Abstract
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.
Highlights
A fundamentally important step in the repair of a broken chromosome by homologous recombination is the identification and use of a homologous donor sequence to repair the double-strand break (DSB) [1,2,3,4,5,6]
To examine intrachromosomal repair in more detail, we constructed a series of 12 strains, in which a DSB was created within a 2-kb LEU2 gene inserted 625 kb from the left end on chromosome 2 (Chr2) and a donor was inserted at different sites across the chromosome (Fig 1A)
The sixteen chromosomes adopt a preferential 3D conformation with centromeres clustered at spindle pole body and telomeres loosely associated at the nuclear envelope, the so-called Rabl configuration [21]
Summary
A fundamentally important step in the repair of a broken chromosome by homologous recombination is the identification and use of a homologous donor sequence to repair the DSB [1,2,3,4,5,6]. DSBs are processed by exonucleases to expose 3’-ended single-stranded regions upon which Rad recombination protein is loaded and forms a nucleoprotein. The Rad filament, like its bacterial RecA counterpart, interrogates other sequences in the genome to locate a homologous segment with which it can promote strand invasion to form a displacement loop and initiate DNA synthesis using the homologous sequence as a template to repair the DSB. How the search for homology—on a sister chromatid, a homologous chromosome or at an ectopic site—is accomplished remains a subject of active investigation. Several lines of evidence suggest that the search is more efficient intrachromosomally—at least over modest distances of 100–200 kb [2,6], but this question needs to be explored in more detail
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