1+1 = 3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers

The methionine salvage pathway.

<p>The enzyme names are from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Sekowska2" target="_blank">[4]</a>, and compound names are from KEGG <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Kanehisa1" target="_blank">[18]</a>. The reactions in black are known in bacteria <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Sekowska2" target="_blank">[4]</a>. The yeast pathway is indicated by blue gene names under the corresponding enzymes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Pirkov1" target="_blank">[9]</a>. Dashed lines indicate variants of the pathway (see text). In <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Ashida1" target="_blank">[5]</a> it was noted that the genes coding for mtnB and mtnC appear to be fused in <i>Arabidopsis thaliana</i>, and the genes for mtnB and mtnD appear to be fused in <i>Tetrahymena thermophila</i>, which indicates that the pathway in these organisms proceeds through the green and red reaction lines, respectively. We identified another fusion gene, between mtnK and mtnA, in <i>Tetrahymena</i> (red line).</p

Screenshot of a tblastn search of the mtnAK enzyme from <i>Tetrahymena</i> (XP_001031773) against the EST sequences from <i>Tetrahymena</i> in GenBank.

<p>The EST sequences TT1BI24TH (acc: FF565362; evalue = 1×10⁻¹²⁵) and TT1BI24TV (acc: FF565363; evalue = 2×10⁻¹²⁵) correspond to the 5′- and 3′-end of a single cDNA clone, indicating that the fusion protein is expressed in <i>Tetrahymena</i>.</p

Complementation experiment of yeast single knockout strains with mtnBD fusion gene from <i>Tetrahymena</i>.

<p>Cells were grown to late exponential phase in −Leu +Met liquid media, transferred to −Leu−Met for an overnight to deplete internal Methionine pool, and serial dilutions for each strain were prepared in a 96-well plate with 1×10⁸ cells/ml, 1×10⁷ cells/ml, 1×10⁶ cells/ml, 1×10⁵, 1×10⁴ cells/ml, and 1×10³ cells/ml. 3 µl of each diluted culture was spotted with a 96-well pin replicator onto (A) −Met−MTA (negative control plate). (B) +Met plates, (positive control). (C) −Met +MTA (5 mM) (experimental plate). The strains assayed are: 1. <i>mtnB</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 2. <i>mtnB</i>Δ + pGREG505; 3. <i>mtnC</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 4. <i>mtnC</i>Δ + pGREG505; 5. <i>mtnD</i>Δ + pGREG505/<i>SYN-MTNBD</i>; 6. <i>mtnD</i>Δ + pGREG505; columns 7–12 are replicates of columns 1 through 6. After four days, none of the six yeast strains grew on the negative control plate. All six strains grew on the positive control plate. Only the strains transformed with pGREG/<i>SYN-MTNBD</i> (columns 1, 3, 5, 7, 9, 11) grew in the experimental plate.</p

Homologs of <i>B. subtillis</i> and yeast methionine salvage pathway enzymes in <i>Tetrahymena</i>.

<p>*Enzyme names and EC numbers are from KEGG (Kyoto Encyclopedia of Genes and Genomes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1000701#pgen.1000701-Kanehisa1" target="_blank">[18]</a>.</p

Growth Inhibition and DNA Damage Induced by X‑Phenols in Yeast: A Quantitative Structure–Activity Relationship Study

Phenolic compounds and their derivatives are ubiquitous constituents of numerous synthetic and natural chemicals that exist in the environment. Their toxicity is mostly attributed to their hydrophobicity and/or the formation of free radicals. In a continuation of the study of phenolic toxicity in a systematic manner, we have examined the biological responses of Saccharomyces cerevisiae to a series of mostly monosubstituted phenols utilizing a quantitative structure–activity relationship (QSAR) approach. The biological end points included a growth assay that determines the levels of growth inhibition induced by the phenols as well as a yeast deletion (DEL) assay that assesses the ability of X-phenols to induce DNA damage or DNA breaks. The QSAR analysis of cell growth patterns determined by IC₅₀ and IC₈₀ values indicates that toxicity is delineated by a hydrophobic, parabolic model. The DEL assay was then utilized to detect genomic deletions in yeast. The increase in the genotoxicity was enhanced by the electrophilicity of the phenolic substituents that were strong electron donors as well as by minimal hydrophobicity. The electrophilicities are represented by Brown’s sigma plus values that are a variant of the Hammett sigma constants. A few mutant strains of genes involved in DNA repair were separately exposed to 2,6-di-<i>tert</i>-butyl-4-methyl-phenol (BHT) and butylated hydroxy anisole (BHA). They were subsequently screened for growth phenotypes. BHA-induced growth defects in most of the DNA repair null mutant strains, whereas BHT was unresponsive

Comparative Genomic Screen in Two Yeasts Reveals Conserved Pathways in the Response Network to Phenol Stress

Living organisms encounter various perturbations, and response mechanisms to such perturbations are vital for species survival. Defective stress responses are implicated in many human diseases including cancer and neurodegenerative disorders. Phenol derivatives, naturally occurring and synthetic, display beneficial as well as detrimental effects. The phenol derivatives in this study, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and bisphenol A (BPA), are widely used as food preservatives and industrial chemicals. Conflicting results have been reported regarding their biological activity and correlation with disease development; understanding the molecular basis of phenol action is a key step for addressing issues relevant to human health. This work presents the first comparative genomic analysis of the genetic networks for phenol stress response in an evolutionary context of two divergent yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Genomic screening of deletion strain libraries of the two yeasts identified genes required for cellular response to phenol stress, which are enriched in human orthologs. Functional analysis of these genes uncovered the major signaling pathways involved. The results provide a global view of the biological events constituting the defense process, including cell cycle arrest, DNA repair, phenol detoxification by V-ATPases, reactive oxygen species alleviation, and endoplasmic reticulum stress relief through ergosterol and the unfolded protein response, revealing novel roles for these cellular pathways