Dynamic Eukaryotic Replication Machines

动态真核复制机器

• 批准号: 7880903
• 负责人: Linda B Bloom
• 金额: $ 28.69万
• 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/7880903
• 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 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; 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）：DNA复制对基因组的复制是生长和发育所需的正常细胞分裂的先决条件。 DNA的合成是由DNA聚合酶催化的，但是，仅这些酶不能有效地使DNA复制整个基因组。 DNA聚合酶加工性因子是滑动夹和夹具装载机，通过将DNA聚合酶链接到复制的模板中，从而提高了DNA复制的效率。这些加工性因子的结构和功能是从细菌到人的。夹具装载机是一种分子机，它使用ATP将环形滑动夹的组装催化到DNA上。该提案的主要目标是阐明真核夹具加载器复制因子C（RFC）通过定义单个组件的功能来在DNA上组装夹具的机制。我们的压倒性假设是，每种相互作用RFC与其结合伙伴（包括个别ATP分子，夹具（PCNA）和DNA）诱导构象变化，从而促进了途径的下一步，这些离散构象变化有利于有序的事件序列以促进有效的夹具负载。我们检验该假设的主要方法是分析纯化的酿酒酵母RFC催化的反应，并使用基于荧光的基于荧光的测定法对蛋白质蛋白质蛋白和蛋白质Indeindna相互作用以及ATP水解进行测量的替代性RAD24-RFC夹具加载器。此外，位于ATP结合位点中的位置定向诱变对保守序列基序将用于评估单个RFC亚基对夹紧负载的贡献。 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.我们的荧光方法的主要优势是，可以直接在解决方案和实时监测该动态夹具载荷反应，以揭示事件的时间顺序，以及导致该顺序的因素。我们广泛的长期目标是定义分子机制，复制机制复制基因组，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聚合酶加工性因子RFC和PCNA的生化活性以及DNA损伤检查点复合物（RAD24-RFC）的生化活性来促进这些目标。对DNA复制的生化基础的基本理解对于使生化缺陷和疾病之间的临床相关性至关重要。 DNA复制领域的基础研究导致了重要的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。