Elsevier

DNA Repair

Volume 9, Issue 1, 2 January 2010, Pages 23-32
DNA Repair

The rad52-Y66A allele alters the choice of donor template during spontaneous chromosomal recombination

https://doi.org/10.1016/j.dnarep.2009.10.001Get rights and content

Abstract

Spontaneous mitotic recombination is a potential source of genetic changes such as loss of heterozygosity and chromosome translocations, which may lead to genetic disease. In this study we have used a rad52 hyper-recombination mutant, rad52-Y66A, to investigate the process of spontaneous heteroallelic recombination in the yeast Saccharomyces cerevisiae. We find that spontaneous recombination has different genetic requirements, depending on whether the recombination event occurs between chromosomes or between chromosome and plasmid sequences. The hyper-recombination phenotype of the rad52-Y66A mutation is epistatic with deletion of MRE11, which is required for establishment of DNA damage-induced cohesion. Moreover, single-cell analysis of strains expressing YFP-tagged Rad52-Y66A reveals a close to wild-type frequency of focus formation, but with foci lasting 6 times longer. This result suggests that spontaneous DNA lesions that require recombinational repair occur at the same frequency in wild-type and rad52-Y66A cells, but that the recombination process is slow in rad52-Y66A cells. Taken together, we propose that the slow recombinational DNA repair in the rad52-Y66A mutant leads to a by-pass of the window-of-opportunity for sister chromatid recombination normally promoted by MRE11-dependent damage-induced cohesion thereby causing a shift towards interchromosomal recombination.

Introduction

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], [2]. 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, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54, RFA1, MRE11 and XRS2 [4], [5]. 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], [8], [9], [10], 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], [12], [13], [14]. Notably, mre11 results in hyper-recombination between interchromosomal heteroalleles, but not between sister chromatids [15], [16], [17], [18]. 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], [20]. 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], [22], [23], [24], [25], [26]. Gene conversion in yeast involves mismatch repair (MMR) of heteroduplex DNA (hDNA) for both meiotic [27], [28] and mitotic events [29], [30], [31], [32], [33]. Thus, the amount of homology at the DSB ends, the direction of mismatch repair and the length of hDNA greatly influence recombinational repair.

The RAD52 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], [5]. 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.

Section snippets

Genetic methods, yeast strains and plasmids

Yeast strains were manipulated using standard genetic techniques and media was prepared as described previously except that twice the amount of leucine was used (60 mg/L) [36]. All strains used in this study are listed in Table 4 and all are RAD5 derivatives of W303 [37], [38]. Other genetic markers have been described previously [39].

The ctf4::kanMX6 allele was amplified from the available yeast gene deletion library strain [40] using primers 5′-CATCCTCTTCATGTACTACTTATGTCCA and

A rad52-Y66A mutant displays context-dependent hyper-recombination

To explore further the mechanism of spontaneous mitotic recombination, we analyzed the hyper-recombination phenotype of a γ-ray sensitive rad52-Y66A mutant [1]. Heteroallelic recombination was measured for diploid wild-type, rad52-Y66A and rad52Δ strains using two previously described non-functional ade2 heteroalleles, ade2-a and ade2-n [45]. Two assays were used to measure the rate of prototroph formation due to recombination between two interchromosomal heteroalleles (C × C) in diploid strains (

Discussion

In the budding yeast S. cerevisiae, recombinational repair during mitotic growth relies on several DNA repair proteins, including those encoded by genes of the RAD52 epistasis group. In this study, we measured spontaneous heteroallelic recombination between two chromosomes (C × C) or between a chromosome and a plasmid (C × P). Interestingly, we find that the two configurations display different requirements. The rate of wild-type C × P recombination is 10-fold lower than the rate of C × C

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by The Danish Agency for Science, Technology and Innovation and the Villum Kann Rasmussen Foundation (ML), the Danish Research Council for Technology and Production Sciences (UHM), and the National Institutes of Health (GM50237 and GM67055 to RR). We thank Marisa Wagner for constructing the pWJ1190 plasmid and Lorraine Symington for sharing strains.

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    Present address: Chiquita Brands International, 2051 S.E. 35th Street, Hollywood, FL 33316, USA.

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