Mechanisms of R-loop-Associated Genome Instability

项目摘要 复制和转录是基本的生理过程，但矛盾的是，它们 都威胁到基因组的稳定性。复制分叉遇到各种类型的内源性和 外源性障碍，使它们无法准确完成DNA复制。最近的研究 表明转录的有害影响可能是R环的结果，三个- 含有RNA-DNA杂合体和单链区域的单链核酸结构 DNA. R环存在于哺乳动物细胞的整个基因组中，并调节哺乳动物细胞的各个方面。 基因表达，但它们的积累会导致DNA损伤，特别是当细胞 进行DNA复制越来越多的证据表明，转录之间的冲突- 相关的R环和复制蛋白复合物是基因组的重要因素 不稳定性，但驱动这种不稳定性的具体机制目前尚不清楚。很长的- 这项研究计划的长期目标是了解细胞是如何区分和解决调节 和有害的R环，以及这在人类疾病中是如何被扰乱的。假设R- 循环是动态结构，当它们积累时变得容易处理， 导致DNA断裂的形成并最终导致基因组不稳定。对象 本申请的目的是定义R环如何在整个细胞中识别和处理 细胞周期，以确定基因组加工发生在哪里以及如何发生 与复制机制的冲突有助于R环处理。在第一个目标中， 将在细胞中探索参与DNA修复的细胞核酸内切酶对R环的加工 使用分子和细胞生物学方法。在第二个目标中，R- 将识别和绘制环路形成和处理。这些研究将利用 尖端的基因组方法，已经开发出映射的空间分布， R环和R环加工产物遍布整个基因组。最后，在第三个目标中， 将研究R环对复制和转录机制之间碰撞的影响 在细胞中。这些研究将利用最近开发的一种新系统来控制这种 复制叉和最近断裂映射背景下的碰撞和R环形成 战略布局

PROJECT SUMMARY Replication and transcription are fundamental physiological processes, yet paradoxically they both threaten genome stability. Replication forks encounter various types of endogenous and exogenous obstacles that keep them from accurately completing DNA replication. Recent studies suggest that the deleterious effects of transcription could be a consequence of R-loops, three- stranded nucleic acid structures containing an RNA-DNA hybrid and a region of single-stranded DNA. R-loops occur throughout the genome of mammalian cells and regulate various aspects of gene expression, but their accumulation leads to DNA damage, particularly when cells are undergoing DNA replication. Increasing evidence suggests that conflicts between transcription- associated R-loops and replication protein complexes are important factors underlying genome instability, but the specific mechanisms driving this instability are currently unknown. The long- term goal of this research program is to understand how cells distinguish and resolve regulatory and deleterious R-loops, and how this is perturbed in human disease. It is hypothesized that R- loops are dynamic structures that become susceptible to processing when they accumulate, leading to the formation of DNA breaks and ultimately resulting in genome instability. The object of this application is to define how R-loops are recognized and processed in the cells throughout the cell cycle, to determine where in the genome processing occurs, and to determine how conflicts with the replication machinery contribute to R-loop processing. In the first aim, the processing of R-loops by cellular endonucleases involved in DNA repair will be explored in cells using molecular and cell biological approaches. In the second aim, the sites and products of R- loop formation and processing will be identified and mapped. These studies will take advantage of cutting-edge genomic approaches that have been developed to map the spatial distribution of R-loops and R-loop processing products throughout the genome. Finally, in the third aim, the impact of an R-loop on collisions between replication and transcription machineries will be studied in cells. These studies will take advantage of a novel system recently developed to control such collisions and R-loop formation in the context of the replication fork and recent break-mapping strategies.