"Cell Cycle Regulation of Eukaryotic DNA Replication"

描述（由申请人提供）：真核生物中遗传物质的精确遗传需要数百至数千个复制起点中的每一个的起始受到精细调控，使得DNA在每个细胞周期中精确复制一次。 当复制控制机制出错时，预计会发生基因组不稳定性，但复制失控对基因组完整性的精确分子后果是完全未知的。此外，还没有定义管理复制的全套监管策略。细胞周期蛋白依赖性激酶（CDK）是刺激DNA复制启动和抑制DNA复制重新启动的关键分子调节因子。为了了解基因组完整性的分子基础，有必要对这些依赖CDK的调控事件进行更深入的了解。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再复制的起源和后果。我们的具体目的如下：（1）我们发现CDK靶向聚合酶α引发酶以阻断再复制，这挑战了CDK抑制再复制的唯一策略是阻止复制前复合物的重组的流行范式。因此，在这个目标中，我们将通过研究CDK如何抑制聚合酶α-引发酶以防止再复制来表征一种新的复制控制机制。(2)我们将继续通过完成我们正在进行的筛选来发现新的复制控制策略，以确定参与触发启动或阻止再复制的新CDK靶点;该筛选已经成功确定了五个这样的靶点，包括聚合酶α引发酶。(3)使用一个强大的拷贝数测定，我们已经获得了第一个证据表明，再复制导致遗传的遗传变化，即基因复制事件，代表基因扩增的早期步骤。我们将利用这一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，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点。

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.