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

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.

Fig. 1

Heteroallelic recombination and DNA repair. (A) Assay for interchromosomal recombination between the ade2-a and ade2-n heteroalleles in diploid cells. -a and -n denote the deletion of the AatII and NdeI enzyme restriction sites, respectively. Three outcomes resulting in adenine prototrophs are illustrated: (i) reciprocal exchange, (ii) conversion of the ade-n allele, and (iii) conversion of the ade2-a allele. (B) Spontaneous recombination between heteroalleles located at the endogenous ADE2 locus (ade2-a) and on a single-copy plasmid (ade2-n). (C) Genomic context of heteroallelic recombination. Patches of prototroph recombinants after replica plating onto SC-Ade plates. (i) MATa RAD52 ade2-a/MATα RAD52 ade2-n, MATa rad52-Y66A ade2-a/MATα rad52-Y66A ade2-n, and MATa RAD52 ade2-a/MATα rad52-Y66A ade2-n diploid strains. (ii) MATa rad52-Y66A ade2-a haploid, MATα rad52-Y66A ade2-a haploid, and MATa rad52-Y66A ade2-a/MATα rad52-Y66A ade2-a transformed with single-copy plasmid carrying the ade2-n heteroallele. (D) Survival after γ-irradiation. Haploid (1n) and diploid (2n) strains were analyzed by counting colonies of surviving cells after 3 days unless otherwise stated. □ RAD52 diploid; ■ RAD52 haploid; ◯ rad52-Y66A diploid; ● rad52-Y66A haploid; △ rad52 null (rad52Δ) diploid; ◇ rad52 null (rad52Δ) diploid after 6 days; and ▲ rad52 null (rad52Δ) haploid. In the rad52 null diploid after 6 days many survivors exhibit loss of the TRP1, LYS2 and/or MAT markers either independently or in combination, indicating that >60% of the survivors are aneuploid (2n-1, 2n-2 and 2n-3), whereas <5% of wild-type survivors are aneuploid. Curves represent the average of 3 trials and error-bars indicate the standard error of the mean.

Fig. 2

UV- and γ-ray sensitivity of rad52-Y66A and msh2Δ mutants. (A) Survival of diploid strains after γ-irradiation. □ RAD52; ◆ msh2 null (msh2Δ); ◯ rad52-Y66A; ◇ rad52-Y66A msh2Δ; and △ rad52Δ. (B) Survival of diploid strains after UV-irradiation. Ten-fold serial dilutions were plated for each strain onto YPD plates and irradiated with 30 or 60 J/m². The unirradiated control (0 J/m²) shows the starting concentration for each strain. Curves represent the average of 3 trials and error-bars indicate the standard error of the mean. (C) Hydroxyurea sensitivity of rad52-Y66A. Ten-fold serial dilutions for each strain were used to test survival after DNA damage induced by hydroxyurea (HU, 25 mM and 50 mM). Plates were scanned after 3 days at 30°C.

Fig. 3

Spontaneous Rad52 foci. Diploid strains rad52Δ/rad52Δ and rad52Δ msh2Δ/rad52Δ msh2Δ transformed with a single-copy plasmid carrying either RAD52-YFP or rad52-Y66A-YFP. (A) Microscopy of cells. Spontaneous Rad52 foci can be seen in the bottom three panels. (B) Percentage of cells carrying a spontaneous Rad52 focus in unbudded (G1, white box) and budded (S/G2/M, grey box) cells. RAD52-YFP MSH2 0/358 unbudded and 67/442 budded cells. rad52-Y66A-YFP MSH2 82/220 unbudded and 346/434 budded cells. RAD52-YFP msh2Δ 3/197 unbudded and 70/432 budded cells. rad52-Y66A-YFP msh2Δ 37/293 unbudded and 219/518 budded cells. (C) Duration of Rad52 foci determined by time-lapse microscopy. Each circle represents a focus. Black circles: focus lasted within the specified category. Grey circles: focus lasted at least this number of minutes. (D) Percentage of cells that formed at least one Rad52 focus per cell cycle. RAD52-YFP MSH2 43/61 cells. rad52-Y66A-YFP MSH2 59/72 cells. RAD52-YFP msh2Δ 32/56 cells. rad52-Y66A-YFP msh2Δ 12/19 cells.

Fig. 4

Model for rad52-Y66A hyper-recombination. (A) Genetic requirements for C × C versus C × P recombination. Centromere regions are tethered to the spindle-pole body (SPB) at the nuclear envelope (NE) thereby restricting the mobility of the plasmid-borne ade2-n allele and reducing its availability for heteroallelic recombination. Loss of centromere function in the ctf4Δ mutant mobilizes the plasmid to become available for heteroallelic recombination. The mre11Δ and rad52-Y66A mutants show defects in DNA damage-induced cohesion or a failure to complete recombination before cohesion is disassembled, respectively, leading to increased C × C recombination. The spatial restriction of the plasmid-borne heteroallele to the SPB is not releaved in mre11Δ and rad52-Y66A mutants. (B) Transient cohesion model. Mre11 facilitates damage-induced cohesion (dashed lines) which favors the sister chromatid as a template for DSB repair (in grey, i and ii). After approximately 20 min, Mre11 dissociates from the DSB, which may be followed by a gradual loss of cohesion. If repair is not accomplished before loss of cohesion, the DSB may be released to interact with the homologue (in black, iii and vi).