Dynamics and regulation of sister chromosome cohesion in E. coli

DNA复制叉的过早或计划外阻滞是DNA损伤的主要原因，包括双链断裂和基因组重排。目前预计叉部阻滞引起的突变将超过外源性来源引起的突变，介导叉部阻滞频率或修复的蛋白质缺陷与从癌症到抗生素耐药性的广泛遗传疾病有关。尽管叉停和致病突变之间有很强的联系，但引起和防止叉停的根本机制尚不清楚。该项目的长期目标是通过建立对模式生物大肠杆菌中叉子如何、何时以及为何发生的全面理解，来提高我们识别和减轻人类和致病菌中复制叉子停滞的主要原因的能力。这一目标将通过两个具体目标来实现。在第一个目标中，在大肠杆菌染色体中自发叉捕获的位置将通过对无法处理捕获叉的菌株进行深度测序来绘制和量化。这些阻滞位点将通过ChIP-Seq与潜在的复制屏障元件（包括核相关蛋白和转录复合物）相关联，并使用新开发的Psora-Seq方法与DNA拓扑特征相关联。在第二个目标中，我们将定义一个停顿叉在体内的进化，包括中间叉结构和复制和重启/修复蛋白的占用。叉阻滞的频率和位置通常在基因组中是偶发的，将使用先前模拟生理叉阻滞的项目中开发的可调复制叉屏障系统来控制。通过确定大肠杆菌中最常见的复制叉停滞的位置和原因，以及叉重新启动的速度和功效，我们期望这个项目的结果将有助于更好地理解促进和减轻人类细胞中叉停滞的机制。

Premature/unscheduled arrest of DNA replication forks is a major cause of DNA damage, including double strand breaks and genome rearrangements. Mutation arising from fork arrest is now predicted to exceed that occurring from exogenous sources, and defects in proteins that mediate the frequency or repair of arrested forks are associated with a wide spectrum of genetic diseases from cancer to antibiotic resistance. Despite the strong connection between fork arrest and disease-causing mutation, the root mechanisms that cause and prevent fork arrest are poorly understood. The long-term goal of this project is to advance our ability to identify and mitigate the primary causes of replication fork arrest in humans and pathogenic bacteria by establishing a comprehensive understanding of how, when, and why fork arrest occurs in the model organism Escherichia coli. This goal will be pursued through two specific aims. In the first aim, the locations of spontaneous fork arrest in the E. coli chromosome will be mapped and quantified by deep sequencing of strains unable to process arrested forks. These arrest sites will be correlated with potential replication barrier elements including nucleoid-associated proteins and transcription complexes by ChIP-Seq, and to DNA topological features using a newly developed method called Psora-Seq. In the second aim, we will define the evolution of an arrested fork in vivo including intermediate fork structures and occupancy by replication and restart/repair proteins. The frequency and location of fork arrest, normally sporadic in the genome, will be controlled using a tunable replication fork barrier system developed in a previous project that emulates physiological fork arrest. By determining in E. coli, the most frequent location and cause of replication fork arrest, and the speed and efficacy of fork restart, we expect that results from this project will lead to a better understanding of the mechanisms that promote and mitigate fork arrest in human cells.