In living cells, DNA damage occurs as a result of cell metabolism, developmental processes and exogenous sources such as chemical agents or radiation. Repair of DNA damage is essential to prevent chromosome loss and cell death. In the budding yeast Saccharomyces cerevisiae
, homologous recombination (HR) is the major pathway for repair of DNA double-strand breaks (DSBs). However, although DSBs are recombinogenic, they do not appear to be the main source of spontaneous mitotic HR [1
]. Hence, mutants exist that recombine at wild-type or higher levels despite the fact that they are defective in DNA DSB repair. The nature of the lesions provoking HR is still poorly defined, but understanding the phenotype of mutants that separate DNA DSB repair from spontaneous HR will likely provide clues to the mechanisms of spontaneous HR. Many of the genes involved in this process were identified in yeast by screening for mutants sensitive to ionizing radiation [3
]. These mutants constitute the RAD52
epistasis group and include RAD50
]. Amongst these genes in S. cerevisiae
, disruption of RAD52
causes the most severe recombination defect.
The Mre11-Rad50-Xrs2 complex (MRX) is one of the earliest proteins detected at a DSB [6
]. The recruitment of MRX to a DSB results in a high local concentration of the complex as visualized by fluorescence microscopy as Mre11 focus formation [6
]. The MRX complex contributes to the initial processing of DSB ends into 3' single-stranded DNA (ssDNA) tails [7
], which are essential for copying genetic information from an intact donor sequence during homologous recombination. Furthermore, recent studies have shown that the MRX complex is required for the postreplicative reestablishment of cohesion in response to genotoxic stress [11
]. Notably, mre11
results in hyper-recombination between interchromosomal heteroalleles, but not between sister chromatids [15
]. Importantly, the association of MRX with a DSB is transient and the dissociation of MRX from the site of DNA damage is concurrent with the appearance of ssDNA and recruitment of the Rad52 mediator protein [6
], which in turn recruits the Rad51 recombinase to catalyze strand-invasion.
DSBs promote mitotic recombination and result in reciprocal exchange or gene conversion events [19
]. Frequencies of gene conversion are highest near DSBs [21
]. Moreover, conversion tracts are usually continuous and if multiple markers at a DNA DSB are involved, a central marker is almost always co-converted if the flanking markers are converted [21
]. Gene conversion in yeast involves mismatch repair (MMR) of heteroduplex DNA (hDNA) for both meiotic [27
] and mitotic events [29
]. Thus, the amount of homology at the DSB-ends, the direction of mismatch repair and the length of hDNA greatly influence recombinational repair.
epistasis group is also important for spontaneous mitotic recombination although this process is less well characterized and the requirements for individual genes depend on the assay suggesting the existence of multiple pathways [4
]. Importantly, Rad52 is essential for all types of spontaneous mitotic recombination, whereas Rad51 function is required only for some types of recombination. Finally, genes outside of the RAD52
epistasis group also affect spontaneous mitotic recombination as illustrated by a recent genome-wide analysis of the genetic control of Rad52 foci [34
]. Some of these genes that affect recombination include factors that contribute to chromosome integrity by maintaining chromatin architecture and organization, regulating cell cycle and spindle checkpoints, and repairing DNA lesions via other pathways.
To gain insight into the mechanism(s) of spontaneous mitotic recombination, we analyzed the phenotype of a rad52-Y66A
mutant that blocks the repair DNA damage induced by γ-irradiation, but is proficient for spontaneous mitotic recombination at a rate higher than wild type. This allele was generated by site-directed mutagenesis in an alanine scan of the conserved N terminus of Rad52 [35
]. It was subsequently shown that rad52-Y66A
cells are deficient in the repair of a single DSB induced during mating-type switching and are sensitive to a top1-T722A
mutation, which causes the accumulation of covalent topoisomerase-DNA intermediates that are frequently converted to DSBs [1
]. Further, the rad52-Y66A
mutant is proficient for UV-induced heteroallelic recombination. The data presented here suggest that the rad52-Y66A
hyper-recombination phenotype may result from a slowdown in DNA repair that leads to a loss of damage-induced cohesion prior to completion of repair, causing a shift from sister chromatid to interchromosomal recombination.