Lee, C.-S. | Search bioRxiv

bioRxiv2018

Live cell monitoring of double strand breaks in S. cerevisiae

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.

molecular biology↗

bioRxiv2017

Position Effects Influencing Intrachromosomal Repair of a Double-Strand Break in Budding Yeast

Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast. The data for interchromosomal recombination suggest that searching for homology is accomplished by a random collision process, strongly influenced by the contact probability of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome and whether there are additional constraints imposed on repair. Specifically, we examined how intrachromosomal repair is affected by the location of the donor sequence along the 812-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (r = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (CEN2, position 238kb) to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently constrained in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.\n\nAuthor SummaryA double-strand break (DSB) on a chromosome can be repaired by recombining with an ectopic homologous donor sequence. Interchromosomal ectopic recombination is strongly influenced by the three-dimensional arrangement of chromosomes in the nucleus of haploid budding yeast, that is strongly influenced by the probability of chemical cross-linking of the donor and recipient sequences. Here we explore how recombination occurs on the same chromosome. We examined how intrachromosomal repair is affected by the location of the donor sequence along the 812-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625kb). Repair correlates well with contact frequencies determined by chromosome conformation capture-based studies (r = 0.85). Moreover, there is a profound constraint imposed by the anchoring of the centromere (CEN2, position 238kb) to the spindle pole body. Sequences at the same distance on either side of CEN2 are equivalently accessible in recombining with a DSB located more distally on one arm, suggesting that sequences on the opposite arm from the DSB are not otherwise constrained in their interaction with the DSB. The centromere constraint can be partially relieved by inducing transcription through the centromere to inactivate CEN2 tethering. In diploid cells, repair of a DSB via its allelic donor is strongly influenced by the presence and the position of an ectopic intrachromosomal donor.

genetics↗