RAD51 Is Required for the Repair of Plasmid Double-Stranded DNA Gaps from Either Plasmid or Chromosomal Templates

Abstract

DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52epistasis group was tested in this system. RAD51,RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, orRAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not onRAD51. The residual repair events in rad51mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms forRAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair.

ACKNOWLEDGMENTS

We thank members of the Symington laboratory, W. K. Holloman, and C. S. H. Young for helpful discussions through the course of this work, and we thank H. Klein and R. Rothstein for yeast strains.

This work was supported by Public Health Service grant NIH GM54099 from the National Institutes of Health.