The dynamics of homologous pairing during mating type interconversion in budding yeast.
Cells repair most double-strand breaks (DSBs) that arise during replication or by environmental insults through homologous recombination, a high-fidelity process critical for maintenance of genomic integrity. However, neither the detailed mechanism of homologous recombination nor the specific roles of critical components of the recombination machinery-such as Bloom and Werner syndrome proteins-have been resolved. We have taken a novel approach to examining the mechanism of homologous recombination by tracking both a DSB and the template from which it is repaired during the repair process in individual yeast cells. The two loci were labeled with arrays of DNA binding sites and visualized in live cells expressing green fluorescent protein-DNA binding protein chimeras. Following induction of an endonuclease that introduces a DSB next to one of the marked loci, live cells were imaged repeatedly to determine the relative positions of the DSB and the template locus. We found a significant increase in persistent associations between donor and recipient loci following formation of the DSB, demonstrating DSB-induced pairing between donor and template. However, such associations were transient and occurred repeatedly in every cell, a result not predicted from previous studies on populations of cells. Moreover, these associations were absent in sgs1 or srs2 mutants, yeast homologs of the Bloom and Werner syndrome genes, but were enhanced in a rad54 mutant, whose protein product promotes efficient strand exchange in vitro. Our results indicate that a DSB makes multiple and reversible contacts with a template during the repair process, suggesting that repair could involve interactions with multiple templates, potentially creating novel combinations of sequences at the repair site. Our results further suggest that both Sgs1 and Srs2 are required for efficient completion of recombination and that Rad54 may serve to dissociate such interactions. Finally, these results demonstrate that mechanistic insights into recombination not accessible from studies of populations of cells emerge from observations of individual cells.
Houston
Peter L
PL
Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA.
Broach
James R
JR
D003433:Crosses, Genetic; D004271:DNA, Fungal / Q000235:genetics; D017353:Gene Deletion; D017930:Genes, Reporter; D014644:Genetic Variation; D016681:Genome, Fungal; D010957:Plasmids; D011995:Recombination, Genetic; D012440:Saccharomyces / Q000235:genetics* / Q000254:growth & development; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004271:DNA, Fungal