Mechanisms of R-loop-Associated Genome Instability

项目摘要(来自父应用程序) 复制和转录是基本的生理过程，但矛盾的是，它们都威胁着 基因组的稳定性。复制分叉遇到了各种类型的内源性和外源性障碍，这些障碍使 它们不能准确地完成DNA复制。最近的研究表明，吸烟的有害影响 转录可能是R-环的结果，R-环是包含RNA-DNA的三链核酸结构 杂交种和单链DNA区域。R-环存在于哺乳动物细胞的整个基因组中， 调节基因表达的各个方面，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导致基因组不稳定的重要因素，但 导致这种不稳定的具体机制目前尚不清楚。这项研究的长期目标是 程序是为了了解细胞如何区分和解决调节和有害的R环，以及这是如何 饱受人类疾病的困扰。假设R-环是动态结构，容易受到 当它们积累时进行处理，导致DNA断裂的形成，最终导致基因组 不稳定。本申请的目的是定义在细胞中如何识别和处理R环 在整个细胞周期中，确定基因组处理发生在哪里，以及如何确定冲突 与复制机械一起有助于R环的加工。在第一个目标中，R-循环的处理通过 参与DNA修复的细胞内切酶将在细胞中利用分子和细胞生物学进行探索 接近了。在第二个目标中，将确定R环形成和加工的地点和产品，并 已映射。这些研究将利用先进的基因组方法，这些方法已经被开发出来 绘制R-环和R-环加工产物在整个基因组中的空间分布。最后，在 第三个目的，将研究R环对复制和转录机制之间的碰撞的影响 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碰撞和 在复制分叉和最近的中断映射策略的背景下形成R-环。

PROJECT SUMMARY (from parent application) 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.