Dynamic Eukaryotic Replication Machines

动态真核复制机器

• 批准号: 7917132
• 负责人: Linda B Bloom
• 金额: $ 23.39万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-18 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7917132
• 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(申请人提供)：通过DNA复制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的合成是由DNA聚合酶催化的，然而，仅靠这些酶不能有效地制造DNA来复制整个基因组。DNA聚合酶加工性因子，一个滑动夹子和夹子加载器，通过将DNA聚合酶与被复制的模板捆绑在一起，提高了DNA复制的效率。这些加工性因子的结构和功能从细菌到人类都是保守的。夹子加载器是一种分子机器，它使用三磷酸腺苷催化将环形滑动夹子组装到DNA上。这项建议的主要目的是阐明真核细胞的钳夹加载器，复制因子C(RFC)，通过定义单个组件的功能来组装DNA上的钳子的机制。我们的最重要的假设是，RFC与其结合伙伴，包括单个ATP分子、钳子(增殖细胞核抗原)和DNA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事件的有序序列，以促进有效的钳制负载。我们检验这一假说的主要方法将是分析纯化的酿酒酵母RFC和替代的Rad24-RFC钳制加载器在体外催化的反应，使用基于荧光的方法来测量蛋白质和蛋白DNA的相互作用以及ATP的水解。此外，对ATP结合位点中保守的序列基序进行定点突变将被用来评估单个RFC亚基对钳制负载的贡献。具体地说，我们的目标是1)确定单个RFC亚基对ATP结合和水解的功能，2)使用替代的钳制加载器Rad24-RFC作为一种工具来确定RFC的大“A亚单位”对增殖细胞核抗原和DNA结合的贡献，3)确定钳制和DNA结合对RFC和Rad24-RFC活性的相互影响。我们的荧光方法的一个主要优点是，可以在溶液中直接实时监测这种动态钳制加载反应，以揭示事件的时间顺序以及导致这种顺序的因素。我们广泛和长期的目标是定义复制机制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聚合酶过程因子RFC和增殖细胞核抗原以及DNA损伤检查点复合体Rad24-RFC的生化活性来为这些目标做出贡献。对DNA复制的生化基础的基本了解对于建立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。