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Molecular mechanism for chromosome stability using Nbs1 knockout mice

Nbs1 敲除小鼠染色体稳定性的分子机制

基本信息

批准号：
12672201
负责人：
MATSUURA Shinya
金额：
$ 2.18万
依托单位：
HIROSHIMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672201/
关键词：
NBS AT NBS1 Radiation dsb ATM telomere focus 放射線高感受性染色体不安定性ノックアウトマウス Nbs1 Atm Ku70 高発癌性免疫不全

项目摘要

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.

Nijmegen断裂综合征中突变的基因NBS1编码一个95 kD的蛋白质，该蛋白质与RAD 50和MRE 11形成复合物，该复合物参与DNA双链断裂修复的起始过程。在小鼠中，Nbs1基因的完全缺失导致早期胚胎死亡和ES细胞存活。因此，已经表明，人类NBS患者不是无效突变体，而是亚型突变体。与这一预测一致，在NBS患者中发现了C末端截短的NBS 1蛋白。在本研究中，我们通过将Nbs1基因外显子3的3'-一半以及外显子4和5的所有外显子替换为LacZ基因和PGKneo盒来靶向破坏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。胚胎死亡时间延迟至着床后8.5 - 9.5 dpc，胚胎成纤维细胞基本上存活。胚胎成纤维细胞的分析表明，Nbs1突变等位基因表达了一个意想不到的少量的C-末端Nbs1蛋白，这可能是类似于人类的C-末端蛋白。与该结果一致，通过去除PGKneo盒，C-末端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-末端Nbs1蛋白减少小鼠Nbs1无效突变体的致死表型的严重程度。