A Biochemical Approach to Understand Nascent Strand Degradation Mechanisms and the Role of EXO1

Abstract

Complete and accurate genome duplication is important for genome stability. During DNA replication, genotoxic stress can induce nascent strand degradation (NSD), a process in which nucleases degrade newly synthesized DNA. NSD is a complex process with numerous nucleases involved in the degradation of both nascent strands at different replication fork structures. While NSD facilitates fork restart and promotes genome stability, the precise mechanisms by which NSD occurs remain poorly understood. A challenge in the field has been the lack of experimental approaches capable of distinguishing between strand-specific degradation events and degradation at different replication fork structures. First, I used the Xenopus egg extract model system to develop a robust biochemical approach to study NSD. This approach allows for strand-specific analysis of NSD at defined replication fork structures. Using this approach, I found that the 5’-3’ nuclease EXO1 is crucial for degradation at uncoupled forks. This degradation is a distinct and parallel response to RAD51-regulated NSD pathways. I also found that both leading and lagging strands undergo 5’-3’ degradation by EXO1. Importantly, leading strand degradation is not initiated from the 3’ end and instead initiates from a 5’ entry point such as the lagging strand of a sister fork. This demonstrates that the degradation of both nascent strands occurs independently of each other in a strand-uncoupled manner. Furthermore, I found that EXO1-mediated degradation of uncoupled forks is crucial for activating the ATR-dependent checkpoint and restrains fork progression through both ATR-dependent and independent mechanisms. Ultimately, my dissertation established an approach which uncovered mechanistic details of NSD and the critical role of EXO1 and its contributions to checkpoint signaling and replication fork dynamics.