Chromatin architecture defines DNA replication origins

描述(由申请人提供)：DNA复制是遗传基因信息所需的基本和受调控的周期事件。在每个细胞周期中，必须选择并激活数以千计的复制起点(起始点)，以确保基因组在S期的范围内只复制一次。如果不能适当地调控DNA复制程序，可能会导致灾难性的基因组不稳定。我们在黑腹葡萄球菌和酿酒酵母中的研究发现，染色质结构和依赖于ATP的染色质重塑活动是起源选择的重要决定因素。我们提出了遗传、基因组和生化方法，以了解当地的染色质结构和依赖于ATP的染色质重塑如何有助于酵母和果蝇复制起点的选择和调控。具体地说，我们将评估染色质动力学(复制无关的组蛋白H3/H4交换)与起始点选择和激活之间的相互作用。我们还将使用全面的基因组方法来测试候选的依赖于ATP的染色质重塑活动在起源选择和激活中的作用。我们最近开发了一种全基因组的‘足迹’分析，将用于解决关于起源结构和关键复制启动因子在单个起源的分布的基本问题。作为modENCODE研究的一部分，我们发现NURF染色质重塑复合体的多个成分对ORC定位具有很强的预测价值。我们将测试NURF在确定果蝇复制起源方面的作用。通过利用酵母和果蝇的实验优势，我们将对染色质结构如何指定真核复制起源产生重要的机械见解。

DESCRIPTION (provided by applicant): DNA replication is an essential and regulated cycle event required for the inheritance of genetic information. Every cell cycle, thousands of replication start sites (origins) must be selected and activated to ensure that the genome is copied exactly once within the confines of S-phase. Failure to properly regulate the DNA replication program may lead to catastrophic genomic instability. Our work in D. melanogaster and S. cerevisiae has identified chromatin architecture and ATP-dependent chromatin remodeling activities as important determinants of origin selection. We are proposing genetic, genomic and biochemical approaches to understand how the local chromatin architecture and ATP-dependent chromatin remodeling contribute to the selection and regulation of replication origins in yeast and Drosophila. Specifically, we will assess the interplay between chromatin dynamics (replication independent histone H3/H4 exchange) and origin selection and activation. We will also test the role of candidate ATP- dependent chromatin remodeling activities on origin selection and activation using comprehensive genomic approaches. We have recently developed a genome-wide 'footprinting' assay that will be used to address fundamental questions regarding origin architecture and the distribution of key replication initiation factors t individual origins. As part of our modENCODE studies, we identified multiple components of the NURF chromatin remodeling complex as having strong predictive value for ORC localization. We will test the role of NURF in specifying Drosophila origins of replication. By exploiting the experimental strengths of both yeast and Drosophila, we will generate important mechanistic insights into how the chromatin architecture specifies eukaryotic origins of replication.