Functional Role of Cellular Proteins in DNA Replication

细胞蛋白质在 DNA 复制中的功能作用

• 批准号: 7049943
• 负责人: MARC S WOLD
• 金额: $ 37.15万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2009-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049943
• 关键词: DNA binding protein; DNA repair; DNA replication; X ray crystallography; intermolecular interaction; molecular genetics; nuclear magnetic resonance spectroscopy; phosphorylation; protein structure function; yeasts

DESCRIPTION (provided by applicant): Defects in cellular DMA metabolism have a direct role in many human disease processes. Impaired responses to DNA damage and defects in basal DNA repair have been implicated as causal factors in life- shortening diseases including cancer, trinucleotide repeat diseases such as Fragile X and Huntington's diseases, and genetic diseases such as Xeroderma Pigmentosum, Cockayne's and Werner's syndromes. Understanding the molecular mechanisms of cellular DNA replication and repair is essential for developing new treatments for these diseases. Replication protein A (RPA) is a multi-functional, single-stranded DMA-binding protein composed of subunits of 70-, 32- and 14-kDa. RPA is essential for DNA replication, DNA repair, recombination and coordination of the cellular response to DNA damage. RPA is phosphorylated on the N-terminus of 32-kDa subunit during S-phase and in response to DNA damage. Recent data demonstrate that RPA hyper- phosphorylation modulates RPA activity and plays a role in the cellular response to DNA damage. The mechanism of regulation of RPA is currently poorly understood. We hypothesize that hyper-phosphorylation of RPA induces a novel conformational change in RPA that alters specific RPA-protein and/or RPA-DNA interactions to regulate DNA metabolism. Studies proposed will test this hypothesis using a combination of biochemical, genetic and structural approaches to elucidate the mechanism of RPA regulation. In three independent Aims, we will: (1) define RPA-DNA interactions modulated by hyper-phosphorylation; (2) define RPA-protein interactions modulated by hyper- phosphorylation and characterize changes in RPA activity in vitro and in vivo caused by hyper- phosphorylation; and (3) carry out structural analysis to define hyper-phosphorylation induced conformational changes in RPA. These studies will both elucidate the mechanism of the RPA regulatory switch and provide insights into RPA function in cellular DNA metabolism.

描述（由申请人提供）：细胞 DMA 代谢缺陷在许多人类疾病过程中具有直接作用。对DNA损伤的反应受损和基础DNA修复缺陷被认为是缩短寿命的疾病的致病因素，包括癌症、脆性X染色体和亨廷顿舞蹈病等三核苷酸重复疾病，以及色素性干皮病、科凯恩综合征和维尔纳综合征等遗传病。了解细胞 DNA 复制和修复的分子机制对于开发这些疾病的新疗法至关重要。复制蛋白 A (RPA) 是一种多功能、单链 DMA 结合蛋白，由 70、32 和 14 kDa 的亚基组成。 RPA 对于 DNA 复制、DNA 修复、重组以及协调细胞对 DNA 损伤的反应至关重要。 RPA 在 S 期和对 DNA 损伤的反应中在 32 kDa 亚基的 N 末端被磷酸化。最近的数据表明，RPA 过度磷酸化可调节 RPA 活性，并在细胞对 DNA 损伤的反应中发挥作用。目前对 RPA 的调节机制知之甚少。我们假设 RPA 的过度磷酸化会诱导 RPA 发生新的构象变化，从而改变特定的 RPA-蛋白质和/或 RPA-DNA 相互作用，从而调节 DNA 代谢。拟议的研究将结合生化、遗传和结构方法来检验这一假设，以阐明 RPA 调节机制。在三个独立的目标中，我们将：（1）定义由过度磷酸化调节的 RPA-DNA 相互作用； (2)定义由过度磷酸化调节的RPA-蛋白质相互作用，并表征由过度磷酸化引起的体外和体内RPA活性的变化； (3) 进行结构分析以确定 RPA 中过度磷酸化诱导的构象变化。这些研究将阐明 RPA 调节开关的机制，并深入了解 RPA 在细胞 DNA 代谢中的功能。