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: 3303976
• 负责人: MARC S WOLD
• 金额: $ 15.14万
• 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 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3303976
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA primase; DNA replication; DNA replication origin; DNA topoisomerases; HeLa cells; affinity chromatography; binding proteins; chemical synthesis; chromatography; cow; crosslink; hydropathy; molecular cloning; monoclonal antibody; peptide chemical synthesis; phosphorylation; posttranslational modifications; protein purification; protein reconstitution; protein sequence; protein structure function; radiopharmacology; radiotracer; ribonuclease III; simian virus 40; site directed mutagenesis; stoichiometry; thymus; tissue /cell culture; western blottings

Control of chromosomal replication is crucial for the growth and development of mammalian cells. During each cell cycle, a dividing cell accurately duplicates its entire complement chromosomes, composed of several billion basepairs. Perturbations of this fundamental process lead to abnormal growth and are associated with various disease states. The long-term objective of this work is to increase our understanding of the molecular mechanisms of chromosomal DNA replication. Because the process of chromosomal replication is complex, simpler viral model systems such as simian virus 40 (SV40) are essential for identifying and purifying replication proteins. By dissecting such systems and studying the properties of individual proteins in detail, it is possible to understand how these proteins function in concert to replicate chromosomal DNA. This proposal focuses on a novel multi-subunit, human, single-stranded DNA- binding protein, Replication Protein-A (RP-A). This protein is absolutely required for SV40 DNA replication and is probably involved in chromosomal replication. The principal goal of these studies is to elucidate the physiological role of RP-A during DNA replication. A SV40-based cell-free replication system will be used as a functional assay. There will be three major experimental components of this research: (1) Both biochemical properties and functional activities, such as single- stranded DNA-binding activity, will be studied to determine how they influence the role of RP-A in DNA replication. (2) The affect of RP-A on the enzymatic activities of other replication proteins will be determined Specific interactions will be analyzed for in immunoprecipitation and cross-linking studies. The mechanistic consequences of such interactions will be examined. (3) Protein affinity chromatography will be used to isolate novel replication proteins. The functional significance of such proteins will be analyzed in the SV40 cell-free system. Previously unidentified proteins will be purified and characterized. In the future, these studies will be expanded to examine the roles of other DNA replication proteins. One future direction will be to establish non- viral model reactions for chromosomal DNA replication.

染色体复制的控制对于生长和发育至关重要 哺乳动物细胞的发育。 在每个细胞周期中，一个正在分裂的细胞 准确地复制其整个补体染色体，由 数十亿个碱基对。 这一基本过程的扰动导致 异常生长并与各种疾病状态相关。 这 这项工作的长期目标是增加我们对 染色体DNA复制的分子机制。 因为过程 染色体复制的过程是复杂的，更简单的病毒模型系统，例如 猿猴病毒 40 (SV40) 对于识别和纯化至关重要 复制蛋白。 通过剖析此类系统并研究 单个蛋白质的详细特性，可以理解 这些蛋白质如何协同作用来复制染色体 DNA。 该提案重点关注一种新型多亚基、人类单链 DNA- 结合蛋白，复制蛋白-A (RP-A)。 这个蛋白质绝对是 SV40 DNA 复制所需，可能参与染色体 复制。 这些研究的主要目标是阐明 RP-A 在 DNA 复制过程中的生理作用。 基于 SV40 的无细胞 复制系统将用作功能测定。 将会有三个 本研究的主要实验组成部分： (1)兼具生化特性和功能活性，如单 链DNA结合活性，将被研究以确定它们如何 影响 RP-A 在 DNA 复制中的作用。 (2) RP-A对其他复制酶活性的影响 将确定蛋白质的具体相互作用将在 免疫沉淀和交联研究。 机械论 将审查这种相互作用的后果。 (3) 蛋白质亲和层析将用于分离新的 复制蛋白。 此类蛋白质的功能意义将是 在 SV40 无细胞系统中进行分析。 以前未鉴定的蛋白质 将被纯化和表征。 未来，这些研究将扩大到检验其他因素的作用 DNA复制蛋白。 未来的一个方向是建立非 染色体 DNA 复制的病毒模型反应。