Mechanisms Linking Genome Stability to RNA Metabolism

将基因组稳定性与 RNA 代谢联系起来的机制

• 批准号: 8464164
• 负责人: Karlene A Cimprich
• 金额: $ 39.66万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8464164
• 关键词: Affect; Biochemical Genetics; Biological; Cells; ChIP-seq; Chromatin; Chromatin Structure; Chromosomal Rearrangement; Complex; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA biosynthesis; Data; Defect; Event; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Hybrids; Individual; Lead; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Mediator of activation protein; Messenger RNA; Metabolism; Molecular; Nature; Phosphorylation; Play; Prevention; Process; Proteins; RNA; RNA Helicase; RNA Polymerase II; RNA Splicing; Research; Role; Site; Small Interfering RNA; Source; Structure; Susceptibility Gene; Testing; Transcription-Coupled Repair; Work; abstracting; acrosome stabilizing factor; developmental disease; genome-wide; helicase; human disease; insight; knock-down; novel; nuclease; nucleic acid structure; prevent; programs

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.

描述（由申请人提供）： 项目概要/摘要本申请的长期目标是了解细胞用于维持基因组稳定性的分子机制。基因组不稳定性是癌症的标志，基因组维护基因的缺陷使个体易患癌症。这些缺陷也会导致神经生成障碍和发育问题。最近，在哺乳动物细胞中完成了siRNA筛选，以鉴定当丢失时导致3 H2 AX磷酸化增加的基因，3 H2 AX是DNA双链断裂（DSB）的标志物。在这些命中中富集的是在RNA代谢和转录中具有既定作用的基因，并且已经表明，对于许多这些命中，H2 AX磷酸化依赖于RNA-DNA杂交体（也称为R环的结构）的形成。目前尚不清楚R环如何导致细胞中的DNA损伤，也不知道阻止这些结构积累的基本生物学机制。在本申请的第一个目的中，将使用在siRNA筛选中鉴定的几个基因来探测这些事件，从而检查R环导致DNA损伤的分子机制。此外，ChIP-Seq方法将用于确定基因组中发生DNA损伤的位置，并评估DNA损伤的位点是否也是R环形成的相同位点。在第二个目标中，将研究在筛选中鉴定的一个基因的分子功能，该基因是一种DExxQ型解旋酶，其在丢失时导致R环形成增加。据推测，这种解旋酶在处理R环中起直接作用，从而保护细胞免受DNA损伤和基因组不稳定性。生物化学和遗传学方法将用于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，并更好地了解RNA代谢缺陷如何影响基因组稳定性。