DNA double strand break repair by NBS1 complex.

NBS1 复合物修复 DNA 双链断裂。

基本信息

批准号：
13116201
负责人：
MATSUURA Shinya
金额：
$ 7.87万
依托单位：
Hiroshima University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research on Priority Areas
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13116201/
关键词：
Nijmegen breakage syndrome Nbs1 Knockout mouse Radiation scnsitivity DT40 cells Homologous recombination Histone H2AX Nuclear foci NBS AT NBS1 電離放射線 DN二重鎖切断 ATM フォーカス dsb テロメア

项目摘要

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder, resulting from the defects in DSB repair. We have isolated the NBS1 gene, mutated in NBS patients. To analyze the NBS1 function in DSB repair, we established mouse Nbs1 knockout mutant. The Nbs1 mutants exhibited embryonic lethality. The embryos died at 8.5-9.5 dpc. On the other hands, the Nbs1-/-primary fibroblast cells were virtually viable. Chromosome analysis of the transformed MEF showed high frequencies of breaks and gaps after irradiation. Clonogenic analysis revealed cellular hypersensitivity to irradiation. These results clearly demonstrated that mouse Nbs1 is also responsible for genomic integrity.Next, we carried out targeted disruption of the NBS1 gene using chicken DT4O cells. Scneo reporter assays revealed that NBS1-disrupted cells showed marked reduction of HR events ollowing generation of DSB. On the other hand, NHEJ assays using linearized plasmid DNA showed that NBS1 mutant DT40 cells were proficient in end-joining activity. These results demonstrated that NBS1 is essential for HR-mediated repair, but not for NHEJ repair. NBS1 might be required to process recombinant intermediates and to suppress inter-chromosomal translocation.Histone H2AX is the first protein, which is phosphorylated by ATM and forms discrete foci immediately after irradiation. NBS1 complex also forms foci on sites of DSBs after irradiation. We reported that NBS1 directly binds to gamma-H2AX, via the FHA/BRCT domain, and form foci to co-localize with that of gamma-H2AX. Such foci formation could facilitate DNA repair and cell cycle monitoring. We proposed the two-step binding model of NBS1 complex for DNA repair and checkpoint control.

Nijmegen Breakage综合征（NBS）是一种常染色体隐性疾病，是由于DSB修复中的缺陷所致。我们已经分离了NBS1基因，在NBS患者中突变。为了分析DSB修复中的NBS1功能，我们建立了小鼠NBS1基因敲除突变体。 NBS1突变体表现出胚胎致死性。胚胎死于8.5-9.5 dpc。另一方面，NBS1 - / - 原发性成纤维细胞几乎是可行的。转化的MEF的染色体分析显示辐射后的断裂和间隙频率很高。克隆分析显示细胞对辐照的超敏反应。这些结果清楚地表明，小鼠NBS1也负责基因组完整性。接下来，我们使用鸡DT4O细胞对NBS1基因进行了靶向破坏。 Scneo报告基因测定法显示，NBS1散布的细胞显示出明显的HR事件降低了DSB的生成。另一方面，使用线性化质粒DNA的NHEJ分析表明，NBS1突变体DT40细胞精通末端结合活性。这些结果表明，NBS1对于HR介导的修复至关重要，但对于NHEJ修复不是必不可少的。可能需要NBS1处理重组中间体并抑制染色体易位。组蛋白H2AX是第一种蛋白质，该蛋白质被ATM磷酸化并在辐照后立即形成离散灶。 NBS1复合物还将重点放在辐照后DSB的位点上。我们报告说，NBS1通过FHA/BRCT结构域直接与γ-H2AX结合，并形成焦点以与γ-H2AX共定位。这种焦点形成可以促进DNA修复和细胞周期监测。我们提出了用于DNA修复和检查点控制的NBS1复合物的两步结合模型。