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 RAD52 epistasis 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, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-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.Transformation of fungi with plasmid DNA has yielded important insights into the mechanisms of recombination and double-strand break repair (DSBR). Early studies by Hinnen et al. (17) provided evidence for integration of circular nonreplicating plasmids by homologous recombination into the yeast genome. In a subsequent study, Orr-Weaver et al. (38) elaborated more thoroughly the way in which circular and linear nonreplicating DNA molecules recombine with homologous chromosomal sequences. They showed that DNA ends are highly recombinogenic and interact directly with homologous sequences. If two restriction cuts are made within a plasmid region homologous to chromosomal DNA, thereby producing a double-strand gap, the resulting deleted linear plasmids transform at a high frequency and are faithfully repaired during the integration process. Using linear replicating plasmids, Orr-Weaver and Szostak (37) reported the recovery of approximately equal numbers of integrated and nonintegrated plasmids and concluded that gene conversion by double-strand gap repair can occur either with or without crossing over. These studies formed the basis for the DSBR model (65). They also observed circularization of linear plasmid DNA, suggesting the presence of additional, recombination-independent repair pathways. Subsequent studies of plasmid gap repair in Saccharomyces cerevisiae and Ustilago maydis indicated a lower association of crossing over with gene conversion (12,44). Studies in Drosophila and mouse cells have also shown a very low association of crossing over (Ͻ5%) during DSBR (35,48).When plasmids capable of autonomous replication are cut within reg...

Sequence Divergence Impedes Crossover More Than Noncrossover Events During Mitotic Gap Repair in Yeast

“…If this is the case, then most COs in this system likely derive from a single rather than a double HJ intermediate. Although there appears to be a consistent role of Rad1-Rad10 in promoting COs in plasmid-based DSB/gap repair assays (Schiestl and Prakash 1988;Bartsch et al 2000), it should be noted that its effect during the repair of an HO-induced chromosomal DSB has been variable (Ira et al 2003;Nicholson et al 2006). This variability could be related to the lengths of the homology that flank the DSB, which would limit the extent and hence stability of heteroduplex intermediates.…”

“…The his3-0,D39 and his3-18,D39 strains (or appropriate mutant derivatives) were then transformed in parallel using the PCR-generated fragment. Variations in transformation efficiencies in different genetic backgrounds (Bartsch et al 2000;Haghnazari and Heyer 2004) were corrected for by mixing an intact LEU2/ARS/CEN plasmid (pRS315; Sikorski and Hieter 1989) with the linear fragment prior to transformation. His 1 and Leu 1 transformants were selected separately, and the efficiency of gap repair was calculated as the ratio of His 1 to Leu 1 transformants.…”

“…Of particular significance was the observation that the repaired plasmid either remains autonomous or integrates into the host genome , outcomes presumed to reflect cleavage of a Holliday junction intermediate to generate NCO or CO products, respectively. Subsequent studies have confirmed that plasmid-based repair assays generally recapitulate the genetic requirements and features of DSB-initiated chromosomal recombination (Bartsch et al 2000) and hence are a useful model for studying basic recombination processes. In the experiments reported here, a gap-repair assay was used to examine the regulation of mitotic recombination fidelity, with an emphasis on how sequence divergence affects the CO-NCO decision in different genetic backgrounds.…”

Role ofRAD52Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

“…Subsequent studies of plasmid gap repair in yeast and Ustilago maydis and chromosomal DSBR following P element excision in Drosophila melanogaster have shown a lower association of crossing over with gene conversion (20,96,257,291). Similarly, mating-type switching in S. cerevisiae, which is a gene conversion event initiated by a site-specific DSB at the MAT locus made by the HO endonuclease (183,360), is rarely associated with crossing over.…”

Mte1 interacts with Mph1 and promotes crossover recombination and telomere maintenance

“…The yeast telomeres are also bound in the double-stranded region by the Ku complex and by Rap1 and its binding partners, Rif1-Rif2. The association of these protein complexes with telomeric DNA forms a protective capping structure that accounts for two crucial outcomes: the full replication of telomeres and protection of telomeres from recognition by the recombination and checkpoint machineries and associated nuclease and helicase activities (Bartsch et al 2000;Raschle et al 2004;Lisby and Geli 2009;Dewar and Lydall 2012). In the absence of telomerase, telomeres shorten progressively with each round of replication.…”