| Abstract: | 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 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 position of the sequences, lying between the tethered ends, influences their ability to repair a DSB.Taking advantage of the Hi-C database, we established a series of yeast strains in which a DSB, induced at a defined location, could be repaired by a short, homologous donor sequence whose location in each strain was chosen for its apparent contact frequency with the recipient. In this way we were able to learn how the relative positions of sequences influences their recombinational potential and whether, as seems to be the case for translocations arising in mammalian cells (8, 9), chromosomal proximity influences the efficiency or rate of repair. We find that there is a strong correlation between the 3D position of a donor sequence within the yeast nucleus and its efficiency in repairing a DSB and that a given locus has very different repair potential depending on the location of the chromosome break. |
