Expression of the Fpg protein of Escherichia coli in Saccharomyces cerevisiae: effects on spontaneous mutagenesis and sensitivity to oxidative DNA damage.
The biological relevance of oxidative DNA damage has been unveiled by the identification of genes such as fpg of E. coli or OGG1 of Saccharomyces cerevisiae. Both Fpg and Ogg1 proteins are DNA glycosylases/AP lyases that excise 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG) from damaged DNA. Although similar, the enzymatic and biological properties of Fpg and Ogg1 proteins are not identical. Furthermore, the Fpg and Ogg1 proteins do not show significant sequence homologies. In this study, we investigated the ability of the Fpg protein of E. coli to complement phenotypes thought to be due to oxidative DNA damage in Saccharomyces cerevisiae. To express Fpg in yeast, the coding sequence of the fpg gene was placed under the control of a strong yeast promoter in the expression vector pCM190 to generate the pFPG240 plasmid. The Ogg1-deficient yeast strain CD138, ogg1::TRP1, was transformed with pFPG240 and the expression of Fpg was measured. Expression of Fpg in yeast harboring pFPG240 was revealed by efficient release of Me-FapyG and cleavage of 8-OxoG-containing duplexes by cell free protein extracts. The production of the Fpg protein in yeast cells was further demonstrated by immunoblotting analysis using anti-Fpg antibodies. Fpg expression suppresses the spontaneous mutator phenotype of ogg1- yeast for the production of canavanin resistant mutants (CanR) and Lys+ revertants. Fpg expression also restores the capacity of plasmid DNA treated with methylene blue plus visible light (MB-light) to transform the yeast ogg1- rad1- double mutant.
Guibourt
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CEA, DSV, Département de Radiobiologie et Radiopathologie, UMR217 CNRS-CEA, Radiobiologie Moléculaire et Cellulaire, Fontenay-Aux-Roses, France.
Boiteux
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D003429:Cross Reactions; D004249:DNA Damage / Q000235:genetics*; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D043382:DNA-Formamidopyrimidine Glycosylase; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D008751:Methylene Blue / Q000494:pharmacology; D016296:Mutagenesis; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection
D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D045643:DNA Repair Enzymes; D008751:Methylene Blue