Live cell monitoring of double strand breaks in S. cerevisiae

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 coalesce and dissociate. 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 disappearance of Rad51-GFP foci parallels the completion of DSB repair. However, in meiosis, Rad51-GFP is proficient when homozygous. Using Ddc2-GFP, we conclude that co-localization of foci following 3 DSBs does not represent formation of a homologous recombination \"repair center,\" as the same distribution of Ddc2-GFP foci was found in the presence or absence of the Rad52 protein. The maintenance of separate DSB foci and much of their dynamics depend on functional microtubules, as addition of nocodazole resulted in a greater population of cells displaying a single focus.\n\nAuthor SummaryDouble strand breaks (DSBs) pose the greatest threat to the fidelity of an organisms genome. While much work has been done on the mechanisms of DSB repair, the arrangement and interaction of multiple DSBs within a single cell remain unclear. Using two live-cell fluorescent DSB markers, we show that cells with 3 site-specific DSBs usually form 2 or 3 foci what can coalesce into fewer foci but also dissociate. The aggregation of DSBs into a single focus does not depend on the Rad52 recombination protein, suggesting that there is no \"repair center\" for homologous recombination. DSB foci are highly dynamic and their dynamic nature is dependent on microtubules.