Molecular mechanism for chromosome stability using Nbs1 knockout mice

奈梅根断裂综合征中突变的基因NBS1编码一个95-kD的蛋白，该蛋白与RAD50和MRE11形成复合物，该复合物参与DNA双链断裂修复的起始过程。在小鼠实验中，Nbs1基因的完全缺失导致胚胎早期死亡和胚胎干细胞活性降低。因此，有人认为人类NBS患者不是零突变体，而是次胚突变体。与这一预测一致的是，在NBS患者中发现了c端截断的NBS1蛋白。在本研究中，我们针对Nbs1基因的破坏，将3’-一半外显子3和4、5的所有外显子替换为LacZ基因和PGKneo盒。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。胚胎死亡时间延迟至植入后8.5 ~ 9.5 dpc，胚胎成纤维细胞基本存活。对胚胎成纤维细胞的分析显示，Nbs1突变等位基因意外地表达了少量的c端Nbs1蛋白，这可能类似于人类的c端蛋白。与这一结果一致的是，通过移除PGKneo磁带，c端Nbs1蛋白的缺失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Nbs1零突变体致死表型的严重程度。

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.