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.

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Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't

Authors

Waterman DP, Zhou F, Li K, Lee CS, Tsabar M, Eapen VV, Mazzella A, Haber JE

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