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.
Premature/unscheduled arrest of DNA replication forks frequently results in DNA damage, including highly mutagenic double strand breaks, and is a major contributing source of mutations that cause genetic disease and antibiotic resistance. By defining the DNA structural and protein binding characteristics of spontaneous replication fork arrest in the highly tractable organism Escherichia coli, this project may reveal specific mechanisms of fork arrest in humans, ultimately leading to improved detection and treatment of replication-related diseases.
