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.

奈梅亨断裂综合征（NBS）是一种常染色体隐性遗传疾病，其特征在于显著的癌症易感性、对电离辐射的敏感性和DNA损伤依赖性细胞周期检查点的缺陷。 由于NBS的细胞表型与共济失调毛细血管扩张症（AT）患者的细胞表型非常相似，因此该综合征被描述为AT变异综合征，尽管它在遗传上是不同的。 我们发现，NBS是由于缺乏内在成员的hMre 11/hRad 50蛋白复合物，在本文中称为p95。 我们已经确定，这种复合物，这是均匀分布在细胞核中的辐射前，重新定位到网站的DNA损伤的细胞核中的电离辐射处理，并恢复到均匀分布后完成DNA修复。 该复合物与DNA损伤的关联，以及对电离辐射的细胞反应被p95缺陷所废除的发现，导致了该复合物的作用是感知和信号辐射细胞中DNA损伤的存在的假设。在这个提议中，我们将研究p95的功能，以解决这一假设，并阐明其潜在的作用，修复电离辐射诱导的DNA损伤的Mre 11/Rad 50复合物的一部分。 我们建议进行表型分析的p95缺陷细胞表达的p95的变体，以定义功能上重要的结构域的蛋白质。 为了检查在培养细胞中不可测定的表型并评估p95的遗传相互作用，将构建p95缺陷型小鼠。 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，这将通过繁殖p95和ATM缺陷小鼠来评估。这些研究将提供关于p95和ATM在细胞和生物体对电离辐射的反应中的相对贡献的重要信息。 在本提案中，从三个互补的角度对p95进行了审查。 第一，在细胞水平，第二，在分子水平，第三，在体内通过munbs 1突变小鼠的衍生。