2025 Theses Doctoral

Investigating the impact of site-specific replication stress on homologous recombination

Triplett, Marina Katherine

Genomic instability is a hallmark of cancer that can be caused by various forms of DNA replication stress. Collision of the replication fork with obstacles ahead of the replication machinery can result in replication fork stalling and collapse. To overcome or bypass these obstacles and resume proper replication fork progression, the cell has various replication restart mechanisms involving homologous recombination (HR) that help preserve genome integrity and ensure cell survival. However, HR can also lead to genome rearrangements, particularly when recombination occurs between repetitive DNA sequences. The tandem duplicator phenotype found in breast and ovarian cancer is an example of a genome-wide instability configuration that has been associated with pathways related to homologous recombination and replication stress, but the exact mechanism of how these duplicated sequences are formed is not fully understood.

To examine the molecular mechanisms regulating genome instability in response to replication stress, we have established a genetic system in Saccharomyces cerevisiae to detect recombination events that result in tandem duplications (TDs) and deletions. Using this system, we investigated the mechanisms of recombination upon site-specific replication fork stalling initiated by a protein-induced replication fork barrier. We have found that a Tus/Ter-induced fork block downstream of direct repeats results in an induction in recombination events resulting in TDs and deletions compared to spontaneous frequencies, and that these recombination events have specific genetic requirements.

Mainly focusing on the recombination mechanisms generating Tus/Ter-induced TDs, we determined that formation of these TDs is dependent on Rad52, Rad51, the Mph1 translocase, and structure-selective endonucleases, and that these events appear to be enhanced by disruption of the MRX complex and sister chromatid cohesion. We also found that genetic requirements for recombination in response to fork stalling by a protein-DNA barrier are distinct from those involved in fork collapse at a nick. Taken together, these studies give insight into the mechanisms governing copy number variation in the context of replication fork stalling, which may ultimately provide a better understanding of how replication stress contributes to cancer and other diseases characterized by genome instability.