"Cell Cycle Regulation of Eukaryotic DNA Replication"

《真核DNA复制的细胞周期调控》

• 批准号: 7990228
• 负责人: JOACHIM J LI
• 金额: $ 13.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-18 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7990228
• 关键词: Accounting; Affect; Biological Assay; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation; Chromatin; Chromosome Segregation; Complex; Cyclin-Dependent Kinases; DNA; DNA Damage; DNA Primase; DNA biosynthesis; DNA polymerase alpha-primase; Eukaryota; Eukaryotic Cell; Event; Funding; G1 Phase; Gene Amplification; Gene Duplication; Genes; Genetic Materials; Genome; Genomic Instability; In Vitro; Investigation; Lead; Light; Mitosis; Modeling; Molecular; Mutation; Nuclear Envelope; Oncogenes; Pathway Analysis; Pathway interactions; Phase; Phosphorylation; Polymerase; Positioning Attribute; Process; Property; Proteins; Regulation; Replication Initiation; Replication Origin; Research Personnel; Role; Saccharomyces cerevisiae; Saccharomycetales; Staging; System; Telomere Maintenance; Testing; base; cancer cell; carcinogenesis; innovation; mutant; novel; novel strategies; prevent; programs; therapeutic target; tool; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): The precise inheritance of genetic material in eukaryotes requires that initiation at each of the hundreds to thousands of replication origins be subject to exquisite regulation so that the DNA is duplicated exactly once per cell cycle. When replication control mechanisms go awry, genomic instability is predicted to occur, but the precise molecular consequences of deregulated replication for genome integrity are completely unknown. Additionally, the full battery of regulatory strategies that govern replication has not been defined. Cyclin dependent kinases (CDKs) are key molecular regulators that both stimulate initiation and inhibit re- initiation of DNA replication. To understand the molecular basis of genome integrity, it is essential to develop a more sophisticated understanding of these CDK-dependent regulatory events. Additionally, it is critical to analyze how disrupting this regulation affects faithful inheritance of the genome. To this end, we have developed several innovative tools that allow us to study the genesis and consequences of re-replication in the budding yeast Saccharomyces cerevisiae. Our Specific Aims are as follows: (1) We have discovered that CDKs target polymerase alpha primase to block re-replication, which challenges the prevailing paradigm that the only strategy used by CDKs to inhibit re-replication is to prevent reassembly of a pre-replicative complex. Thus in this aim, we will characterize a novel replication control mechanism by investigating how CDKs inhibit polymerase alpha-primase to prevent re-replication. (2) We will continue to uncover new strategies for replication control by completing our ongoing screen to identify new CDK targets involved in either triggering initiation or preventing re-replication; this screen has already successfully identified five such targets, including polymerase alpha primase. (3) Using a robust copy number assay, we have obtained the first evidence that re-replication causes a heritable genetic change, namely a gene duplication event that represents an early step of gene amplification. We will exploit this unprecedented opportunity to examine the mechanisms by which re-replication promotes gene amplification. Because gene amplification is a primary means of activating oncogenes in cancer cells, these studies will shed light on the molecular triggers of tumorigenesis, and potentially identify targets of therapeutic significance.

描述（由申请人提供）：真核生物中遗传物质的精确遗传要求数百至数千个复制起点中每一个的起始都受到精确的调控，以便DNA在每个细胞周期精确复制一次。 当复制控制机制出现问题时，预计会发生基因组不稳定，但复制失调对基因组完整性的精确分子后果尚不清楚。此外，管理复制的全套监管策略尚未定义。细胞周期蛋白依赖性激酶 (CDK) 是刺激 DNA 复制起始和抑制 DNA 复制重新起始的关键分子调节因子。为了了解基因组完整性的分子基础，有必要对这些 CDK 依赖性调控事件有更深入的了解。此外，分析破坏这种调节如何影响基因组的忠实遗传也至关重要。为此，我们开发了几种创新工具，使我们能够研究芽殖酵母酿酒酵母中重新复制的起源和后果。我们的具体目标如下：（1）我们发现CDK靶向聚合酶α引物酶来阻止再复制，这挑战了流行的范式，即CDK抑制再复制的唯一策略是防止预复制的重新组装。复制复合体。因此，为了这个目标，我们将通过研究 CDK 如何抑制聚合酶 α-引物酶以防止再复制来表征一种新的复制控制机制。 (2) 我们将通过完成我们正在进行的筛选来继续发现新的复制控制策略，以确定涉及触发启动或防止重新复制的新 CDK 目标；该筛选已成功识别出五个此类靶标，包括聚合酶α引物酶。 (3)使用强大的拷贝数测定，我们获得了第一个证据，证明再复制会导致可遗传的基因变化，即代表基因扩增早期步骤的基因复制事件。我们将利用这个前所未有的机会来研究再复制促进基因扩增的机制。由于基因扩增是激活癌细胞中癌基因的主要手段，因此这些研究将揭示肿瘤发生的分子触发因素，并可能确定具有治疗意义的靶标。