RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae

Abstract

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails.

Fig. 1

Translocation assay. a A truncated his3-Δ3′ allele is located on one copy of chromosome XV and a truncated his3-Δ5′ allele is located on one copy of chromosome III. The his3-Δ200 allele at the HIS3 locus on the other copy of chromosome XV cannot contribute to the generation of an intact HIS3 gene by HR. Upon addition of galactose, HO endonuclease is expressed and cutting occurs adjacent to each substrate. Note that in the “one-break” assay cutting only occurs adjacent to the his3-Δ5′ substrate and in the “spontaneous” assay, HO endonuclease is not expressed. b Following DSB formation, end processing generates 3′ single-stranded DNA tails that can anneal, c via the 60 or 311 bp of overlapping sequence homology between the two substrates. d Terminal non-homology is then removed from the annealed intermediate allowing for ligation and creation of a tXV:III translocation chromosome that renders the cell prototrophic for histidine. The reciprocal tIII:XV translocation, which is generated by a mechanism that utilizes minimal shared homology between the chromosome fragments (G. Manthey and A. Bailis, unpublished data) is occasionally observed on chromosome and genomic Southern blots (Pannunzio et al. 2008)

Fig. 2

Alignment of amino acid residues conserved between Rad59, ScRad52 and HsRad52. Portions of the Rad59, Saccharomyces cerevisiae Rad52 (ScRad52) and Homo sapiens Rad52 (HsRad52) amino acid sequences were aligned using the Multalin program. The Rad59 residues that were mutated to alanine in this study are highlighted with a gray box. In order, these are Tyr92, Lys166, Lys174, and Phe180. The in vitro or in vivo effects of these mutations in ScRad52 (Mortensen et al. 2002) or HsRad52 (Kagawa et al. ; Lloyd et al. 2005) are indicated

Fig. 3

Western blot analysis of the levels of Rad52 and Rad59 in wild type and rad59 mutant cells. a Protein extracts were prepared from strains expressing FLAG-tagged versions of the indicated proteins. Anti-FLAG and anti-β-actin were used as the primary antibodies and an HRP conjugated goat anti-mouse was used for the secondary. b The graph displays the mean fold-difference from wild-type Rad59 from four independent determinations of Rad52 or Rad59 normalized to the level of β-actin in wild-type or rad59 mutant cells

Fig. 4

The effect of rad59 missense mutations on translocation formation by SSA. Translocation frequency using his3 substrates with either 311 (white bars) or 60 bp (gray bars) of overlapping homology was measured in each of the indicated strains. Median frequencies and 95% confidence intervals were determined from a minimum of 10 independent trials. The fold-reduction from wild type is indicated above each bar. Strains: ABX1711 (RAD59/RAD59, 311 bp), ABX2184 (RAD59/RAD59, 60 bp), ABX1698 (rad59Δ/rad59Δ, 311 bp), ABX1689 (rad59Δ/rad59Δ, 60 bp), ABX2303 (rad59-K174A/rad59-K174A, 311 bp), ABX2304 (rad59-K174A/rad59-K174A, 60 bp), ABX2368 (rad59-Y92A/rad59-Y92A, 311 bp), ABX2369 (rad59-Y92A/rad59-Y92A, 60 bp), ABX2725 (rad59-F180A/rad59-F180A, 311 bp), ABX2708 (rad59-F180A/rad59-F180A, 60 bp), ABX2758 (rad59-K166A/rad59-K166A, 311 bp), ABX2757(rad59-K166A/rad59-K166A, 60 bp)

Fig. 5

The effect of rad59 missense mutations on spontaneous DRR. The frequency of DRR between truncated his3 alleles on chromosome XV that share 223 bp of overlapping sequence homology was measured in each of the indicated strains. Median frequencies and 95% confidence intervals were determined from a minimum of 10 independent trials. Strains: ABX192-1A (RAD59), ABX2427-2A (rad59Δ), ABX2430-3C (rad59-K174A), ABX2437-2A (rad59-Y92A), ABX2711-1C (rad59-F180A), ABX2745-13A (rad59-K166A)

Fig. 6

The effect of rad59 missense mutations on spontaneous translocation rate. The spontaneous translocation rate using his3 substrates with 311 bp of overlapping homology was measured in each of the indicated strains. Rates were determined by the method of the median (Lea and Coulson 1949). Median rate and 95% confidence intervals were determined from a minimum of 10 independent trials. Strains: ABM130 (RAD59/RAD59), ABX1708 (rad59Δ/rad59Δ), ABX2844 (rad59-K174A/rad59-K174A), ABX2845 (rad59-Y92A/rad59-Y92A), ABX2846 (rad59-K166A/rad59-K166A), ABX2847 (rad59-F180A/rad59-F180A)

Fig. 7

Gamma-ray sensitivity of rad59 missense mutants. Indicated strains were exposed to increasing does of ionizing radiation. Mean plating efficiencies were determined from a minimum of six independent trials. Strains: W961-5A (wild type), ABX552-2A (rad52Δ), ABX512-37A (rad59Δ), ABX2279-2B (rad59-K174A), ABX2351-1D (rad59-Y92A), ABX2705-15A (rad59-F180A), ABX2745-13A (rad59-K166A)

Fig. 8

RAD1 epistasis analysis with RAD59 missense mutants. Translocation frequency using his3 substrates sharing either 60 or 311 bp of overlapping homology was measured in each of the indicated strains following the creation of DSBs. Fold change from wild type is indicated above each bar on the graph. Strains: ABX1711 (wild type, 311 bp), ABX2184 (wild type, 60 bp), ABX1771 (rad1Δ/rad1Δ, 311 bp), ABX1244 (rad1Δ/rad1Δ, 60 bp), ABX1698 (rad59Δ/rad59Δ, 311 bp), ABX1689 (rad59Δ/rad59Δ, 60 bp), ABX2093 (rad1Δ/rad1Δ, rad59Δ/rad59Δ, 311 bp), ABX2095 (rad1Δ/rad1Δ, rad59Δ/rad59Δ, 60 bp), ABX2613 (rad1Δ/radΔ, rad59-Y92A/rad59-Y92A, 311 bp), ABX2614 (rad1Δ/radΔ, rad59-Y92A/rad59-Y92A, 60 bp), ABX2831 (rad1Δ/rad1Δ, rad59-F180A/rad59-F180A, 311 bp), ABX2867 (rad1Δ/rad1Δ, rad59-F180A/rad59-F180A, 60 bp), ABX2827 (rad1Δ/rad1Δ, rad59-K166A/rad59-K166A, 311 bp), ABX2866 (rad1Δ/rad1Δ, rad59-K166A/rad59-K166A, 60 bp)

Fig. 9

MSH2 and RAD59 epistasis analysis. Translocation frequency using his3 substrates sharing either 60 or 311 bp of overlapping homology was measured in each of the indicated strains following the creation of DSBs. Fold change from wild type is indicated above each bar on the graph. Strains: ABX1711 (wild type, 311 bp), ABX2184 (wild type, 60 bp), ABX1698 (rad59Δ/rad59Δ, 311 bp), ABX1689 (rad59Δ/rad59Δ, 60 bp), ABX1660 (msh2Δ/msh2Δ, 311 bp), ABM148 (msh2Δ/msh2Δ, 60 bp), ABX2264 (msh2Δ/msh2Δ, rad59Δ/rad59Δ, 311 bp), ABX2263 (msh2Δ/msh2Δ, rad59Δ/rad59Δ, 60 bp)