DNA double strand break repair by NBS1 complex.

奈梅金氏断裂综合征(NBS)是一种常染色体隐性遗传病，由DSB修复缺陷引起。我们已经分离出在NBS患者中突变的NBS1基因。为了分析NBS1在DSB修复中的作用，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。胚胎在8.5-9.5DPC时死亡。另一方面，NBS1-/-原代成纤维细胞实际上是活的。染色体分析表明，转化的MEF在照射后出现了高频率的断裂和缝隙。克隆形成分析显示细胞对辐射超敏。这些结果清楚地表明，小鼠Nbs1也对基因组的完整性负责。接下来，我们利用鸡DT4O细胞进行了NBS1基因的靶向干扰。Scneo报告分析显示，NBS1破坏的细胞在DSB产生后HR事件显著减少。另一方面，用线性化的质粒DNA进行NHEJ检测表明，NBS1突变的DT40细胞具有熟练的末端连接活性。这些结果表明，NBS1是HR介导的修复所必需的，而不是NHEJ修复所必需的。NBS1可能是处理重组中间体和抑制染色体间易位所必需的。组蛋白H2AX是第一个蛋白质，被ATM磷酸化，在照射后立即形成离散的焦点。照射后，NBS1复合体也在DSB的部位形成焦点。我们报道了NBS1通过FHA/BRCT结构域直接与伽马-H_2AX结合，并形成焦点与伽马-H_2AX共定位。这种病灶的形成可以促进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复合体的两步结合模型。

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.