Homology-directed repair involves multiple strand invasion cycles in fission yeast

Mol Biol Cell. 2022 Apr 1;33(4):ar30. doi: 10.1091/mbc.E20-07-0433. Epub 2022 Jan 26.

Abstract

Homology-directed repair of DNA double-strand breaks (DSBs) represents a highly faithful pathway. Non-crossover repair dominates in mitotically growing cells, likely through a preference for synthesis-dependent strand annealing (SDSA). How homology-directed repair mechanism choice is orchestrated in time and space is not well understood. Here, we develop a microscopy-based assay in living fission yeast to determine the dynamics and kinetics of an engineered, site-specific interhomologue repair event. We observe highly efficient homology search and homology-directed repair in this system. Surprisingly, the initial distance between the DSB and the donor sequence does not correlate with the duration of repair. Instead, we observe that repair often involves multiple site-specific and Rad51-dependent colocalization events between the DSB and donor sequence. Upon loss of the RecQ helicase Rqh1 (BLM in humans) we observe rapid repair possibly involving a single strand invasion event, suggesting that multiple strand invasion cycles antagonized by Rqh1 could reflect ongoing SDSA. However, failure to colocalize with the donor sequence and execute repair is also more likely in rqh1Δ cells, possibly reflecting erroneous strand invasion. This work has implications for the molecular etiology of Bloom syndrome, caused by mutations in BLM and characterized by aberrant sister chromatid crossovers and inefficient repair.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • DNA Breaks, Double-Stranded
  • DNA Helicases / metabolism
  • DNA Repair
  • DNA Replication
  • Humans
  • Recombinational DNA Repair
  • Schizosaccharomyces pombe Proteins* / metabolism
  • Schizosaccharomyces* / metabolism

Substances

  • Schizosaccharomyces pombe Proteins
  • DNA Helicases
  • Rqh1 protein, S pombe