DNA double strand break repair by NBS1 complex.

奈梅亨断裂综合征（NBS）是一种常染色体隐性遗传疾病，由DSB修复缺陷引起。我们已经分离出NBS患者中突变的NBS 1基因。为了分析NBS 1在DSB修复中的功能，我们建立了小鼠NBS 1基因敲除突变体。Nbs 1突变体表现出胚胎致死性。胚胎在8.5-9.5 dpc死亡。另一方面，Nbs 1-/-原代成纤维细胞实际上是活的。染色体分析表明，辐射后的转换MEF断裂和差距的频率很高。克隆形成分析显示细胞对辐射超敏反应。这些结果清楚地表明，小鼠的NBS 1也是负责基因组的完整性。接下来，我们进行了有针对性的破坏NBS 1基因使用鸡DT 40细胞。Scneo报告基因分析显示，NBS 1破坏的细胞在DSB产生后HR事件显著减少。另一方面，使用线性化质粒DNA的NHEJ测定显示NBS 1突变体DT 40细胞精通末端连接活性。这些结果表明，NBS 1是HR介导的修复所必需的，但不是NHEJ修复所必需的。组蛋白H2 AX是第一个被ATM磷酸化的蛋白质，在照射后立即形成离散的焦点。NBS 1复合物也在照射后的DSB位点上形成焦点。我们报道了NBS 1通过FHA/BRCT结构域直接与γ-H2 AX结合，并形成与γ-H2 AX共定位的灶。这种灶的形成可以促进DNA修复和细胞周期监测。我们提出了NBS 1复合物对DNA修复和检查点控制的两步结合模型。

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.