Multiple DNA repair pathways contribute to cell lethality in checkpoint mutants

Abstract

The checkpoint kinases Mec1 and Rad53 play a critical role in stabilising stalled DNA replication forks. We have conducted a genetic screen to identify mutants that render checkpoint-defective yeast cells more resistant to fork-stalling agents (e.g. hydroxyurea, MMS, etc). We screened the yeast deletion library for mutants with heightened resistance to hydroxyurea when the checkpoint has been compromised. From this screen we identified several mutants in gene products involved in Base Excision Repair, Nucleotide Excision Repair, and Mismatch Repair. Mutants were retested for resistance to HU and MMS in rad53Δ background and they all showed increased resistance to low doses of these genotoxic agents, suggesting that multiple repair pathways contribute to lethality after fork stalling in the absence of a functional checkpoint. HU-resistance is increased when combining different repair mutations, however, none of these multiple mutants is able to rescue viability completely, indicating that there are other requirements to maintain fork stability in the absence of rad53. We are currently trying to determine whether these mutants affect restart of stalled replication forks.