Takahiro Shiraishi

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11–Rad50–Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.

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

NBS1 (p95), the protein responsible forNijmegen 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 vivodirect 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.

The FHA domain of NBS1 is essential for nuclear foci formation after irradiation, but not essential for hRAD50/hMRE11/NBS1 complex DNA repair activity

NBS1 (p95), the protein responsible forNijmegen 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 vivodirect 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.

Studies of mutagenesis caused by low dose rate tritium radiation using a novel hyper-sensitive detection system

ABSTRACT A novel hyper-sensitive detection system was developed to detect Hprt-deficient mutations using Hprt deficient hamster fibroblast cells which carry a normal human X-chromosome. The system has been found to be 100-fold more sensitive for detecting mutations than the conventional system which uses an internal Hprt gene. The mutation frequency induced by 1 Gy of tritium radiation at different dose rates (0.9, 0.4, 0.04, and 0.018 Gy/h) was measured. No significant differences in mutation frequencies were observed within the range of dose rates used, suggesting that if a reverse dose-rate effect exists, it may not be observable with tritium radiation at dose rates over 0.018 Gy/h. Interestingly, molecular analysis of the Hprt locus in Hprt-deficient mutants induced by tritium showed that deletion sizes observed in the hamster cell’s human X-chromosome under these conditions are much smaller in cells exposed at 0.04 (and 0.018 Gy/h) than in cells exposed at 0.9 Gy/h. This phenomenon seems to be specific for tritium radiation because it was not apparent after exposure to γ-rays. The novel hyper-sensitive detection system used here is useful for analysis of the mutagenic effects of low doses of tritium radiation delivered at low dose rates.