Responding to different types of damaged telomeres

Abstract

Telomeres are bound by multiple ‘capping’ proteins, which protect chromosome ends from fusion and degradation. Mutations of telomeric proteins of Saccharomyces cerevisiae, cdc13-1 and yku70Δ, induce a telomere uncapping, generation of ssDNA by nucleases, cell cycle arrest and the DNA Damage Response (DDR). In other context the Mec1 checkpoint kinase is activated by Ddc1, Dpb11 and Dna2 in response to DNA damage. I tested how Ddc1, Dpb11 and Dna2 affect cdc13-1 and yku70Δ fitness. DPB11 and DNA2 are essential genes, therefore I analysed checkpoint defective alleles of DPB11, DNA2 and DDC1. I showed that Dpb11 and Dna2 have no effect on yku70Δ fitness in contrast to Ddc1, which slightly affects yku70Δ fitness and might have a role in cell cycle arrest of yku70Δ mutants. I confirmed that Ddc1 strongly, whereas Dpb11 slightly, affects cdc13-1 fitness. Dpb11 may contribute to the checkpoint function of Ddc1. I also found that the cdc13- 1 defect was suppressed by the dna2-W128A,Y130A allele when on a plasmid, but enhanced when integrated into the genome, and these effects might be due to variable dna2-W128A,Y130A copy number (in the genome versus on a plasmid). Surprisingly, in the course of my experiments, I found that deletion of DDC1 suppressed dna2Δ lethality. I confirmed that deletion of RAD9 suppresses dna2Δ lethality, and I found that deletion of RAD17, CHK1, MEC1 and POL32 also allows dna2Δ viability. I observed that elimination of Tel1, Rad53, Exo1, Mre11 or Rad27 do not suppress dna2Δ lethality. Based on the observation that checkpoint gene deletions suppress dna2Δ, whereas they exacerbate defects in other core DNA replication proteins, I propose that the essential function of Dna2 is in the telomeric Okazaki fragment processing. I also found that deletions of HCM1, XBP1 and ULS1, genes selected from a genome-wide screen, suppress growth defect of cdc13-1 and yku70Δ mutants, and ULS1 suppresses stn1-13 defect. Finally, I showed that thermo-sensitive cdc13-1 and cdc15-2 strains adapt to chronic low-dose stress. Adapted cells are fitter if more stress is applied, but are less fit in the absence of stress. I found the consequences of adaptation to be reversible.