Premature arrest of DNA replication forks generates the most severe types of DNA damage that occur in cells, including DNA double strand breaks and chromosome rearrangements. Genetic change arising from spontaneous fork arrest is now predicted to exceed that occurring from exogenous sources, and thus is a major source of the genome instability that causes disease in humans and antibiotic resistance in bacteria. Despite the potential effects of arrested forks on human health, the root mechanisms that cause and prevent fork arrest are poorly understood. The long-term goal of this research 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 E. coli. The objective of this proposal is to determine the dependence of spontaneous replication fork arrest and recovery in E. coli on local DNA helical strain (supercoiling), and identify the mechanisms that regulate it. The central hypothesis is that replication fork arrest occurs primarily by a topological mechanism in which DNA helical strain between the replication fork and bound protein blocks strand unwinding, rather than by direct steric interference between the replisome and protein. The rationale for this proposal is that understanding the mechanism of fork arrest is critical, as sterical and topological mechanisms would differ in subsequent effects and regulation. The objective of this proposal will be achieved through the following specific aims: (1) Determine the dynamics and genetic requirements of replication fork blockage and restart at DNA-bound protein complexes.
This aim will define the spatial and temporal dynamics of replisome stalling in front of a barrier, replication protein disassembly, replication fork reversal, and fork restart. Artificial and natural protein barriers will be examined by whole genome sequencing. (2) Identify mechanisms that promote fork stability through replication barriers.
This aim will be carried out by screening an E. coli overexpression library for enhanced and retarded replication at an artificial barrier, as well as direct testing of how physical association of leftward and rightward replisomes (replication factory) affects replication progression through barriers. (3) Define the relationship between sister chromosome catenation (cohesion), DNA supercoiling, and replication progression through protein barriers. Preliminary evidence indicates that replisome stalling is dependent on a build-up of positive supercoils between the fork and protein barrier. Using a novel method to measure chromosome supercoiling in vivo (PsoraSeq), we will examine how supercoils are diffused along the chromosome, and whether chromosome catenation behind the fork promotes (or hinders) diffusion of restrictive supercoils. Our studies will take advantage of a cell synchronization method we developed, the baby cell machine.
This research is relevant to public health because it will seek to understand the mechanisms behind premature DNA replication fork stoppage, a major source of DNA damage in all cells that is known to promote genetic diseases such as cancer in humans. The proposal is relevant with the NIH's mission and goal to foster fundamental creative discoveries, innovative research strategies, and their applications as a basis for ultimately protecting and improving health.
