Unravelling replication stress responses

Abstract

Faithful DNA replication is essential for cell survival and genome stability. However, DNA replication faces continual challenges from DNA damage, as well as intrinsic and extrinsic stressors, which could cause replication forks to stall. Failure to restart stalled replication forks can lead to a collapse of these structures and result in the formation of DNA double-strand breaks, which, if left unresolved can lead to genome instability and the onset of cancer. To counteract replication fork stalling and collapse, cells activate a replication stress response that induces many process including cell cycle arrest, upregulation of DNA damage response (DDR) genes, and stabilization and repair of stalled or collapsed forks. In this thesis we aimed to deepen our understanding of the replication stress response by identifying and characterizing novel regulatory factors in Saccharomyces cerevisiae. To uncover such factors, we employed several screening approaches, including a genetic interaction screen focused on cohesion and DDR components under hydroxyurea treatment, as well as a genome-wide screen using DNA polymerase occupancy at a barcoded origin tracked by ChIP. Mechanistic follow-up studies elucidated how several newly identified factors (Chl1, Lge1, Rox1 and Mediator complex subunits Soh1 and Srb2) contribute to the cellular response to replication stress.