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. 2018 Jul 9;7:e32744. doi: 10.7554/eLife.32744

Figure 1. Mutations in the Rad51-binding domain of Rad52 suppress MMS and γ-ray sensitivity of Srs2-deficient cells.

(A) Primary structure of Rad52. The conserved N-terminus as well as the RPA and the Rad51 binding domains (BD) are shown. (B) The mutations selected from random and directed mutagenesis are indicated on the alignment of the region 350-389aa of S. cerevisiae Rad52 with that of Rad52 orthologs in 12 fungal species. The amino acid colors refer to the chemical character of the residues. The rad52-P381S and rad52-Y376A mutations are underlined. (C) Serial 10-fold dilutions of haploid strains with the indicated genotypes were spotted onto rich media (YPD) containing different MMS concentrations. The data presented are a selection from those in Figure 1—figure supplement 1. (D,E,F) Survival curves of haploid cells exposed to γ-ray. rad52-YEKF∆ cells were from two different genetic backgrounds: FF18733 (FF) and W303. Data are presented as the mean ± SEM of at least three independent experiments.

Figure 1—source data 1. Survival (%) of haploid cells exposed to γ-ray for Figure 1D,E and F.
elife-32744-fig1-data1.xlsx (44.7KB, xlsx)
DOI: 10.7554/eLife.32744.005

Figure 1.

Figure 1—figure supplement 1. Mutations in the Rad51-binding domain of Rad52 suppress MMS and γ-ray sensitivity of Srs2-deficient cells.

Figure 1—figure supplement 1.

(A,B,C) Serial 10-fold dilutions of haploid strains that were isolated by random mutation screening (green) or that harbor Rad52 mutations created by directed mutagenesis (red). The tested strains are derivative of rad52Δ or rad52Δ srs2Δ null mutants transformed with empty Ycplac111 centromeric plasmid or with wild type or mutated versions of RAD52. (D,E) Survival curves of haploid cells bearing mutations from our collection. Cells were in the log phase of growth when exposed to γ-rays. Data are presented as the mean ± SEM of at least three independent experiments. The rad52-A371T, rad52-S374A and rad52-S387A mutations cannot restore MMS resistance in Srs2-deficient cells, but they can reduce γ-ray sensitivity of Srs2-deficient cells. This difference could be explained by the constant formation of lesions in cells growing in MMS-containing plates, while exposure to γ-rays for a limited period of time creates only a definite number of lesions.

Figure 1—figure supplement 2. The rad52-Y376A, but not the rad52-P381S mutation suppresses the effect of mutations that are synthetically lethal with srs2Δ.

Figure 1—figure supplement 2.

(A) Tetrad analysis of crosses between haploid rad52-P381S srs2Δ strains and haploid mutants that are synthetically lethal with srs2Δ. White diamonds indicate synthetic lethal combinations with or without rad52-P381S. This mutation does not affect the interaction between Rad52 and Rad51 enough to suppress the synthetic lethality. (B) Same crosses as in (A) but with rad52-Y376A. Double mutant spores, which do not contain rad52-Y376A, are indicated by white squares. The white circles mark viable triple mutants.