Mechanisms that ensure the completion of DNA synthesis

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PROJECT SUMMARY Failure to replicate even a short stretch of DNA leads to chromosome missegregation and activation of error prone DNA repair pathways. It is therefore crucial to ensure completion of DNA synthesis. However, the completion of DNA synthesis is not under surveillance by cell cycle checkpoints. Additionally, while defects in earlier stages of replication can be overcome by new initiation events, this is essentially impossible when short stretches of unreplicated DNA remain. Thus, exquisitely effective DNA synthesis mechanisms are needed to ensure the completion of DNA synthesis. The long-term goal of my lab is to elucidate and understand these mechanisms. In the current proposal, we focus on the following mechanisms that normally support completion of DNA synthesis, as well as mechanisms that restart DNA replication in response to DNA damage: (1) Replication termination occurs when two converging replication forks meet on the same stretch of DNA and is how DNA synthesis is normally completed. We recently found that topological stress can cause converging replication forks to stall and that this is overcome by both topoisomerase II-dependent and - independent mechanisms. However, it is unclear why other topoisomerases cannot compensate for loss of topoisomerase II and how these different mechanisms are efficiently engaged to ensure rapid fork convergence. We will determine the roles that different topoisomerases play during DNA replication and how their respective roles contribute to replication termination. We will also investigate how the different proteins that promote fork convergence recognize their targets to ensure that obstacles to termination are efficiently overcome. (2) Replication fork reversal and Nascent Strand Degradation (NSD) are thought to allow replication forks to bypass DNA lesions in an essentially error-free manner. However, it is unclear how exactly NSD contributes to restart of reversed forks. We recently developed a new approach to induce efficient fork reversal and NSD and identified new steps involved in this process. We will leverage our approach and insights to determine the role that NSD plays in restart of reversed forks. (3) Break-Induced Replication (BIR) restarts DNA synthesis from a double-stranded DNA end. However, it is unclear how BIR ultimately leads to completion of DNA synthesis and what the full set of required proteins is. Additionally, Mitotic DNA Synthesis (MiDAS) is a BIR-like process that operates during mitosis but it is unclear how the choice between MiDAS and BIR is determined. We have developed an approach to induce and monitor BIR, which makes us uniquely equipped to address these questions. We will determine how BIR completes synthesis and the proteins involved. We will also determine how the choice between BIR and MiDAS is determined.