RESUMO
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.
Assuntos
Cádmio/toxicidade , Núcleo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Oncogênicas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Animais , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Camundongos , Proteínas Oncogênicas/genética , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
Recombinação Homóloga/genética , Proteína 1 Homóloga a MutL/genética , Proteínas MutL/genética , Mapas de Interação de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Troca Genética , Reparo de Erro de Pareamento de DNA/genética , Genoma Fúngico , Meiose/genética , Saccharomyces cerevisiae/genética , Relação Estrutura-AtividadeRESUMO
Fungi living in heavy metal polluted soils have evolved different cellular and molecular systems to adapt and survive in these harsh environments. Oidiodendron maius Zn is an ericoid mycorrhizal fungus previously shown to be highly tolerant to zinc thanks to antioxidative enzymes and membrane transporters. A novel gene, OmFCR1, was recently identified from this fungus because it conferred strong cadmium tolerance when expressed in yeast. OmFCR1 codes for a protein belonging to the PLAC8 family and physically interacts in yeast with the mismatch repair system, involved in DNA damage repair. The O. maius Zn genome also contains another gene - named OmFCR2 - that codes for a protein sharing with OmFCR1, the PLAC8 domain and other sequence similarities. In this work, we analyzed gene expression of OmFCR1 and OmFCR2 in the fungus O. maius Zn when exposed to cadmium, the ability of OmFCR2 to confer cadmium tolerance when expressed in yeast, and the growth of OmFCR1-null mutants of O. maius Zn upon cadmium exposure. Although OmFCR2 was also able to confer some cadmium tolerance to yeast, the different expression pattern of these two genes would suggest different roles in O. maius Zn.