of the protein must be associated with the crucial phenotype of NBS, which may depend on nuclear localization of hMRE11⅐hRAD50 (2). Recently, we reported that expression of the full-length NBS1 protein complements multiple NBS phenotype characteristics such as radiation sensitivity, the G 2 checkpoint, and focus formation in nucleus after irradiation (8). These findings enable us to analyze the functional domain of NBS1 using deletion mutants of NBS1 transfected in NBS cells. By this approach, we localized an essential domain at C terminus region of N...

Cleavage of the BRCT tandem domains of nibrin by the 657del5 mutation affects the DNA damage response less than the Arg215Trp mutation

“…Thus, the 70-kDa NBN fragment is somehow able to localize at the DSB, bringing on the damaged site also to the MRN complex, even if with a delayed time course. This result agrees with data indicating that the NBN fragment comprising amino acids 401-754 is able to interact with MRE11 (32,39). the 70-kDa NBN fragment levels and the lymphoma risk (40).…”

“…3A indicate that both the 26-and the 70-kDa NBN fragments coimmunoprecipitate with c-H2AX. Although it is well known that the FHA/BRCT1 domains, present in the 26-kDa NBN fragment, are essential for IRIF formation (32,33), these data represent the first evidence for the interaction of the 70-kDa NBN fragment (containing only the second BRCT domain) to c-H2AX. Remarkably, the Arg215Trp NBN mutant does not coimmunoprecipitate with c-H2AX (27).…”

“…The absence of a nuclear localization signal (NLS) within the p26 fragment seems not to be so relevant for its localization within the nucleus (26,33), as demonstrated by the evidence that C-terminal truncation mutants of mouse NBN fused to GFP and transfected into NIH-3T3 cells where able to localize within the nucleus (37). Although the FHA and BRCT1 domains are pivotal for both IRIF formation (32,36,38) and for the interaction with c-H2AX (33), data reported here indicate that the 70-kDa NBN fragment, containing only the BRCT2 domain, binds to c-H2AX. Thus, the 70-kDa NBN fragment is somehow able to localize at the DSB, bringing on the damaged site also to the MRN complex, even if with a delayed time course.…”

“…2A). Indeed, in NBS fibroblasts MRE11 localizes mainly in the cytoplasm and to a lesser extent into the nucleus (26,32), whereas in R215W cells MRE11 localizes completely into the cytoplasm. Remarkably, in NBS fibroblasts, and more severely in the R215W cells, an altered pattern and time course of IRIF formation occurs after IR treatment (Fig.…”

p16 gene transfer increases cell killing with abnormal nucleation after ionising radiation in glioma cells

“…To obtain uniform and reproducible irradiation conditions, cells were trypsinised, counted, placed in the glass tube, and irradiated using a 60 Co source at a dose rate of 0.65 Gy min À1 , as described elsewhere (Tauchi et al, 2001). Known numbers of cells were then replated in culture dishes and returned to the incubator.…”