Molecular mechanism for chromosome stability using Nbs1 knockout mice

该基因在奈梅根断裂综合征NBS1中突变，编码一个95kD的蛋白质，与Rad50和Mre11形成复合体，该复合体参与DNA双链断裂修复的启动过程。在小鼠中，Nbs1基因的完全缺失会导致早期胚胎死亡和ES细胞死亡。因此，有人认为人类NBS患者不是零突变，而是亚型突变。与这一预测一致的是，在NBS患者中发现了C末端截短的NBS1蛋白。在本研究中，我们通过将外显子3的3‘-一半以及外显子4和5的所有外显子替换到LacZ基因和PGKneo盒中来破坏Nbs1基因。我们的Nbs1突变体也表现出胚胎致死性，但表型比以前报道的要温和得多。胚胎死亡时间延至着床后8.5~9.5dpc，胚胎成纤维细胞基本存活。对胚胎成纤维细胞的分析表明，Nbs1突变等位基因意外地表达了少量的C端Nbs1蛋白，这可能与人类的C端蛋白类似。与这一结果一致的是，C端Nbs1蛋白的缺失，通过去除PGKneo盒，提前了胚胎死亡的时间。因此，我们得出结论，C端Nbs1蛋白降低了小鼠Nbs1缺失突变体的致死表型严重程度。

The gene mutated in Nijmegen breakage syndrome, NBS1, encodes a 95-kD protein that forms a complex with RAD50 and MRE11, and the complex is involved in the initiation process of DNA double strand break repair. In mouse, complete loss of Nbs1 gene results in early embryonic lethality and ES cells inviality. Therefore, it has been suggested that human NBS patients are not null mutants but hypomorphic mutants. Consistent with this prediction, C-terminal truncated NBS1 protein was identified in NBS patients. In this study, we targeted for disruption of Nbs1 gene by replacing 3'-half of exon 3 and all exons of 4 and 5 into LacZ gene and PGKneo cassette. Our Nbs1 mutants also exhibited embryonic lethality, but the phenotypes were much milder than those reported previously. The timing of embrynic death delayed to the post-implantation stage of 8.5-9.5 dpc and the embryonic fibroblast cells were virtually viable. Analysis of embryonic fibroblasts revealed that the Nbs1 mutant allele expressed an unexpected small quantity of C-terminal Nbs1 protein, which may be analogous to the human C-terminal protein. Consistent with this result, the absence of C-terminal Nbs1 protein, by removing the PGKneo cassete, advanced the timing of embrynic death. We, therefore, concluded that the C-terminal Nbs1 protein diminished severity of lethal phenotype in mouse Nbs1 null mutants.