Initiation of DNA Replication at Cell Origins in Yeast

描述(由申请人提供)： 本研究的目的是确定真核细胞中DNA复制的启动机制和调控。显然，为了从一代细胞到下一代细胞保持基因组的完整性，DNA及其相关的染色质结构必须以高度受控和准确的方式复制。这些调控的中断可能会促进基因组的不稳定，导致体细胞的肿瘤性转化，或者导致生殖系突变，从而导致许多不同的疾病。此外，DNA复制蛋白是治疗干预和诊断癌细胞增殖和其他增殖性疾病的有形靶点。启动子蛋白(ORC)与一系列DNA复制蛋白，包括CDC6、CDT1和MCM2-7六聚体合作，在DNA复制的起始处建立一个复制前复合体(Pre-RC)，促进每个起始点的DNA合成。最近的进展使前RC与纯化的蛋白在体外组装成为可能。本申请中拟议的研究将使用酿酒酵母来研究DNA复制的启动是如何在Pre-RC组装之后发生的，以及这一过程如何受到CDC7-Dbf4(DDK)蛋白激酶和MCM2-7复合体Mcm4亚单位内DNA复制启动的内在抑制物的调控。这项拟议的研究还将调查核小体中的核心组蛋白是如何在DNA复制过程中被破坏并转移到新合成的DNA的领先和落后链上的。核小体是真核细胞中染色质的基本结构单位。 公共卫生相关性： DNA复制是在有丝分裂或减数分裂过程中，在分离产生的姐妹染色单体之前复制基因组的过程。在有丝分裂退出或G1期，DNA复制的每个起始点都组装了一个复制前复合体，使每个起始点能够启动后续的DNA复制。这个项目将研究DNA合成的启动是如何发生的，它是如何被调控的，以及染色质是如何遗传的。

DESCRIPTION (provided by applicant): The goal of this research is to determine the mechanism and regulation of the initiation of DNA replication in eukaryotic cells. It is clear that for maintenance of the integrity of the genome frm one cell generation to the next, DNA and its associated chromatin structures must be duplicated in a highly controlled and accurate manner. Interruption of these controls may promote genome instability and lead to neoplastic transformation in somatic cells or result in mutations in the germ line that can cause many different disorders. Moreover, the DNA replication proteins represent tangible targets for therapeutic intervention and diagnosis of proliferation of cancer cells, and other proliferative disorders. The initiator protein (ORC) cooperates with a series of DNA replication proteins, including Cdc6, Cdt1 and the MCM2-7 hexamer to establish at origins of DNA replication a pre-Replicative Complex (pre-RC) that facilitates later initiation of DNA synthesis at each origin. Recent progress has enabled the assembly of the pre-RC in vitro with purified proteins. The proposed research in this application will investigate, using the yeast S. cerevisiae, how the initiation of DNA replication occurs following pre-RC assembly and how this process is regulated by the Cdc7-Dbf4 (DDK) protein kinase and by an intrinsic inhibitor of initiation of DNA replication within the Mcm4 subunit of the MCM2-7 complex. The proposed research will also investigate how the core histones within nucleosomes, the fundamental structural unit of chromatin in eukaryotic cells, are disrupted during DNA replication and transferred to the leading and lagging strands of the newly synthesized DNA. PUBLIC HEALTH RELEVANCE: DNA replication is the process whereby the genome is duplicated prior to segregation of the resulting sister chromatids during mitosis or meiosis. During mitotic exit or during G1 phase, a pre-Replicative Complex is assembled each origin of DNA replication that renders each origin competent for subsequent initiation of DNA replication. This project will examine how the initiation of DNA synthesis occurs and how it is regulated, as well as investigate how chromatin is inherited.