Mechanisms of replication fork degradation in vertebrates

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PROJECT SUMMARY DNA replication is constantly challenged by a variety of genotoxins that arise from the environment. These genotoxins can be directly produced by the environment (e.g. UV and ionizing radiation) or can arise indirectly in response to environmental agents (e.g. polycyclic aromatic hydrocarbons, reactive oxygen species). Nascent strand degradation (NSD) and fork reversal promote genome stability in response to genotoxins by facilitating replication fork restart. Despite the importance of nascent strand degradation and fork reversal, there are many open questions about this pathway. For example, too much or too little degradation results in genome stability. It is therefore important to understand how nascent strand degradation is efficiently triggered when needed but with enough specificity that spurious degradation is avoided. However, we do not currently understand how nascent strand degradation is triggered. Additionally, current models for nascent strand degradation are too limited to explain the dozens of proteins currently implicated. Inherited defects in several of these proteins are directly implicated in human diseases (e.g. SMARCAL1, BRCA1, BRCA2) suggesting that defects in this pathway may alter individuals’ susceptibility to environmental genotoxins. Thus, it is crucial to develop a robust paradigm for nascent strand degradation and fork reversal to establish exactly how this pathway leads to replication restart and genome stability. Current approaches to study nascent strand degradation and fork reversal lack the specificity and sensitivity to address these questions. To overcome these limitations, we have developed a new site-specific, highly sensitive, and synchronous approach to study nascent strand degradation and fork reversal in vitro using Xenopus egg extracts. This system contains the full set of cellular proteins involved in DNA replication and DNA repair and provides unparalleled opportunities to observe and manipulate these processes. Our new approach has already revealed key insights into the requirements for nascent strand degradation and the mechanism by which it takes place. The proposed work will combine biochemical and single molecule approaches, both in Xenopus egg extracts and human cells. We will leverage our existing insights and exploit the power of our new system to determine how nascent strand degradation and fork reversal are triggered and the underlying molecular mechanisms involved in these processes. This work will enhance our understanding of one of the major cellular pathways that responds to environmentally sourced genotoxins and allow us to better understand how defects in this pathway may alter individuals’ susceptibility to environmental genotoxins.