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.

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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...

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The carcinoma SC42 was transplanted into the liver of its syngeneic mice DS, and the immunological integrity of the spleen and the effects of splenectomy on the growth and pulmonary metastasis of the liver tumor were assessed. On day 7 after liver tumor transplantation, the natural killer (NK) activity of the splenocytes was significantly elevated; it subsequently decreased at a later stage of the tumor. The response of the splenocytes to PHA and Con-A decreased significantly from the early stage of the tumor. However, the mixed lymphocyte-tumor cell reaction increased significantly from day 14 to day 28. The survival rate of the mice, which had undergone simultaneous splenectomy and liver tumor transplantation, was significantly lower than that of sham-operated control mice. The number of pulmonary metastases in splenectomized mice was significantly greater than in the control mice. There was, however, no difference between the two groups in the weight of the liver tumor. By contrast, splenectomies performed 14 days before or 14 days after tumor transplantation had no significant influence on the survival of the mice. Splenectomies performed on day 0 and on day 3 after tumor transplantation significantly increased the number of pulmonary metastases. Furthermore, the intravenous injection of anti-asialo GM1 antisera on day 0 and day 3 significantly increased the number of pulmonary metastases, but injection of anti-Thy 1.2 antisera had no effect. These results suggest that splenic NK cells may play an important role in the suppression of pulmonary metastasis at early stages of the liver tumor.