Repair of a chromosomal double-strand break (DSB) by gene conversion depends on the ability of the broken ends to encounter a donor sequence. To understand how chromosomal location of a target sequence affects DSB repair, we took advantage of genome-wide Hi-C analysis of yeast chromosomes to create a series of strains in which an induced site-specific DSB in budding yeast is repaired by a 2-kb donor sequence inserted at different locations. The efficiency of repair, measured by cell viability or competition between each donor and a reference site, showed a strong correlation (r = 0.85 and 0.79) with the contact frequencies of each donor with the DSB repair site. Repair efficiency depends on the distance between donor and recipient rather than any intrinsic limitation of a particular donor site. These results further demonstrate that the search for homology is the rate-limiting step in DSB repair and suggest that cells often fail to repair a DSB because they cannot locate a donor before other, apparently lethal, processes arise. The repair efficiency of a donor locus can be improved by four factors: slower 5′ to 3′ resection of the DSB ends, increased abundance of replication protein factor A (RPA), longer shared homology, or presence of a recombination enhancer element adjacent to a donor.homologous recombination | double-strand break repair | chromosome conformation | homology search | donor location H omologous recombination is the predominant mechanism to repair chromosome breaks and preserve genome integrity. In eukaryotes, the broken double-strand break (DSB) ends undergo extensive 5′ to 3′ resection, promoting the binding of the Rad51 recombinase to form a nucleoprotein filament that can search the genome for a homologous sequence with which it can effect repair. Donor template sequences can be located on a sister chromatid, a homologous chromosome or an ectopic location. When ectopic sequences are used, repair results in nonallelic replacements of sequences. In budding yeast Saccharomyces cerevisiae it is possible to monitor the sequence of DSB repair events in real time by Southern blots, PCR or chromatin immunoprecipitation (1).Haploid yeast chromosomes are arranged in a Rabl orientation, with the 16 centromeres all clustered at the spindle-pole body (SPB) whereas the telomeres are associated in loose clusters at the nuclear envelope (2). These observations have been extended by the use of chromosome conformation capture approaches (3, 4) to map the relative positions of loci along each chromosome based on their frequencies of crosslinking (contact frequencies) with many other sites in the genome. Previous studies have shown that telomere-associated sequences preferentially recombine with other telomere-associated loci whereas centromere-linked sites selectively recombine with other centromere-linked loci (5-7). However, such preferences, presumably caused by the constraints of tethering, may not reflect the general behavior of most sequences undergoing homologous recombination. It is not known how the ...

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 813-kb chromosome 2 (Chr2), with a site-specific DSB created on the right arm (position 625 kb). 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 238 kb) 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.

8Repair of a double-strand break (DSB) by an ectopic homologous donor sequence is 1 9 subject to the three-dimensional arrangement of chromosomes in the nucleus of haploid 2 0 budding yeast. The data for interchromosomal recombination suggest that searching 2 1 for homology is accomplished by a random collision process, strongly influenced by the 2 2 contact probability of the donor and recipient sequences. Here we explore how constraints imposed on repair. Specifically, we examined how intrachromosomal repair (Chr2), with a site-specific DSB created on the right arm (position 625kb). Repair . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/114850 doi: bioRxiv preprint first posted online Mar. 7, 2017;