Abstract
This dissertation examines the cellular mechanisms by which Saccharomyces cerevisiae detects, signals, and repairs DNA double-strand breaks (DSBs), with a focus on histone modifications, genome-wide genetic screens, and replication fork-associated proteins.
Chapter 1 provides a comprehensive introduction to DSB repair pathways—homologous recombination (HR), non-homologous end joining (NHEJ), single-strand annealing (SSA), and break-induced replication (BIR)—and reviews the DNA damage checkpoint (DDC) that coordinates repair with cell cycle arrest. The chapter concludes with an overview of the replication stress checkpoint and the Mrc1–Tof1–Csm3 complex that mediates checkpoint signaling at stalled replication forks.
Chapter 2 investigates the role of histone H2B phosphorylation at threonine 129 (γ-H2B) in the DSB response. Using chromatin immunoprecipitation, adaptation assays, and viability assays in multiple repair contexts, we review that γ-H2B is Mec1- and Tel1-dependent and show that it may promote Rad9 accumulation at the break, phenocopying γ-H2A. Loss of H2B-T129 phosphorylation reduces viability in single stranded annealing and gene conversion survival assays, accelerates checkpoint adaptation, implicating γ-H2B in both DSB repair efficiency and checkpoint maintenance.
Chapter 3 presents a genome-wide CRISPRi screen to systematically identify factors required for DSB survival. Using a barcoded guide RNA library in a strain carrying a single galactose-inducible DSB, we quantified guide RNA abundance before and after DSB induction. Known DNA damage response genes were depleted, validating the screen. Novel hits—including spt21Δ, ack1Δ, ubc13Δ, san1Δ, yng1Δ, and yet3Δ—were validated by individual deletion and plating assays. Unexpectedly, guides targeting checkpoint components were consistently enriched after DSB induction, reflecting a proliferative advantage conferred by checkpoint bypass in pooled competitive assays.
Chapter 4 establishes a Mec1-independent DSB survival pathway and identifies the replication checkpoint mediator Mrc1 as a novel DSB repair factor. We show that mec1Δ cells survive DSB at approximately 50% of wild-type efficiency through a pathway requiring Tel1, Rad53, Mre11 and the 9-1-1 clamp, and that Rad53 contributes via a kinase-independent mechanism. Mrc1, but not its partners Tof1 or Csm3, promotes DSB survival independently of its canonical Mec1 phosphorylation sites. Mechanistically, Mrc1 promotes physical tethering of the two DSB ends and limits 5′-to-3′ resection during S phase; both functions require its C-terminal domain, which mediates interaction with Ctf4. Deletion of Ctf4 phenocopies mrc1Δ for end tethering, linking the replisome scaffold to DSB end protection. Mrc1, but not Tof1 or Csm3, is also required for normal heterochromatic silencing.
Chapter 5 outlines work contributed to Dr. Felix Zhou’s paper “Arp2/3 and Type-I myosins control chromosome mobility and end-resection at double-strand breaks in S. cerevisiae”. Where we uncover a novel role for Arp2/3 in budding yeast DSB repair. Overall, these results suggest that the Arp2/3 complex is important for DSB end resection and end resection is closely tied to DSB mobility which impacts cell’s ability to repair a DSB.
Collectively, this dissertation expands our understanding of the factors and pathways that protect genome integrity following DSBs, from chromatin-level histone modifications and genome-wide screens for novel repair factors to replication fork-associated complexes with previously unrecognized roles in DSB repair.