Nucleosome remodelling during resection of genomic DSBs and deprotected telomeres in yeast

Abstract

DNA end resection is a decisive step in the repair of DNA double-strand breaks (DSBs): degradation of the 5′-terminated strands generates 3′ single-stranded DNA (ssDNA) and commits repair to homologous recombination. The core resection enzymes are well characterised, but how resection proceeds through chromatin remains incompletely understood. This thesis investigated how nucleosome remodellers influence long-range resection at AsiSI-induced DSBs and at deprotected telomeres in budding yeast. Strand-specific sequencing was used to measure resection at AsiSI-induced DSBs in yeast strains deficient in nucleosome editing. To compare the mutant strains, cutting efficiencies need to be similar. Therefore, a nanopore-based workflow was used to quantify AsiSI cutting efficiency in parallel. The arp8Δ strain, in which INO80 activity is impaired, showed similar cutting efficiency compared to the control and no significant changes in resection were observed. Data of a strain deficient in SWR1 complex activity and H2A.Z remains inconclusive,as DSB induction was insufficient. Upon complete loss of nucleosome editing activity, cutting efficiency was restored and resection was found to be similar as in control cells. Therefore, nucleosome editing seems to be dispensable for resection. To analyse resection independently of restriction-enzyme cutting, the temperature sensitive cdc13-1 system was used. In cdc13-1 mutants, telomeres get deprotected at restrictive temperatures, leading to activation of the DNA damage response. By quantification of singlestranded DNA (QAOS), we showed that besides Exo1 also Dna2 contributes to long-range resection at telomeres. Thus, long-range resection appeared to be similar at telomeres and DSBs and we investigated the influence of nucleosome remodeller mutants on long-range resection at deprotected telomeres. Depletion of the nucleosome-eviction complexes RSC or SWI/SNF strongly impaired resection, whereas impaired INO80 function or combined loss of SWR1 and H2A.Z had no significant effect. Together, these findings indicate that nucleosome eviction, rather than histone-variant exchange, is critical for efficient long-range resection and establish cdc13-1 telomere deprotection as a complementary model for studying long-range resection.