P95--LINKING DSB REPAIR AND CELL CYCLE CHECKPOINTS

P95——连接 DSB 修复和细胞周期检查点

• 批准号: 6182188
• 负责人: John HJ Petrini
• 金额: $ 28.57万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182188
• 关键词: DNA damage; DNA repair; cell growth regulation; chromosome disorders; gene targeting; genetic models; genetically modified animals; laboratory mouse; model design /development; phenotype; radiation genetics; radiation sensitivity; syndrome

Nijmegen Breakage Syndrome (NBS) is an autosomal recessive disorder characterized by marked cancer predisposition, sensitivity to ionizing radiation, and defects in DNA damage dependent cell cycle checkpoints. Because the cellular phenotypes of NBS strongly resemble those of cells established from ataxia telangiectasia (AT) patients, this syndrome has been described as an AT variant syndrome although it is genetically distinct. We showed that NBS is attributable to deficiency in an intrinsic member of the hMre11/hRad50 protein complex, referred to herein as p95. We have established that this complex, which is uniformly distributed in the nucleus prior to irradiation, relocalizes to sites of DNA damage in the nuclei of cells treated with ionizing radiation, and is restored to uniform distribution upon completion of DNA repair. The association of the complex with DNA damage, in conjunction with the finding that cellular responses to ionizing radiation are abrogated by p95 deficiency lead to the hypothesis that the complex acts to sense and signal the presence of DNA damage in irradiated cells. In this proposal, we will examine the function of p95 to address this hypothesis, and to elucidate its potential role the repair of ionizing radiation-induced DNA damage as part of the Mre11/Rad50 complex. We propose to carry out phenotypic analyses of p95 deficient cells expressing variants of p95 to define functionally important domains of the protein. To examine phenotypes that are not assayable in cultured cells and to assess genetic interactions of p95, p95 deficient mice will be constructed. Of particular interest in this regard are the genetic and functional interactions between p95 and ATM which will be assessed by breeding of p95 and ATM deficient mice. These studies will provide important information regarding the relative contributions of p95 and ATM in the cellular and organismal responses to ionizing radiation. In this proposal, p95 is examined from three complementary perspectives. First, at the cellular level, second, at the molecular level, and third, in vivo through the derivation of a munbs1 mutant mouse.

Nijmegen Breakage综合征（NBS）是一种常染色体隐性疾病，其特征是癌症易感性明显，对电离辐射的敏感性以及DNA损伤依赖性细胞周期检查点的缺陷。 由于NBS的细胞表型强烈类似于毛细血管炎（AT）患者的细胞的细胞表型，因此该综合征被描述为AT变体综合征，尽管它在遗传上是不同的。 我们表明，NBS归因于HMRE11/HRAD50蛋白复合物的固有成员的缺乏，此处称为p95。 我们已经确定，这种复合物在辐照之前均匀地分布在细胞核中，重新定位于用电离辐射处理的细胞核中DNA损伤部位，并在完成DNA修复后恢复为均匀分布。 复合物与DNA损伤的关联，结合了以下发现，即通过p95缺乏对细胞对电离辐射的反应消除，导致假说，即该复合物在辐照细胞中具有感知和信号的DNA损伤。在此提案中，我们将研究p95来解决该假设的功能，并阐明其潜在作用，以修复电离辐射诱导的DNA损伤，这是MRE11/RAD50复合物的一部分。 我们建议对表达p95变体的p95缺陷细胞进行表型分析，以定义蛋白质的功能重要结构域。 为了检查在培养细胞中无法检测到的表型并评估p95的遗传相互作用，将构建p95不足的小鼠。 在这方面特别感兴趣的是p95和ATM之间的遗传和功能相互作用，这些相互作用将通过p95和ATM缺乏小鼠的繁殖来评估。这些研究将提供有关P95和ATM在细胞和有机体对电离辐射的反应中相对贡献的重要信息。 在此提案中，从三个互补角度检查了p95。 首先，在细胞水平，其次，在分子水平，第三，通过衍生的MUNBS1突变小鼠在体内。