Initiation of DNA Replication of Yeast Chromosomes

酵母染色体 DNA 复制的起始

• 批准号: 8514624
• 负责人: Stephen P. Bell
• 金额: $ 26.95万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514624
• 关键词: Address; Biological Assay; Cell division; Cells; Characteristics; Chromosomal Breaks; Chromosomes; Commit; Complex; DNA; DNA Primase; DNA biosynthesis; DNA damage checkpoint; DNA replication origin; DNA-Directed DNA Polymerase; Defect; Development; Ensure; Enzymes; Eukaryotic Cell; Event; Fluorescence; Funding; G1 Phase; Generations; Genome; Genomic Instability; Head; Lead; Malignant Neoplasms; Mediating; Modeling; Organism; Process; Protein Kinase; Proteins; Recruitment Activity; Replication Initiation; Replication Origin; Research; S Phase; Saccharomyces cerevisiae; Single-Stranded DNA; Site; Systems Analysis; Testing; Time; Yeasts; abstracting; base; ds-DNA; helicase; in vitro Assay; in vivo; melting; mutant; novel; prevent; single molecule; tumorigenesis

DESCRIPTION (provided by applicant): Project Summary/Abstract DNA replication is essential to maintain the genome of all organisms. During each round of cell division, eukaryotic cells must establish hundreds to thousands of replication forks that coordinately replicate each chromosome. The events that assemble the multi-enzyme replisomes that act at each replication fork are tightly regulated to ensure that each chromosome is replicated exactly once. Consistent with their importance, defects in or misregulation of replication initiation proteins are known to lead to cancer and developmental abnormalities. It is critical to understand how the essential event of replication initiation occurs and how this event leads to the appropriate assembly of the replication machinery. In recent years, significant advances have been made in our understanding of the initial event of this process, the selection of the sites of replication initiation (origins of replication) through the loading of the replicative DNA helicase. In contrast, we know significantly less about the events that activate these loaded helicases and the subsequent assembly of the DNA replication machinery. The proposed research will exploit a novel in vitro assay that recapitulates the events of DNA replication initiation from a defined origin of replication. We will use this assay and new assays derived from it to address fundamental questions concerning the initiation of replication. We will primarily focus on the committed step of replication initiation, DNA helicase activation. In specific aim one, we will develop assays to address how the loaded helicase is primed for helicase activation but maintained in an inactive state. In the second aim, we will use novel DNA substrates and assays to determine how the numerous proteins required for helicase activation contribute to this event, with a focus on the known helicase-activating protein, Cdc45. In the last aim, we will address how helicase activation and DNA unwinding are coordinated with the initiation of DNA synthesis to prevent the generation of excess single-stranded DNA and activation of DNA-damage checkpoint.

描述(由申请人提供)： 项目摘要/摘要DNA复制对维持所有生物体的基因组是必不可少的。在每一轮细胞分裂中，真核细胞必须建立数百到数千个复制叉子来协调复制每条染色体。组装作用于每个复制分叉的多酶复制体的事件受到严格调控，以确保每个染色体只复制一次。与它们的重要性一致的是，复制起始蛋白的缺陷或调控不当被认为会导致癌症和发育异常。了解复制启动的基本事件是如何发生的，以及该事件如何导致复制机制的适当组装是至关重要的。近年来，我们对这一过程的初始事件，即通过装载复制DNA解旋酶来选择复制起始位置(复制起点)的理解已经取得了重大进展。相比之下，我们对激活这些加载的解旋酶的事件以及随后组装DNA复制机制的了解要少得多。这项拟议的研究将利用一种新的体外分析方法，从一个确定的复制起点概括DNA复制启动的事件。我们将使用这一检测方法和由此衍生的新检测方法来解决与复制启动有关的基本问题。我们将主要集中在复制启动的承诺步骤，DNA解旋酶激活。在特定的目标一，我们将开发分析方法，以解决如何加载的解旋酶是准备解旋酶激活，但保持在不活跃的状态。在第二个目标中，我们将使用新的DNA底物和分析来确定解旋酶激活所需的众多蛋白质如何对这一事件做出贡献，重点是已知的解旋酶激活蛋白CDC45。在最后一个目标中，我们将讨论解旋酶的激活和DNA解离如何与DNA合成的启动相协调，以防止过量单链DNA的产生和DNA损伤检查点的激活。