Dynamics and regulation of sister chromosome cohesion in E. coli

DNA复制叉的过早/非计划性停滞是DNA损伤的主要原因，包括双链断裂和基因组重排。现在预测由叉停滞引起的突变将超过由外源性来源引起的突变，并且介导停滞叉的频率或修复的蛋白质中的缺陷与从癌症到抗生素抗性的广泛遗传疾病相关。尽管分叉停滞和致病突变之间有很强的联系，但引起和防止分叉停滞的根本机制却知之甚少。该项目的长期目标是通过建立对模式生物大肠杆菌中如何，何时以及为什么发生叉逮捕的全面了解，提高我们识别和减轻人类和病原菌复制叉逮捕的主要原因的能力。将通过两个具体目标来实现这一目标。在第一个目标中，E.大肠杆菌染色体将通过对不能处理捕获的叉的菌株进行深度测序来定位和定量。这些逮捕网站将与潜在的复制屏障元件，包括核相关蛋白和转录复合物的ChIP-Seq，并使用新开发的方法称为Psora-Seq的DNA拓扑特征。在第二个目标中，我们将定义一个被捕的叉在体内，包括中间叉结构和复制和重启/修复蛋白的占用的演变。叉逮捕的频率和位置，通常零星的基因组中，将控制使用可调复制叉屏障系统在以前的项目中开发的模拟生理叉逮捕。通过测定E.大肠杆菌中，最常见的位置和原因的复制叉逮捕，和速度和效率的叉重新启动，我们希望从这个项目的结果将导致更好地了解机制，促进和减轻叉逮捕在人类细胞。

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.