Mechanisms of R-loop-Associated Genome Instability

项目总结 复制和转录是基本的生理过程，但矛盾的是，它们 两者都威胁着基因组的稳定。复制分叉遇到各种类型的内源性和 阻止它们准确完成DNA复制的外源障碍。最新研究 提示转录的有害影响可能是R-环的结果，三个- 含有RNA-DNA杂交体和一个单链区域的链状核酸结构 DNAR-环广泛存在于哺乳动物细胞的基因组中，调节着 基因表达，但它们的积累会导致DNA损伤，特别是当细胞 正在进行DNA复制。越来越多的证据表明，转录之间的冲突- 相关的R环和复制蛋白复合体是构成基因组的重要因素 目前尚不清楚导致这种不稳定的具体机制。长的- 这项研究计划的学期目标是了解细胞如何区分和解决调控 以及有害的R-环，以及这在人类疾病中是如何受到干扰的。假设R- 循环是动态结构，当它们累积时变得容易被处理， 导致DNA断裂的形成，最终导致基因组不稳定。该对象 该应用的目的是定义在整个细胞中如何识别和处理R环 细胞周期，以确定基因组处理发生在哪里，并确定如何 与复制机制的冲突会导致R循环处理。第一个目标是， 将在细胞中探索参与DNA修复的细胞内切酶对R环的处理 使用分子和细胞生物学方法。在第二个目标中，R-的网站和产品- 环路的形成和处理将被识别和映射。这些研究将利用 已经开发的尖端基因组方法已经被开发来绘制出 基因组中的R-环和R-环加工产物。最后，在第三个目标中， 将研究R环对复制和转录机器之间冲突的影响 在牢房里。这些研究将利用最近开发的一种新系统来控制这种 在复制分叉和最近中断映射的上下文中的碰撞和R-环形成 战略。

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.