DNA double strand break repair by NBS1 complex.

NBS1 复合物修复 DNA 双链断裂。

• 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

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.

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 NBS1 基因，该基因在 NBS 患者中发生突变。为了分析NBS1在DSB修复中的功能，我们建立了小鼠Nbs1敲除突变体。 Nbs1 突变体表现出胚胎致死性。胚胎在 8.5-9.5 dpc 时死亡。另一方面，Nbs1-/-原代成纤维细胞实际上是存活的。转化 MEF 的染色体分析显示，辐射后断裂和间隙频率较高。克隆分析揭示细胞对辐射过敏。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 报告基因检测显示，NBS1 破坏的细胞在 DSB 产生后表现出 HR 事件显着减少。另一方面，使用线性化质粒 DNA 的 NHEJ 测定表明 NBS1 突变体 DT40 细胞具有熟练的末端连接活性。这些结果表明 NBS1 对于 HR 介导的修复至关重要，但对于 NHEJ 修复则不然。 NBS1 可能需要处理重组中间体并抑制染色体间易位。组蛋白 H2AX 是第一个蛋白质，它被 ATM 磷酸化，并在辐射后立即形成离散的病灶。辐射后，NBS1 复合物还在 DSB 位点上形成焦点。我们报道NBS1通过FHA/BRCT结构域直接结合gamma-H2AX，并形成与gamma-H2AX共定位的焦点。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 复合物的两步结合模型。

项目成果

Tauchi, H., et al.: "The forkhead-associatecl domain of NBS1 is essential for nuclear foci formation after Irradiation but not essential for hRAD5O-hMRE11-NBS1 complex DNA repair"J.Biol.Chem.. 276. 12-15 (2001)

Tauchi, H., 等人：“NBS1 的叉头相关结构域对于辐射后核灶的形成至关重要，但对于 hRAD5O-hMRE11-NBS1 复合物 DNA 修复不是必需的”J.Biol.Chem.. 276. 12-15

Hama S.: "p16 gene transfer increased cell killing with abnormal nucleation after ionizing radiation in glioma cells."Br. J. Cancer. 89. 1802-1811 (2003)

Hama S.：“p16 基因转移增加了神经胶质瘤细胞电离辐射后细胞杀伤和异常成核。”Br。

Tauchi, H., et al.: "Nijmegen breakage syndrome gene, NBS1, awl molecular links to factors for genome stability."Oncogene. 21. 8967-8980 (2002)

Tauchi, H. 等人：“奈梅亨断裂综合征基因、NBS1、锥子分子与基因组稳定性因素的联系。”癌基因。

Yamada, M., et al.: "Combined immunodeficiency, chromosomal instability, and postnatal growth deficiency in a Japanese girl"Am.J.Med.Genet.. 100. 9-12 (2001)

Yamada, M., et al.：“日本女孩的联合免疫缺陷、染色体不稳定和产后生长缺陷”Am.J.Med.Genet.. 100. 9-12 (2001)

Tauchi, H., et al.: "Nbs1 is essential for DNA repair via homologous recombination in higher vertebrate cells."Nature. 420. 93-98 (2002)

Tauchi, H. 等人：“Nbs1 对于高等脊椎动物细胞中通过同源重组进行 DNA 修复至关重要。”《自然》。