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.

Cadmium 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. 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. 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 a PLAC8 protein in physically coupling 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 of cell division as compared to control cells.

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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.

22Protein domains are structurally and functionally distinct units responsible for particular protein 23 functions or interactions. Although protein domains contribute to the overall protein function(s) and 24 can be used for protein classification, about 20% of protein domains are currently annotated as 25 "domains of an unknown function" (DUFs). DUF 614, a cysteine-rich domain better known as 26 PLAC8 (Placenta-Specific Gene 8), occurs in proteins found in the majority of Eukaryotes. containing proteins play important yet diverse roles in different organisms, such as control of cell 28 proliferation in animals and plants or heavy metal resistance in plants and fungi. For example, 29 Onzin from Mus musculus is a key regulator of cell proliferation, whereas FCR1 from the 30 ascomycete Oidiodendron maius confers cadmium resistance. Onzin and FCR1 are small, single-31 domain PLAC8 proteins and we hypothesized that, despite their apparently different role, a 32 common molecular function of these proteins may be linked to the PLAC8 domain. To address this 33 hypothesis, we compared these two PLAC8-containing proteins by heterologous expression in the 34 PLAC8-free yeast Saccharomyces cerevisiae. When expressed in yeast, both Onzin and FCR1 35 improved cadmium resistance, reduced cadmium-induced DNA mutagenesis, localized in the 36 nucleus and induced similar transcriptional changes. Our results support the hypothesis of a 37 common ancestral function of the PLAC8 domain that may link some mitochondrial biosynthetic 38 pathways (i.e. leucine biosynthesis and Fe-S cluster biogenesis) with the control of DNA damage, 39 thus opening new perspectives to understand the role of this protein domain in the cellular biology 40 of Eukaryotes.41 42 Author Summary 43Protein domains are the functional units of proteins and typically have distinct structure and 44 function. However, many widely distributed protein domains are currently annotated as "domains of 45 unknown function" (DUFs). We have focused on DUF 614, a protein domain found in many 46 Eukaryotes and better known as PLAC8 (Placenta-Specific Gene 8). The functional role of DUF 47 614 is unclear because PLAC8 proteins seem to play important yet different roles in taxonomically 48 distant organisms such as animals, plants and fungi. We used S. cerevisiae to test whether these 49 apparently different functions, namely in cell proliferation and metal tolerance, respectively 50 reported for the murine Onzin and the fungal FCR1, are mediated by the same molecular 51 mechanisms. Our data demonstrate that the two PLAC8 proteins induced the same growth 52 phenotype and transcriptional changes in S. cerevisiae. In particular, they both induced the 53 biosynthesis of the amino acid leucine and of the iron-sulfur cluster, one of the most ancient protein 54 cofactors. These similarities support the hypothesis of an ancestral function of the DUF 164 55 domain, whereas the transcriptomic data open new perspectives to understand the role of PLAC8-56 proteins in Eukaryotes.57...