Open AccessLive cell monitoring of double strand breaks in S. cerevisiae
Author(s) -
David P. Waterman,
Felix Zhou,
Kevin Li,
ChengSheng Lee,
Michael Tsabar,
Vinay V. Eapen,
Allison Mazzella,
James E. Haber
Publication year - 2019
Publication title -
plos genetics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.587
H-Index - 233
eISSN - 1553-7404
pISSN - 1553-7390
DOI - 10.1371/journal.pgen.1008001
Subject(s) - rad51 , rad52 , homologous recombination , biology , dna repair , mitotic crossover , microbiology and biotechnology , green fluorescent protein , mitosis , dna , genetics , gene
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.