Initiation of DNA Replication of Yeast Chromosomes

描述（由申请人提供）： DNA复制对于维持所有生物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，以协调复制每条染色体。组装多酶复制体的事件在每个复制叉处起作用，这些事件受到严格调控，以确保每条染色体精确复制一次。与它们的重要性一致，已知复制起始蛋白的缺陷或失调会导致癌症和发育异常。关键是要了解复制启动的基本事件是如何发生的，以及这个事件如何导致复制机制的适当组装。 近年来，我们对这一过程的初始事件的理解取得了重大进展，即通过加载复制性DNA解旋酶来选择复制起始位点（复制起点）。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复制机制的组装知之甚少。 拟议的研究将利用一种新的体外试验，概括了从一个确定的复制起点的DNA复制启动的事件。我们将使用这种检测方法和新的检测方法来解决有关复制起始的基本问题。我们将主要集中在复制起始的关键步骤，DNA解旋酶激活。在具体目标一中，我们将开发测定以解决加载的解旋酶如何引发解旋酶活化但维持在非活性状态。在第二个目标中，我们将使用新的DNA底物和测定来确定解旋酶激活所需的众多蛋白质如何促成这一事件，重点是已知的解旋酶激活蛋白Cdc45。在最后一个目标中，我们将讨论解旋酶激活和DNA解旋如何与DNA合成的起始协调，以防止过量单链DNA的产生和DNA损伤检查点的激活。

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.