FUNCTIONAL ROLES OF CELLULAR PROTEINS IN DNA REPLICATION

细胞蛋白质在 DNA 复制中的功能作用

• 批准号: 2444761
• 负责人: MARC S WOLD
• 金额: $ 20.07万
• 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 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2444761
• 关键词: DNA binding protein; DNA replication; affinity chromatography; gel mobility shift assay; intermolecular interaction; molecular cloning; phosphorylation; posttranslational modifications; protein structure function; simian virus 40; site directed mutagenesis; tissue /cell culture

Each cell cycle, a human cell must specifically initiate DNA replication at multiple sites and accurately duplicate the entire 4x10-9 base pair human genome. Perturbations of chromosomal DNA replication lead to abnormal growth and are associated with various disease states. The goal of this research is to elucidate molecular mechanisms and regulatory processes of chromosomal DNA replication. Because the process of chromosomal replication is complex, we will use the virus, simian virus 40 (SV40) as a model system for examining the mechanism of eukaryotic DNA replication. With the exception of the virally encoded initiator large T antigen, SV40 relies completely on the host replication apparatus, so it provides an excellent system for the biochemical analysis of cellular replication proteins and processes. This proposal focuses on the human multi-subunit, single-stranded DNA- binding protein, Replication Protein A (hRPA). This protein is essential for DNA replication. In addition, hRPA is involved in DNA repair and recombination, and may be a target for regulation in these processes. hRPA binds tightly to single-strand DNA, affects the activity of other replication proteins and interacts specifically with large T antigen, DNA polymerase alpha, the tumor suppressor p53, several transcriptional initiators (e.g. GAL4 and VP16). The physiological importance of the activities attributed to hRPA and the function of hRPA in the cell are currently not understood. The objective of this research is to determine the specific role of hRPA in DNA replication. A combination of biochemical assays and mutational analysis will be used to elucidate the function of hRPA. The specific functions of each of the three subunits of hRPA and the structure of hRPA will be determined. The interactions of hRPA with both DNA and other proteins will be studied and quantitated to determine their importance for hRPA function. hRPA becomes phosphorylated at the beginning of S-phase. The role of post-translational modifications on hRPA function in DNA replication will be determined.

每个细胞周期，人类细胞必须特异性启动DNA复制 在多个位点准确复制整个 4x10-9 碱基对 人类基因组。 染色体DNA复制的扰动导致 异常生长并与各种疾病状态相关。目标 这项研究的目的是阐明分子机制和调控 染色体DNA复制过程。因为这个过程 染色体复制很复杂，我们会用到病毒，猿猴病毒40 （SV40）作为检查真核 DNA 机制的模型系统 复制。 除了病毒编码的引发剂大T SV40 抗原，完全依赖于宿主复制装置，因此它 为细胞生化分析提供了一个优秀的系统 复制蛋白质和过程。 该提案重点关注人类多亚基、单链 DNA- 结合蛋白，复制蛋白 A (hRPA)。这种蛋白质是必需的 用于DNA复制。此外，hRPA 还参与 DNA 修复和 重组，并且可能是这些过程中监管的目标。 hRPA 与单链 DNA 紧密结合，影响其他 DNA 的活性 复制蛋白并与大 T 抗原、DNA 特异性相互作用 聚合酶 α、肿瘤抑制因子 p53、多种转录因子 引发剂（例如 GAL4 和 VP16）。 的生理重要性 hRPA 的活性和 hRPA 在细胞中的功能是 目前不明白。这项研究的目的是确定 hRPA 在 DNA 复制中的具体作用。 的组合 生化分析和突变分析将用于阐明 hRPA 的功能。三个亚基各自的具体功能 hRPA 和 hRPA 的结构将被确定。的相互作用 将研究并定量含有 DNA 和其他蛋白质的 hRPA 确定它们对 hRPA 功能的重要性。 hRPA 被磷酸化 S期开始时。翻译后修饰的作用 将确定 hRPA 在 DNA 复制中的功能。