Live cell monitoring of double strand breaks in S. cerevisiae

PLoS Genet. 2019 Mar 1;15(3):e1008001. doi: 10.1371/journal.pgen.1008001. eCollection 2019 Mar.

Abstract

We have used two different live-cell fluorescent protein markers to monitor the formation and localization of double-strand breaks (DSBs) in budding yeast. Using GFP derivatives of the Rad51 recombination protein or the Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on different chromosomes, usually display 2 or 3 foci that may coalesce and dissociate. This motion is independent of Rad52 and microtubules. Rad51-GFP, by itself, is unable to repair DSBs by homologous recombination in mitotic cells, but is able to form foci and allow repair when heterozygous with a wild type Rad51 protein. The kinetics of formation and disappearance of a Rad51-GFP focus parallels the completion of site-specific DSB repair. However, Rad51-GFP is proficient during meiosis when homozygous, similar to rad51 "site II" mutants that can bind single-stranded DNA but not complete strand exchange. Rad52-RFP and Rad51-GFP co-localize to the same DSB, but a significant minority of foci have Rad51-GFP without visible Rad52-RFP. We conclude that co-localization of foci in cells with 3 DSBs does not represent formation of a homologous recombination "repair center," as the same distribution of Ddc2-GFP foci was found in the absence of the Rad52 protein.

Publication types

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

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics*
  • Cell Cycle Proteins / genetics*
  • DNA Breaks, Double-Stranded*
  • DNA Damage / genetics
  • DNA-Binding Proteins / genetics
  • Green Fluorescent Proteins / genetics
  • Homologous Recombination / genetics
  • Kinetics
  • Meiosis / genetics
  • Rad51 Recombinase / genetics*
  • Rad52 DNA Repair and Recombination Protein / genetics*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / genetics*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • LCD1 protein, S cerevisiae
  • RAD52 protein, S cerevisiae
  • Rad52 DNA Repair and Recombination Protein
  • Saccharomyces cerevisiae Proteins
  • Green Fluorescent Proteins
  • Rad51 Recombinase