Premature arrest of DNA replication forks is a major cause of the most severe kinds 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 sources and mechanisms of spontaneous replication fork arrest in E. coli, and to identify the cellular mechanisms that prevent 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 location and source of spontaneous RFBs in the E. coli genome. Utilizing synchronized cells and a novel chromosome supercoiling assay, this aim will produce an aerial view of how and where fork pausing occurs over the chromosome, and will investigate the role of DNA topology in fork arrest. Spontaneous fork pausing will be correlated with binding of major nucleoid proteins and transcription by ChIP-seq. (2) Define and differentiate replication arrest and restart in models of two canonical RFBs. Using physiological engineered barriers consisting of either a stable DNA-bound protein complex or an actively transcribing gene oriented head-on with replication, this aim will provide a highly quantitative investigation of how fork arrest and recovery differs between the two major identified classes of RFBs. (3) Identify cellular mechanisms that increase the stability and progression of replication forks at RFBs.
This aim will test two models of replication fork stability through reduction in helical stress at the fork; factory replication and chromosome cohesion (catenation of sister duplexes behind the fork). It will also reveal new and unexpected mechanisms of fork stability through an unbiased screen.
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.
