Yeast expression of mammalian Onzin and fungal FCR1 suggests ancestral functions of PLAC8 proteins in mitochondrial metabolism and DNA repair

The cysteine-rich PLAC8 domain of unknown function occurs in proteins found in most Eukaryotes. PLAC8-proteins play important yet diverse roles in different organisms, such as control of cell proliferation in animals and plants or heavy metal resistance in plants and fungi. Mammalian Onzin can be either pro-proliferative or pro-apoptotic, depending on the cell type, whereas fungal FCR1 confers cadmium tolerance. Despite their different role in different organisms, we hypothesized common ancestral functions linked to the PLAC8 domain. To address this hypothesis, and to investigate the molecular function of the PLAC8 domain, murine Onzin and fungal FCR1 were expressed in the PLAC8-free yeast Saccharomyces cerevisiae. The two PLAC8-proteins localized in the nucleus and induced almost identical phenotypes and transcriptional changes when exposed to cadmium stress. Like FCR1, Onzin also reduced DNA damage and increased cadmium tolerance by a DUN1-dependent pathway. Both proteins activated transcription of ancient mitochondrial pathways such as leucine and Fe-S cluster biosynthesis, known to regulate cell proliferation and DNA repair in yeast. These results strongly suggest a common ancestral function of PLAC8 proteins and open new perspectives to understand the role of the PLAC8 domain in the cellular biology of Eukaryotes

A PLAC8-containing protein from an endomycorrhizal fungus confers cadmium resistance to yeast cells by interacting with Mlh3p

<p>admium is a genotoxic pollutant known to target proteins that are involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing mutagenic and carcinogenic effects.</p> <p>We have identified a PLAC8 domain-containing protein, named OmFCR, by a yeast functional screen aimed at identifying genes involved in cadmium resistance in the endomycorrhizal fungus Oidiodendron maius. OmFCR shows a remarkable specificity in mediating cadmium resistance. Both its function and its nuclear localization in yeast strictly depend on the interaction with Mlh3p, a subunit of the mismatch repair (MMR) system.</p> <p>Although proteins belonging to the PLAC8 family are widespread in eukaryotes, they are poorly characterized and their biological role still remains elusive. Our work represents the first report about the potential role of PLAC8 protein as an MMR-associated protein that physically couples DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways. On the basis of cell survival assays and yeast growth curves, we hypothesize that, upon cadmium exposure, OmFCR might promote a higher rate in cell division as compared to control cells.</p

Data from: Mlh3 mutations in baker's yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide

Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker's yeast 60 new mlh3 alleles. Five alleles specifically disrupted MMR, whereas one (mlh3-32) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR+, crossover-) and Mlh1-mlh3-45 (MMR-, crossover+) displayed wild-type endonuclease activities in vitro. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR+ crossover-, MMR- crossover+, endonuclease defective and null mlh3 mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the mlh3 mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3's enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated

<i>mlh3</i> mutations in baker’s yeast alter meiotic recombination outcomes by increasing noncrossover events genome-wide

<div><p>Mlh1-Mlh3 is an endonuclease hypothesized to act in meiosis to resolve double Holliday junctions into crossovers. It also plays a minor role in eukaryotic DNA mismatch repair (MMR). To understand how Mlh1-Mlh3 functions in both meiosis and MMR, we analyzed in baker’s yeast 60 new <i>mlh3</i> alleles. Five alleles specifically disrupted MMR, whereas one (<i>mlh3-32</i>) specifically disrupted meiotic crossing over. Mlh1-mlh3 representatives for each class were purified and characterized. Both Mlh1-mlh3-32 (MMR⁺, crossover^-) and Mlh1-mlh3-45 (MMR^-, crossover⁺) displayed wild-type endonuclease activities <i>in vitro</i>. Msh2-Msh3, an MSH complex that acts with Mlh1-Mlh3 in MMR, stimulated the endonuclease activity of Mlh1-mlh3-32 but not Mlh1-mlh3-45, suggesting that Mlh1-mlh3-45 is defective in MSH interactions. Whole genome recombination maps were constructed for wild-type and MMR⁺ crossover^-, MMR^- crossover⁺, endonuclease defective and null <i>mlh3</i> mutants in an S288c/YJM789 hybrid background. Compared to wild-type, all of the <i>mlh3</i> mutants showed increases in the number of noncrossover events, consistent with recombination intermediates being resolved through alternative recombination pathways. Our observations provide a structure-function map for Mlh3 that reveals the importance of protein-protein interactions in regulating Mlh1-Mlh3’s enzymatic activity. They also illustrate how defective meiotic components can alter the fate of meiotic recombination intermediates, providing new insights for how meiotic recombination pathways are regulated.</p></div

Total interhomolog events and distribution of gene conversion tract lengths associated with NCO and CO events in wild-type, <i>mlh3-23</i>, <i>mlh3-32</i>, <i>mlh3-D523N</i> and <i>mlh3Δ</i> mutants.

<p>A. Total average inter-homolog events (IH; <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s016" target="_blank">S9 Table</a>), and each event type (E1-E7) as a fraction of the total, in <i>MLH3</i>, <i>mlh3-23</i>, <i>mlh3-32</i>, <i>mlh3-D523N</i>, and <i>mlh3Δ</i> mutants. * p ≤ 0.05 compared to wild-type (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s002" target="_blank">S2 File</a>). B. Average number of tracts ordered by size for simple CO (E2) and simple NCO (E1) events. Median CO and NCO tract sizes are also presented, and events were assigned as described by Oke et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref034" target="_blank">34</a>] (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s002" target="_blank">S2 File</a>). The Y axis represents pooled data from all tetrads, normalized by dividing the number of tetrads.</p

Cumulative genetic distance and spore viability of <i>mlh3</i> separation of function mutants.

<p>A. Distribution of genetic markers on chromosome XV used to determine genetic distances in the EAY1112/EAY2413 background (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s008" target="_blank">S1 Table</a>). The solid circle indicates the centromere. The distances between markers are not drawn to scale. The actual physical and genetic distances in the wild-type diploid are given numerically for each interval and for the entire region between <i>CENXV</i> and <i>HIS3</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref007" target="_blank">7</a>]. B. Cumulative genetic distances between <i>URA3</i> and <i>HIS3</i> markers from tetrads of <i>MLH3</i> and indicated <i>mlh3</i> variants. Each bar is further divided into sectors that correspond to the four genetic intervals that span <i>URA3-HIS3</i> (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s011" target="_blank">S4 Table</a>). C. Spore viabilities are plotted vs. genetic map distances from panel B for <i>MLH3</i> (dark blue), <i>mlh3Δ</i> (red), and the separation of function mutants (light blue). Yellow diamonds represent data from Sonntag Brown et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref006" target="_blank">6</a>] (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s002" target="_blank">S2 File</a>).</p

Site directed mutagenesis of <i>MLH3</i>.

<p>A. Functional organization of Mlh3 based on sequence homology and secondary structure prediction [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref051" target="_blank">51</a>]. The vertical bars indicate the approximate position of the <i>mlh3</i> mutations (except <i>mlh3-60</i>) analyzed in this study and described in panel B. <i>mlh3-39</i>, <i>-40</i>, <i>-57</i>, <i>-58</i>, and <i>-59</i> colored in red are based on highly conserved residues in the endonuclease motifs of Pms1 which were shown in the crystal structure of Mlh1-Pms1 to form a single metal binding site [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref051" target="_blank">51</a>] described in panel C. B. Amino acid positions of charged-to-alanine substitutions presented in red on the primary sequence of <i>Saccharomyces cerevisiae</i> Mlh3. Each cluster of underlined residues represents one allele corresponding to the vertical bars in panel A. <i>mlh3-39</i>, <i>-40</i>, <i>-57</i>, <i>-58</i>, and <i>-59</i> are colored in red as in panel A. <i>mlh3-60</i> represents the last 11 residues of Pms1 which constitute patch II of the heterodimerization interface of Mlh1-Pms1 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref051" target="_blank">51</a>]. C. Metal binding site of Pms1 (left panel) from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.ref051" target="_blank">51</a>] comprised of the five highlighted residues (H703, E707, C817, C848, and H850) were found to be highly conserved in Mlh3 (right panel) based on sequence alignment and structural modeling (H525, E529, C670, C701, and H703) and were targeted in the mutagenesis described in this study (alleles represented in red in A and B).</p

Mlh1-mlh3-32 and Mlh1-mlh3-45 display wild-type endonuclease activities that are differentially stimulated by Msh2-Msh3.

<p>A. SDS-PAGE analysis of purified Mlh1-Mlh3, Mlh1-mlh3-32 and Mlh1-mlh3-45. Coomassie Blue R250-stained 8% Tris-glycine gel. 0.5 μg of each protein is shown. MW = Molecular Weight Standards from top to bottom- 200, 116, 97, 66, 45 kD). B, C. Mlh1-Mlh3, Mlh1-mlh3-32 and Mlh1-mlh3-45 (18, 37, 70 nM) were incubated with 2.2 nM supercoiled pBR322 DNA, and analyzed in agarose gel electrophoresis (C) and the endonuclease activity was quantified (average of 6 independent experiments presented +/-SD) as described in the Methods (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006974#pgen.1006974.s002" target="_blank">S2 File</a>). Ladder: 1 kb DNA ladder (New England BioLabs). Migration of closed circular (cc), nicked (nc) and linear (l) pBR322 DNA is indicated. D. Endonuclease assays were performed as in B., but contained 20 nM of the indicated wild-type or mutant Mlh1-Mlh3 complex and 40 nM Msh2-Msh3 when indicated. Reactions were performed in triplicate, samples were resolved on agarose gels, and the fraction of nicked DNA was quantified, averaged, and the standard deviation between experiments was calculated. The average fraction of supercoiled substrate cleaved is presented +/-S.D. below the gel. (bkg) background, (cc) closed circular DNA, (nc) nicked DNA.</p