Live cell monitoring of double strand breaks in S. cerevisiae

• DOI: 10.1371/journal.pgen.1008001
• Publication Date: 2019-03-01T18:34:39Z
• Authors: David P. Waterman; Felix Zhou; Kevin Li; Cheng-Sheng Lee; Michael Tsabar; Vinay V. Eapen; Allison Mazzella; James E. Haber
• Keywords: RAD52; Mitotic crossover

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 Rad51 recombination or Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on chromosomes, usually display 2 3 foci may coalesce dissociate. This motion is independent Rad52 microtubules. Rad51-GFP, by itself, unable repair DSBs homologous mitotic cells, but able form allow when heterozygous a wild type protein. The kinetics disappearance Rad51-GFP focus parallels completion DSB repair. However, proficient during meiosis homozygous, similar rad51 "site II" mutants can bind single-stranded DNA not complete strand exchange. Rad52-RFP co-localize same DSB, significant minority without visible Rad52-RFP. conclude co-localization does represent "repair center," as distribution Ddc2-GFP was found absence