Dynamic Eukaryotic Replication Machines

动态真核复制机器

• 批准号: 7527173
• 负责人: Linda B Bloom
• 金额: $ 27.24万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7527173
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Area; Bacteria; Basic Science; Binding; Binding Sites; Biochemical; Biological Assay; Biological Process; Boxing; Cell division; Clinical; Complex; Conserved Sequence; Coupled; DNA; DNA Binding; DNA Damage; DNA biosynthesis; DNA chemical synthesis; DNA damage checkpoint; DNA-Directed DNA Polymerase; Defect; Development; Diagnostic; Disease; Dissociation; Enzymes; Event; Family; Fluorescence; Genome; Goals; Growth and Development function; Human; Hydrolysis; In Vitro; Individual; Measures; Medical; Molecular; Molecular Machines; Monitor; Mutation; Normal Cell; Nucleotides; Pathway interactions; Rate; Reaction; Saccharomyces cerevisiae; Shapes; Site; Site-Directed Mutagenesis; Slide; Solutions; Specificity; Structure; Substrate Specificity; Testing; Therapeutic Agents; Time; Walkers; activator 1 protein; base; defined contribution; ds-DNA; enhancing factor; insight; man; member; pathogen; preference; tool

DESCRIPTION (provided by applicant): Duplication of the genome by DNA replication is a prerequisite for normal cell division required for growth and development. Synthesis of DNA is catalyzed by DNA polymerases, however, these enzymes alone cannot make DNA efficiently enough to duplicate the entire genome. DNA polymerase processivity factors, a sliding clamp and clamp loader, enhance the efficiency of DNA replication by tethering a DNA polymerase to the template being copied. The structure and function of these processivity factors are conserved from bacteria to man. The clamp loader is a molecular machine that uses ATP to catalyze the assembly of ring-shaped sliding clamps onto DNA. The major goal of this proposal is to elucidate the mechanism by which the eukaryotic clamp loader, replication factor C (RFC), assembles clamps on DNA by defining functions for individual components. Our overriding hypothesis is that each interaction RFC makes with its binding partners, including individual ATP molecules, the clamp (PCNA), and DNA, induces conformational changes that facilitate the next step in the pathway, and these discrete conformational changes favor an ordered sequence of events to promote efficient clamp loading. Our major approach to testing this hypothesis will be to analyze reactions catalyzed by purified Saccharomyces cerevisiae RFC and an alternative Rad24-RFC clamp loader in vitro using fluorescence-based assays to measure proteinprotein and proteinDNA interactions as well as ATP hydrolysis. In addition, site-directed mutagenesis to conserved sequence motifs in ATP binding sites will be used to evaluate the contributions of individual RFC subunits to clamp loading. Specifically, our aims are 1) to define functions for ATP binding and hydrolysis by individual RFC subunits, 2) to use the alternative clamp loader, Rad24-RFC, as a tool to identify contributions that the large "A-subunit" of RFC makes to PCNA and DNA binding, 3) to identify reciprocal effects of clamp and DNA binding on the activities of RFC and Rad24- RFC. A major strength of our fluorescence approach is that this dynamic clamp loading reaction can be monitored directly in solution and in real time to uncover the temporal order of events, and factors that give rise to this order. Our broad and long-term objectives are to define molecular mechanisms by which the replication machinery duplicates genomes, and to define mechanisms by which these enzymes respond to DNA damage that is encountered during replication. This project will contribute to those objectives by characterizing the biochemical activities of DNA polymerase processivity factors, RFC and PCNA, and of a DNA damage checkpoint complex, Rad24-RFC. A fundamental understanding of the biochemical basis of DNA replication is essential to making clinical correlations between biochemical defects and disease. Basic research in the area of DNA replication has led to the development of important medical diagnostic tools as well as the development of therapeutic agents that inhibit replication of pathogens.

描述（由申请人提供）：通过 DNA 复制来复制基因组是生长和发育所需的正常细胞分裂的先决条件。 DNA 的合成由 DNA 聚合酶催化，然而，仅靠这些酶无法有效地制造 DNA 来复制整个基因组。 DNA 聚合酶持续合成因子（滑动夹和夹钳加载器）通过将 DNA 聚合酶束缚到正在复制的模板上来提高 DNA 复制的效率。这些持续因子的结构和功能从细菌到人类都是保守的。夹钳装载机是一种分子机器，利用 ATP 催化环形滑动夹组装到 DNA 上。该提案的主要目标是通过定义各个组件的功能来阐明真核夹具装载机复制因子 C (RFC) 在 DNA 上组装夹具的机制。我们最重要的假设是，RFC 与其结合伙伴（包括单个 ATP 分子、钳子 (PCNA) 和 DNA）进行的每次相互作用都会诱导构象变化，从而促进途径的下一步，而这些离散的构象变化有利于有序的序列促进高效夹紧加载的事件。我们测试这一假设的主要方法是使用基于荧光的测定法来分析纯化的酿酒酵母 RFC 和替代的 Rad24-RFC 夹钳在体外催化的反应，以测量蛋白质蛋白质和蛋白质 DNA 相互作用以及 ATP 水解。此外，对 ATP 结合位点中保守序列基序的定点诱变将用于评估单个 RFC 亚基对钳负载的贡献。具体来说，我们的目标是 1) 定义单个 RFC 亚基的 ATP 结合和水解功能，2) 使用替代夹钳装载机 Rad24-RFC 作为工具来确定 RFC 的大“A 亚基”所做的贡献PCNA 和 DNA 结合，3) 确定钳和 DNA 结合对 RFC 和 Rad24-RFC 活性的相互影响。我们的荧光方法的主要优势在于，可以在溶液中直接实时监测这种动态钳加载反应，以揭示事件的时间顺序以及引起该顺序的因素。我们广泛而长期的目标是定义复制机器复制基因组的分子机制，并定义这些酶对复制过程中遇到的 DNA 损伤做出反应的机制。该项目将通过表征 DNA 聚合酶持续合成因子 RFC 和 PCNA 以及 DNA 损伤检查点复合物 Rad24-RFC 的生化活性来实现这些目标。对 DNA 复制的生化基础的基本了解对于建立生化缺陷与疾病之间的临床关联至关重要。 DNA复制领域的基础研究促进了重要医学诊断工具的开发以及抑制病原体复制的治疗剂的开发。