Peroxiredoxin Tsa1 is the key peroxidase suppressing genome instability and protecting against cell death in Saccharomyces cerevisiae

Abstract

Peroxiredoxins (Prxs) constitute a family of thiol-specific peroxidases that utilize cysteine (Cys) as the primary site of oxidation during the reduction of peroxides. To gain more insight into the physiological role of the five Prxs in budding yeast Saccharomyces cerevisiae, we performed a comparative study and found that Tsa1 was distinguished from the other Prxs in that by itself it played a key role in maintaining genome stability and in sustaining aerobic viability of rad51 mutants that are deficient in recombinational repair. Tsa2 and Dot5 played minor but distinct roles in suppressing the accumulation of mutations in cooperation with Tsa1. Tsa2 was capable of largely complementing the absence of Tsa1 when expressed under the control of the Tsa1 promoter. The presence of peroxidatic cysteine (Cys(47)) was essential for Tsa1 activity, while Tsa1(C170S) lacking the resolving Cys was partially functional. In the absence of Tsa1 activity (tsa1 or tsa1(CCS) lacking the peroxidatic and resolving Cys) and recombinational repair (rad51), dying cells displayed irregular cell size/shape, abnormal cell cycle progression, and significant increase of phosphatidylserine externalization, an early marker of apoptosis-like cell death. The tsa1(CCS) rad51- or tsa1 rad51-induced cell death did not depend on the caspase Yca1 and Ste20 kinase, while the absence of the checkpoint protein Rad9 accelerated the cell death processes. These results indicate that the peroxiredoxin Tsa1, in cooperation with appropriate DNA repair and checkpoint mechanisms, acts to protect S. cerevisiae cells against toxic levels of DNA damage that occur during aerobic growth.

Figure 1. Comparative study of the five Prxs.

(A) Sensitivity of S. cerevisiae prxs mutant strains to H₂O₂. Equal numbers of cells were serially diluted (10 fold per dilution) and spotted onto SC medium or SC medium containing indicated concentration of H₂O₂. (B) Effect of inactivation of RAD51 upon viability and growth of each prx mutant. A rad51 strain (MEHY694) was crossed with tsa1, tsa2, ahp1, prx1, and dot5 single mutants to form heterozygous diploids. After sporulation of these heterozygous diploids, tetrads were dissected and grown on YPD at 30°C for 3 days. Spore genotypes were determined by replica plating on appropriate media. Representative tetrads are presented. Circles indicate either the inferred or determined prx and rad51 double mutants.

Figure 2. Construction of the strains expressing desired genes under control of the TSA1 promoter.

The S. cerevisiae strains expressing TSA2 or mutated forms of TSA1 (tsa1^C47S, tsa1^C170S, and tsa1^CCS) under control of the endogenous TSA1 promoter were constructed as described in Materials and Methods. The desired coding sequence was tailed by PCR with sequence homologous to regions flanking the chromosomal TSA1 to allow targeted recombination.

Figure 3. Effect of TSA2 expression under the control of the TSA1 promoter.

(A) Analysis of mRNA expression by quantitative RT-PCR. For each experiment, the relative levels of TSA1 or TSA2 relative to ADH were calculated with the ADH level set as 1. Columns, mean of 3 independent experiments; bars, SD. (B) Analysis of protein level by Western blot. 3HA-tagged native Tsa1, native Tsa2 and tsa1::TSA2 produced Tsa2 were detected by anti-HA antibody staining with short (upper panel) and long (middle panel) exposures presented and Adh levels (encoded by ADH1) as a loading control (bottom panel). (C) The tsa1 (RDKY5502) and tsa1::TSA2 (MEHY1710) mutants were crossed with rad51 (MEHY694) and rad6 (MEHY575) mutants and the spore clones obtained by tetrad dissections of the resulting heterozygous diploids (MEHY101 tsa1/TSA1 rad51/RAD51, MEHY1838 tsa1::TSA2/TSA1 rad51/RAD51, MEHY591 tsa1/TSA1 rad6/RAD6 and MEHY1841 tsa1::TSA2/TSA1 rad6/RAD6) are shown. Circles indicate either the inferred or determined tsa1 rad51 or tsa1::TSA2 rad51 double mutants (upper panel) and tsa1 rad6 or tsa1::TSA2 rad6 double mutants (bottom panel).

Figure 4. Effect of Cys substitutions on Tsa1 function in suppressing cell death.

The tsa1^C47S, tsa1^C170S, and tsa1^CCS mutants were crossed with a rad51 single mutant, tetrads of resulting heterozygous diploids (MEHY1844 tsa1^C47S/TSA1 rad51/RAD51, MEHY1847 tsa1^C170S/TSA1 rad51/RAD51, and MEHY1850 tsa1^CCS/TSA1 rad51/RAD51) were dissected and then incubated under aerobic or anaerobic conditions for 3 or 5 days respectively. Circles indicate either the inferred or determined tsa1^C47S rad51, tsa1^C170S rad51, or tsa1^CCS rad51 double mutants grown under aerobic (upper panel) or under anaerobic conditions (bottom panel).

Figure 5. Properties of a tsa1^CCS rad51 double mutant under aerobic growth.

(A) Cultures of wild-type and tsa1^CCS rad51 cells from anaerobic growth conditions were diluted in fresh medium and samples were taken at time 0, 8 and 24 hr after shifting to aeration to perform FACS analysis and microscopic observation by phase contrast and after DAPI staining. The same magnification (×1000) was used for microscopic analysis of both strains. The FACS profile and morphology of rad51 and tsa1^CCS cells were similar to that of wild-type (data not shown). (B) Representative microscopic images of tsa1^CCS rad51 and tsa1^CCS rad51 rad9 spore colonies (magnification×400) 48 hr after tetrad dissection of a tsa1^CCS/TSA1 rad51/RAD51 rad9/RAD9 heterozygous diploid (MEHY2089). The genotype of the microcolonies was inferred from the segregation patterns of the different mutations present in the diploid strain. (C) FACS analysis of tsa1^CCS rad51 rad9 and rad9 cells at 0, 8, and 24 hr after shifting to aeration.

Figure 6. Quantification of phosphatidylserine externalization and loss of membrane integrity.

(A) Exponentially growing wild-type cells were treated with 1 mM and 5 mM H₂O₂ for 200 min and assayed using FACS based measurement of annexin V/PI co-staining. The percentage of intact cells (double negative), early apoptotic cells (annexin V positive and PI negative) and late apoptotic/necrotic cells (double positive) is presented. (B) The indicated strains were similarly characterized at different time points after shifting from anaerobic to aerobic growth. The values presented are the mean±SD of the values from at least four independent experiments.