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Dynamic Eukaryotic Replication Machines

动态真核复制机器

基本信息

批准号：
8102155
负责人：
Linda B Bloom
金额：
$ 28.36万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-09-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8102155
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 DNA-Protein Interaction 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 Proteins 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

项目摘要

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，这些酶本身不能有效地制造DNA来复制整个基因组。DNA聚合酶加工因子，一个滑动钳和钳装载器，通过将DNA聚合酶拴在被复制的模板上，提高DNA复制的效率。这些加工因子的结构和功能从细菌到人类都是保守的。夹子装载器是一种分子机器，它使用ATP催化环状滑动夹子在DNA上的组装。本研究的主要目的是阐明真核夹加载器复制因子C （RFC）通过定义单个组分的功能在DNA上组装夹的机制。我们最重要的假设是，RFC与其结合伙伴（包括单个ATP分子、夹子（PCNA）和DNA）的每次相互作用都会诱导构象变化，从而促进通路的下一步，这些离散的构象变化有利于有序的事件序列，以促进有效的夹子加载。我们测试这一假设的主要方法是分析纯化的酿酒酵母RFC和另一种Rad24-RFC夹紧加载器在体外催化的反应，使用基于荧光的测定方法来测量蛋白质和蛋白质dna相互作用以及ATP水解。此外，对ATP结合位点上保守序列基序的定点突变将用于评估单个RFC亚基对箝位加载的贡献。具体来说，我们的目标是1)定义单个RFC亚基对ATP结合和水解的功能，2)使用可选的箝位加载器Rad24-RFC作为确定RFC大“a -亚基”对PCNA和DNA结合的贡献的工具，3)确定箝位和DNA结合对RFC和Rad24-RFC活性的相互作用。我们的荧光方法的一个主要优势是，这种动态箝位加载反应可以直接在溶液中实时监测，以揭示事件的时间顺序，以及导致这种顺序的因素。我们的广泛和长期目标是确定复制机制复制基因组的分子机制，并确定这些酶对复制过程中遇到的DNA损伤作出反应的机制。该项目将通过描述DNA聚合酶加工因子（RFC和PCNA）以及DNA损伤检查点复合物（Rad24-RFC）的生化活性，为实现这些目标做出贡献。对DNA复制的生化基础有一个基本的了解，是建立生化缺陷与疾病的临床相关性所必需的。DNA复制领域的基础研究导致了重要医疗诊断工具的发展以及抑制病原体复制的治疗剂的发展。