Mapping yeast mitotic 5' resection at base resolution reveals the sequence and positional dependence of nucleases in vivo

Nucleic Acids Res. 2021 Dec 16;49(22):12607-12621. doi: 10.1093/nar/gkab597.

Abstract

Resection of the 5'-terminated strand at DNA double-strand breaks (DSBs) is the critical regulated step in the transition to homologous recombination. Recent studies have described a multi-step model of DSB resection where endonucleolytic cleavage mediated by Mre11 and Sae2 leads to further degradation mediated by redundant pathways catalyzed by Exo1 and Sgs1/Dna2. These models have not been well tested at mitotic DSBs in vivo because most methods used to monitor resection cannot precisely map early cleavage events. Here we report resection monitoring with high-throughput sequencing using molecular identifiers, allowing exact counting of cleaved 5' ends at base resolution. Mutant strains, including exo1Δ, mre11-H125N and exo1Δ sgs1Δ, revealed a major Mre11-dependent cleavage position 60-70 bp from the DSB end whose exact position depended on local sequence. They further revealed an Exo1-dependent pause point approximately 200 bp from the DSB. Suppressing resection extension in exo1Δ sgs1Δ yeast exposed a footprint of regions where cleavage was restricted within 119 bp of the DSB. These results provide detailed in vivo views of prevailing models of DSB resection and extend them to show the combined influence of sequence specificity and access restrictions on Mre11 and Exo1 nucleases.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alleles
  • Base Sequence
  • DNA / chemistry
  • DNA Breaks, Double-Stranded*
  • DNA End-Joining Repair
  • Exodeoxyribonucleases / genetics
  • Exodeoxyribonucleases / metabolism*
  • Fungal Proteins / metabolism*
  • Fungal Proteins / physiology
  • Gene Deletion
  • MRE11 Homologue Protein / metabolism*
  • MRE11 Homologue Protein / physiology
  • Mitosis / genetics*
  • RecQ Helicases / genetics
  • Recombinational DNA Repair*
  • Saccharomycetales / enzymology
  • Saccharomycetales / genetics

Substances

  • Fungal Proteins
  • DNA
  • Exodeoxyribonucleases
  • MRE11 Homologue Protein
  • exodeoxyribonuclease I
  • RecQ Helicases