Okazaki Fragment Synthesis and Chromatin Assembly

描述（由申请人提供）： 染色质具有重要的细胞功能。染色质和调节其结构的因子的缺陷与包括癌症在内的许多人类疾病相关。研究DNA复制过程中产生的新生DNA上的染色质组装提供了研究染色质结构如何建立和维持的机会。然而，复制叉处染色质组装的分析因该过程的高度动态性而混淆。我们已经发现，滞后链合成和核小体相互影响。在这个项目中，我们使用冈崎片段作为工具来询问染色质组装途径，并获得DNA复制的详细图片。该项目有三个目标：目标1。我们将分析从芽殖酵母中纯化的冈崎片段，以测试哪些组蛋白伴侣在滞后链上装载核小体。我们将探索ATP依赖性染色质重塑酶的作用，并测试组蛋白沉积时是否发生核小体重新定位。此外，我们将研究主要的滞后链聚合酶，聚合酶，如何与核小体相互作用，以建立冈崎片段的末端。这将是有史以来第一次系统地分析真核生物冈崎片段的实验。目标2.我们将通过深度测序绘制冈崎片段，以询问整个基因组的DNA复制模式。我们将在全基因组范围内绘制复制起点的起始位点和终止位点。此外，我们将确定每个复制起点的发射特性，使我们能够告诉何时，多久一次，以及一个特定的起点是活跃的。编译我们的数据，我们将产生任何真核生物的DNA复制的最详细的地图，使基因组复制的基本方面得到理解。目标3：我们将测试，如果冈崎加工和复制动态观察芽殖酵母的属性是保守的其他真核生物。利用我们在芽殖酵母方面的经验，我们将开发出精确表征S.粟球。我们将定义复制起点和整个基因组DNA复制的动力学。此外，我们将测试冈崎片段是否来自S。粟酒裂殖子受核小体位置和染色质结构的影响。我们的跨学科工作将解决以前难以解决的关键问题。这些研究将直接相关的研究计划，并将转化为更好地了解生物过程的基本保持基因组的完整性和染色质状态。 公共卫生相关性： 每次细胞分裂时，它必须产生一个准确的基因组拷贝。这个过程称为DNA复制，是必不可少的，DNA复制中的缺陷可能导致突变，染色体重排和癌症。该项目将使用新技术来了解控制DNA复制的分子机制。)

DESCRIPTION (provided by applicant): Chromatin serves an essential cellular function. Defects in chromatin and factors that modulate its structure are associated with a number of human diseases including cancer. Studying chromatin assembly on nascent DNA produced during DNA replication offers the opportunity to investigate how chromatin structure is established and maintained. Nevertheless, the analysis of chromatin assembly at the replication fork is confounded by the highly dynamic nature of the process. We have found that lagging strand synthesis and nucleosomes impact each other. In this project, we use Okazaki fragments as a tool to interrogate chromatin assembly pathways and gain a detailed picture of DNA replication. This project has three aims: Aim 1. We will analyze purified Okazaki fragments from budding yeast to test which histone chaperones are load nucleosomes on the lagging strand. We will explore the role of ATP dependent chromatin remodeling enzymes and test if nucleosome repositioning occurs as histones are being deposited. In addition, we will investigate how the predominant lagging strand polymerase, polymerase ¿, interacts with nucleosomes to establish the ends of Okazaki fragments. These will be the first experiments ever to systematically analyze eukaryotic Okazaki fragments. Aim 2. We will map Okazaki fragments by deep sequencing to interrogate DNA replication patterns across the genome. We will map sites of initiation at replication origins as well as sites of termination genome wide. In addition, we will determine the firing characteristics of each replication origin allowing us to tell when, how often, and for how long a particular origin is active. Compiling our data, we will produce the most detailed map of DNA replication for any eukaryote, allowing fundamental aspects of genomic replication to be understood. Aim 3. We will test if the properties of Okazaki processing and replication dynamics observed in budding yeast are conserved in other eukaryotes. Using our experience with budding yeast, we will develop methods to precisely characterize Okazaki fragments in S. pombe. We will define replication origins and the dynamics of DNA replication across the genome. In addition, we will test if Okazaki fragments from S. pombe are influenced by nucleosome positions and chromatin structure. Our interdisciplinary work will address key problems that were previously intractable. These studies will be directly relevant to may research programs and will translate into a better understanding of the biological processes fundamental to maintaining genomic integrity and chromatin states. PUBLIC HEALTH RELEVANCE: Project narrative Each time a cell divides it must produce an accurate copy of its genome. This process, termed DNA replication, is essential and defects in DNA replication can lead to mutations, chromosomal rearrangements and cancer. This project will use new technology to understand the molecular mechanisms that control DNA replication. )