Mechanisms Linking Genome Stability to RNA Metabolism

描述(由申请人提供)： 项目概述/摘要这项应用的长期目标是了解细胞用来维持基因组稳定的分子机制。基因组不稳定是癌症的一个标志，基因组维护基因的缺陷使个人容易患癌症。这种缺陷还会导致神经发生障碍和发育问题。最近，在哺乳动物细胞中完成了siRNA筛选，以确定当丢失时会导致DNA双链断裂(DSB)的标志物3H2 AX磷酸化增加的基因。在HITS中，丰富的是在RNA新陈代谢和转录中具有既定作用的基因，研究表明，对于许多HITS，H2AX的磷酸化依赖于RNA-DNA杂交物的形成，这种结构也被称为R-环。目前尚不清楚R-环是如何导致细胞DNA损伤的，也不知道阻止这些结构积累的基本生物学机制。在这项应用的第一个目标中，将使用siRNA筛选中确定的几个基因来探测这些事件，以检查R-环导致DNA损伤的分子机制。此外，芯片-序列方法将用于确定基因组中发生DNA损伤的位置，并评估DNA损伤位置是否也与R-环形成的位置相同。在第二个目标中，将研究在筛选中发现的一个基因的分子功能，该基因是一种DExxQ型解旋酶，当丢失时会导致R环形成增加。据推测，这种解旋酶在处理R-环过程中发挥直接作用，从而保护细胞免受DNA损伤和基因组的不稳定。将使用生化和遗传方法来确定解旋酶结构域在防止DNA损伤中的作用，并评估解旋酶作用于的结构类型。这些研究将帮助我们阐明一种新的基因组维持基因的功能，并更好地理解RNA代谢缺陷如何影响基因组稳定性。

DESCRIPTION (provided by applicant): Project Summary/Abstract The long-term goal of this application is to understand the molecular mechanisms a cell uses to maintain genome stability. Genome instability is a hallmark of cancer, and defects in genome maintenance genes predispose individuals to cancer. Such defects also cause neurogenerative disorders and developmental problems. Recently, an siRNA screen was completed in mammalian cells to identify genes which, when lost, lead to increased phosphorylation of 3H2AX, a marker for DNA double-strand breaks (DSBs). Enriched among the hits are genes with established roles in RNA metabolism and transcription, and it was shown that for many of these hits, H2AX phosphorylation is dependent on the formation of RNA-DNA hybrids, structures also known as R-loops. It is not clear how R-loops lead to DNA damage in cells, and the basic biological mechanisms that prevent the accumulation of these structures are not known. In the first aim of this application, the molecular mechanisms by which R-loops lead to DNA damage will be examined, using several of the genes identified in the siRNA screen to probe these events. In addition, ChIP-Seq approaches will be used to determine where DNA damage occurs in the genome, and to assess whether the sites of DNA damage are also the same sites where R-loops form. In the second aim, the molecular function of one gene identified in the screen, a DExxQ- type helicase that leads to increased R-loop formation when lost, will be studied. It is hypothesized that this helicase plays a direct role in processing R-loops thereby protecting cells against DNA damage and genome instability. Biochemical and genetic approaches will be used to determine the role of the helicase domain in preventing DNA damage and to assess the types of structures on which the helicase acts. These studies will help us elucidate the function of a novel genome maintenance gene and provide a better understanding of how defects in RNA metabolism can affect genome stability.