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复制对于维持所有生物的基因组至关重要。在每一轮细胞分裂期间，真核细胞必须建立数百至数千个复制的叉子，以协调每个染色体。密切调节了在每个复制叉上组装多酶重新酶的事件，以确保每个染色体精确地重复一次。与其重要性一致，已知复制起始蛋白的缺陷或失调会导致癌症和发育异常。重要的是要了解复制启动的基本事件以及该事件如何导致复制机制的适当组装。 近年来，在我们理解此过程的最初事件时，已经取得了重大进展，即通过加载复制性DNA解旋酶的加载来选择复制起始（复制的起源）。相比之下，我们对激活这些加载的解旋酶的事件以及随后的DNA复制机械组装的了解明显较少。 拟议的研究将利用一种新型的体外测定法，该测定法将DNA复制启动的事件从定义的复制起源。我们将使用此测定法和从中得出的新测定法来解决有关复制启动的基本问题。我们将主要关注所做的复制启动步骤，DNA解旋酶激活。在特定目标中，我们将开发测定方法，以解决载荷解旋酶的启动以进行解旋酶激活，但保持在不活跃状态。在第二个目标中，我们将使用新型的DNA底物和测定方法来确定解旋酶激活所需的众多蛋白质如何有助于此事件，重点是已知的解旋酶激活蛋白Cdc45。在最后一个目的中，我们将解决解旋酶激活和DNA放松如何与DNA合成的启动，以防止产生过量的单链DNA和DNA破坏检查点的激活。