Dynamics and regulation of sister chromosome cohesion in E. coli

DNA复制叉过早/意外停止是造成DNA损伤的主要原因，包括双链断裂和基因组重排。现在预测，叉子停滞引起的突变将超过外部来源产生的突变，而介导停滞叉子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。尽管叉子停滞和致病突变之间有很强的联系，但导致和防止叉子停顿的根本机制却知之甚少。该项目的长期目标是通过全面了解模式生物大肠杆菌中复制叉子停止的方式、时间和原因，提高我们识别和缓解人类和病原菌复制叉子停止的主要原因的能力。这一目标将通过两个具体目标来实现。在第一个目标中，将通过对无法处理受阻分叉的菌株的深度测序来定位和量化自发性分叉在大肠杆菌染色体中的位置。这些阻止位点将通过CHIP-SEQ与潜在的复制障碍元件，包括核相关蛋白和转录复合体相关联，并使用一种新开发的称为Psora-Seq的方法与DNA拓扑特征相关联。在第二个目标中，我们将定义体内受阻叉子的进化，包括通过复制和重新启动/修复蛋白的中间叉子结构和占位。叉子停止的频率和位置通常在基因组中是零星的，将使用在先前的项目中开发的模拟生理性叉子停止的可调复制叉子屏障系统来控制。通过在大肠杆菌中确定复制分叉停滞最频繁的位置和原因，以及分叉重新启动的速度和有效性，我们预计该项目的结果将有助于更好地理解促进和减轻人类细胞分叉停滞的机制。

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.