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

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

A S-adenosylmethionine methyltransferase-like domain within the essential, Fe-S-containing yeast protein Dre2

Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two \u3b1-helices and a \u3b2-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo

A S-adenosylmethionine methyltransferase-like domain within the essential, Fe-S-containing yeast protein Dre2

Yeast Dre2 is an essential Fe-S cluster-containing protein that has been implicated in cytosolic Fe-S protein biogenesis and in cell death regulation in response to oxidative stress. Its absence in yeast can be complemented by the human homologous antiapoptotic protein cytokine-induced apoptosis inhibitor 1 (also known as anamorsin), suggesting at least one common function. Using complementary techniques, we have investigated the biochemical and biophysical properties of Dre2. We show that it contains an N-terminal domain whose structure in solution consists of a stable well-structured monomer with an overall typical S-adenosylmethionine methyltransferase fold lacking two α-helices and a β-strand. The highly conserved C-terminus of Dre2, containing two Fe-S clusters, influences the flexibility of the N-terminal domain. We discuss the hypotheses that the activity of the N-terminal domain could be modulated by the redox activity of Fe-S clusters containing the C-terminus domain in vivo

Mutation rates of peroxiredoxin mutants.

<p>The numbers in parentheses indicate the low and high values for the 95% confidence interval for each rate obtained.</p><p>ND, not precisely determined. We did not analyze enough events to determine the GCR rates of these mutants but estimated that they were similar to that of the wild-type strain.</p

Comparative study of the five Prxs.

<p>(A) Sensitivity of <i>S. cerevisiae prxs</i> 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 <i>RAD51</i> upon viability and growth of each <i>prx</i> mutant. A <i>rad51</i> strain (MEHY694) was crossed with <i>tsa1</i>, <i>tsa2</i>, <i>ahp1</i>, <i>prx1</i>, and <i>dot5</i> 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 <i>prx</i> and <i>rad51</i> double mutants.</p

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

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

Construction of the strains expressing desired genes under control of the <i>TSA1</i> promoter.

<p>The <i>S. cerevisiae</i> strains expressing <i>TSA2</i> or mutated forms of <i>TSA1</i> (<i>tsa1^C47S</i>, <i>tsa1^C170S</i>, and <i>tsa1^CCS</i>) under control of the endogenous <i>TSA1</i> promoter were constructed as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000524#s4" target="_blank">Materials and Methods</a>. The desired coding sequence was tailed by PCR with sequence homologous to regions flanking the chromosomal <i>TSA1</i> to allow targeted recombination.</p

Properties of a <i>tsa1^CCS rad51</i> double mutant under aerobic growth.

<p>(A) Cultures of wild-type and <i>tsa1^CCS rad51</i> 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 <i>rad51</i> and <i>tsa1^CCS</i> cells were similar to that of wild-type (data not shown). (B) Representative microscopic images of <i>tsa1^CCS rad51</i> and <i>tsa1^CCS rad51 rad9</i> spore colonies (magnification×400) 48 hr after tetrad dissection of a <i>tsa1^CCS</i>/<i>TSA1 rad51/RAD51 rad9/RAD9</i> 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 <i>tsa1^CCS rad51 rad9</i> and <i>rad9</i> cells at 0, 8, and 24 hr after shifting to aeration.</p