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。染色质缺陷和调节其结构的因素与包括癌症在内的许多人类疾病有关。研究DNA复制过程中产生的新生DNA上的染色质组装为研究染色质结构是如何建立和维持的提供了机会。然而，在复制叉处对染色质组装的分析被该过程的高度动态性质所混淆。我们发现，滞后的链合成和核小体相互影响。在这个项目中，我们使用Okazaki片段作为工具来询问染色质组装路径，并获得DNA复制的详细图像。本项目有三个目标：目标1.分析发芽酵母中纯化的Okazaki片段，以测试哪些组蛋白伴侣蛋白是在滞后链上负载核小体的。我们将探索依赖于三磷酸腺苷的染色质重塑酶的作用，并测试核小体在组蛋白沉积时是否发生重新定位。此外，我们将研究主要的滞后链聚合酶，聚合酶δ，如何与核小体相互作用，以确定冈崎片段的末端。这将是有史以来系统分析真核冈崎片段的第一次实验。目的2.我们将通过深度测序来绘制冈崎片段的图谱，以询问整个基因组的DNA复制模式。我们将绘制全基因组复制起始点和终止点的图谱。此外，我们还将确定每个复制点的触发特征，从而告诉我们某个特定点处于活动状态的时间、频率和持续时间。汇编我们的数据，我们将为任何真核生物制作最详细的DNA复制图谱，从而使人们能够了解基因组复制的基本方面。目的3.我们将测试在萌芽酵母中观察到的冈崎加工和复制动力学的特性是否在其他真核生物中保守。利用我们在发芽酵母方面的经验，我们将开发方法来准确地表征S.pombe中的Okazaki片段。我们将定义复制起点和跨基因组的DNA复制动态。此外，我们还将测试S.pombe的Okazaki片段是否会受到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。这些研究将与可能的研究计划直接相关，并将转化为对维持基因组完整性和染色质状态基本的生物过程的更好理解。

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 δ, 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.