All strains used in this study were isogenic and are listed in Additional file
1: Table S1. Standard techniques for yeast strain construction and growth were used
. Construction of the rad27::LEU2, rad51::LEU2, rad59::LEU2, rad59-Y92A, rad59-K166A, rad59-K174A, rad59-F180A and srs2::TRP1 alleles have been described previously
[27, 58–60]. The rad27::LEU2 allele can be followed in crosses by PCR, using the forward primer 5′-GCG TTG ACA GCA TAC ATT-3′, and reverse primer 5′-CGT ACA AAC CAA ATG CGG-3′. The rad59::LEU2 allele is followed by PCR using the forward primer 5′-GCC ACA GTT TGG CAA GGG-3′, and the reverse primer 5′-GGG TTT GTT GCC ATC TGC G-3′. The rad59 missense alleles were followed in crosses by allele-specific PCR
. Unique forward primers were used to detect rad59-Y92A (5′-GCT AAT GAA ACA TTC GGG GC-3′), rad59-K166A (5′-AAT GTT ATA ACA GGT CGA AAG C-3′), rad59-K174A (5′-AAG GGT TAC GTA GAG GAG AAG-3′), and rad59-F180A (5′-AAG AAG GCG TTA TTG AGC GC-3′). All allele-specific PCRs use the same reverse primer (5′-TAT ATA AGT ACG TGA GAT CTA TTT G-3′). Presence of the rad59-K174A allele is scored by digesting the PCR product with MseI restriction endonuclease. DNA was purified for PCR analysis using a standard method
Diploid yeast strains heterozygous for each of the rad59 alleles (rad59/RAD59) and the rad27::LEU2 allele (rad27::LEU2/RAD27) were sporulated and dissected. After 72 h, five representative tetrads from each diploid were selected. The presence of rad27 and rad59 mutant alleles in each of the colonies that arose from the spores was scored using PCR as described above.
At least 10, five-milliliter YPD (1% yeast extract, 2% peptone, 2% dextrose) cultures were inoculated with colonies arising from the spores of freshly dissected tetrads and grown overnight at 30°. These were sub-cultured into Klett tubes containing five milliliters of YPD medium that were incubated at 30° while shaking. Cell density was measured by monitoring culture turbidity with a Klett-Summerson colorimeter each hour over a 10 h period. Doubling times were calculated using a standard algorithm
. The 95% confidence intervals and Mann–Whitney values were determined using the Prism statistics package (GraphPad, La Jolla, CA).
At least five, five-milliliter YPD cultures were inoculated with colonies arising from freshly dissected tetrads and grown overnight at 30°. Overnight cultures were sub-cultured into five milliliters of YPD medium and grown to mid-log phase at 30° defined by growth curve using a Klett-Summerson colorimeter. Cells were processed for flow cytometry using the following adaptation of a published method
. The cell density was determined by hemacytometer count and aliquots containing 107 cells were pelleted, resuspended in 70% ice-cold ethanol, and fixed while rotating at 4° overnight. Fixed cells were pelleted, resuspended in 1 ml of citrate buffer (50 mM Na citrate, pH 7.2), and sonicated (Misonix 3000, Farmingdale, NY). Sonicated cells were pelleted, resuspended in citrate buffer and treated with 25 μl of 10 mg/ml RNase A, at 50° for one h, followed by treatment with 50 μl of 20 mg/ml Proteinase K and incubation at 50° for one h. Cells were pelleted and resuspended in 1 ml of citrate buffer, and either rotated overnight at 4°, or stained immediately by adding 16 μl of 1 mg/ml propidium iodide and rotating for 45 min at room temperature in the dark before processing by flow cytometry (Beckman Coulter CyAn ADP 9color, Miami FL). Fractions of cells in the G1, S and G2/M phases of the cell cycle were determined using FlowJo v.7.6.5 image processing software (Tree Star, Ashland, OR). The ratio of cells in G1 vs. S + G2/M were calculated for each trial and the median value for each strain used for comparing cell cycle distributions in different strains. The Mann–Whitney test was used to assess the statistical significance of differences between strains.
Spontaneous ectopic gene conversion
Spontaneous ectopic gene conversion in haploid strains was assayed as described previously
, but using substrates described in a separate analysis
. All strains contained the sam1-ΔBgl II-HOcs allele at the SAM1 locus on chromosome XII, the sam1-ΔSal I allele adjacent to the HIS3 locus on chromosome XV, and a HIS3 gene replacing the SAM2 coding sequence at the SAM2 locus (sam2::HIS3) on chromosome IV. The sam1-∆Bgl II-HOcs allele has a 117 bp fragment of the MAT locus disrupting the Bgl II site in the SAM1 coding sequence, while the sam1-ΔSal I allele has a 4 bp insertion at the Sal I site
. The sam1-ΔSal I allele lacks a promoter, preventing conversion events at this locus from generating AdoMet+ recombinants. The sam1-∆Bgl II-HOcs and sam1-ΔSal I alleles are also in opposite orientations relative to their centromeres, preventing the isolation of single crossover recombinants.
At least ten freshly dissected haploid segregants of each strain were inoculated into five-milliliter YPD cultures supplemented with 100 μg/ml of S-adenosylmethionine (AdoMet) and grown to saturation at 30°. Appropriate dilutions of each culture were plated onto YPD + AdoMet plates to determine the number of viable cells, and onto YPD plates lacking AdoMet to determine the number of AdoMet prototrophic recombinants.
All rates were determined by the method of the median
. Rates and 95% confidence intervals were calculated as described previously
Spontaneous hetero-allelic recombination
Rates of spontaneous hetero-allelic recombination were determined as for ectopic gene conversion except that different substrates were used in diploid cells. All strains contained the sam2-ΔEcoR V-HOcs allele at the SAM2 locus on one copy of chromosome IV, the sam2-ΔSal I allele on the other, and a LEU2 marker replacing the SAM1 coding sequence at the SAM1 locus on both copies of chromosome XII. The sam2-ΔEcoR V-HOcs allele has a 117 bp fragment of the MAT locus disrupting the EcoR V site, while the sam2-ΔSal I allele has a 4 bp insertion disrupting the Sal I site
Rates of mutation at the CAN1 locus were examined using a previously published assay
[8, 10, 18]. At least ten freshly dissected segregants were used to inoculate one-milliliter YPD cultures that were grown to saturation at 30°. Appropriate dilutions were plated onto YPD to determine viability and synthetic medium lacking arginine but containing 60 μg/ml of canavanine to select for mutants.
Unequal sister chromatid recombination (USCR)
Rates of USCR were determined using a previously published assay
[8, 10, 67]. At least ten freshly dissected segregants containing the USCE construct at the TRP1 locus on chromosome IV and the his3∆200 allele at the HIS3 locus on chromosome XV, were struck out to single colonies on YPD. After three days of growth at 30°, single colonies were used to inoculate one-milliliter YPD cultures, and grown to saturation at 30°. Appropriate dilutions were plated onto YPD to assess viability and onto medium lacking histidine to determine the number of histidine prototrophic recombinants.
Loss of heterozygosity (LOH)
Rates of spontaneous LOH by three different mechanisms were assessed using a previously published assay
. Freshly dissected haploid segregants containing either the hxt13::URA3, CAN1, and HOM3 alleles, or the HXT13, can1-100, and hom3-10 alleles on chromosome V were crossed and the resulting diploids struck out to single colonies on YPD. At least 12 independent colonies were inoculated into one-milliliter YPD liquid cultures and grown to saturation at 30°. Appropriate dilutions were plated onto YPD for viability and synthetic medium lacking arginine, but containing 60 μg/ml of canavanine to select for clones resistant to canavanine. After three days of growth at 30° canavanine-resistant (CanR) colonies were replica plated onto synthetic medium lacking either uracil or threonine to assay for the presence of the hxt13::URA3 (Ura+) and HOM3 (Thr+) alleles, respectively. Rates of interstitial LOH, terminal LOH, and CL were determined from the number of Ura+CanRThr+, Ura-CanRThr+, and Ura-CanRThr- recombinant colonies, respectively.
Modeling the Rad59 protein
The crystal structure of the N-terminus of human Rad52
 was obtained from the RSCB Protein Data Bank (http://www.rcsb.org/pdb/). This structure was imaged using the molecular modeling program, SYBYL, and the amino acids corresponding to those mutated in the rad59 missense alleles were identified, and highlighted.
Availability of supporting data
The data sets supporting the results of this article are included within the article and in Additional file