A spontaneous mutator strain ofEscherichia coli (fpg mutY) was used to clone the OGG] gene of Saccharomyces cerevisiae, which encodes a DNA glycosylase activity that excises 7, (fpg) harboring pYSB110 was used to purify the Oggl protein of S. cerevisiae to apparent homogeneity. The Oggl protein possesses a DNA glycosylase activity that releases 8-OxoG and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. TheOggl protein preferentially incises DNA that contains 8-OxoG opposite cytosine (8-OxoG/C) or thymine (8-OxoG/T). In contrast, Oggl protein does not incise the duplex where an adenine is placed opposite 8-OxoG (8-OxoG/A). The mechanism of strand cleavage by Oggl protein is probably due to the excision of 8-OxoG followed by a a-elimination at the resulting apurinic/apyrimidinic site.Reactive oxygen species formed in cells as by-products of aerobic metabolism or during oxidative stress have been suggested to play a role in biological processes such as mutagenesis, carcinogenesis, and aging (1, 2). Hydroxyl radical (OH') and singlet oxygen (102) are highly reactive and produce complex patterns of DNA modifications (3, 4). Several lines of evidence suggest that an oxidatively damaged form of guanine,7,, is highly mutagenic in vitro and in vivo (5, 6). Escherichia coli possesses two DNA glycosylase activities that prevent spontaneous mutagenesis by 8-OxoG: the Fpg protein, which excises 8-OxoG residues in DNA (4, 7-9), and the MutY protein, which excises the adenine residues incorporated opposite 8-OxoG (7, 10, 11). The Fpg protein also releases imidazole ring-opened purines such as 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Fapy) (9). Inactivation of both the fg (mutM) and the mutY (micA) genes of E. coli results in a strong GC --TA spontaneous mutator phenotype (7,12,13

Saccharomyces cerevisiae SCF Met30 ubiquitin-protein ligase controls cell cycle function and sulfur amino acid metabolism. We report here that the SCF Met30 complex mediates the transcriptional repression of the MET gene network by triggering degradation of the transcriptional activator Met4p when intracellular S-adenosylmethionine (AdoMet) increases. This AdoMet-induced Met4p degradation is dependent upon the 26S proteasome function. Unlike Met4p, the other components of the specific transcriptional activation complexes that are assembled upstream of the MET genes do not appear to be regulated at the protein level. We provide evidence that the interaction between Met4p and the F-box protein Met30p occurs irrespective of the level of intracellular AdoMet, suggesting that the timing of Met4p degradation is not controlled by its interaction with the SCF Met30 complex. We also demonstrate that Met30p is a short-lived protein, which localizes within the nucleus. Furthermore, transcription of the MET30 gene is regulated by intracellular AdoMet levels and is dependent upon the Met4p transcription activation function. Thus Met4p appears to control its own degradation by regulating the amount of assembled SCF Met30 ubiquitin ligase.

The OGG1 gene of Saccharomyces cerevisiae encodes a DNA glycosylase that excises 7,8-dihydro-8-oxoguanine (8-OxoG) and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine. To investigate the biological role of the OGG1 gene, mutants were constructed by partial deletion of the coding sequence and insertion of marker genes, yielding ogg1::TRP1 and ogg1::URA3 mutant strains. The disruption of the OGG1 gene does not compromise the viability of haploid cells, therefore it is not an essential gene. The capacity to repair 8-OxoG has been measured in cell-free extracts of wild-type and ogg1 strains using a 34mer DNA fragment containing a single 8-OxoG residue paired with a cytosine (8-OxoG/C) as a substrate. Cell-free extracts of the wild-type strain efficiently cleave the 8-OxoG-containing strand of the 8-OxoG/C duplex. In contrast, cell-free extracts of the Ogg1-deficient strain have no detectable activity that can cleave the 8-OxoG/C duplex. The biological properties of the ogg1 mutant have also been investigated. The results show that the ogg1 disruptant is not hypersensitive to DNA-damaging agents such as ultraviolet light at 254 nm, hydrogen peroxide or methyl methanesulfonate. However, the ogg1 mutant exhibits a mutator phenotype. When compared to those of a wild-type strain, the frequencies of mutation to canavanine resistance (CanR) and reversion to Lys+ are sevenfold and tenfold higher for the ogg1 mutant strain, respectively. Moreover, using a specific tester system, we show that the Ogg1-deficient strain displays a 50-fold increase in spontaneously occurring G x C-->T x A transversions compared to the wild-type strain. The five other base substitution events are not affected by the disruption of the OGG1 gene. These results strongly suggest that endogeneous reactive oxygen species cause DNA damage and that the excision of 8-OxoG catalyzed by the Ogg1 protein contributes to the maintenance of genetic stability in S. cerevisiae.

The ubiquitin system has been recently implicated in various aspects of transcriptional regulation, including proteasome-dependent degradation of transcriptional activators. In yeast, the activator Met4 is inhibited by the SCF(Met30) ubiquitin ligase, which recognizes and oligo-ubiquitylates Met4. Here, we demonstrate that in minimal media, Met4 is ubiquitylated and rapidly degraded in response to methionine excess, whereas in rich media, Met4 is oligo-ubiquitylated but remains stable. In the latter growth condition, oligo-ubiquitylated Met4 is not recruited to MET gene promoters, but is recruited to the SAM genes, which are required for production of S-adenosylmethionine, an unstable metabolite that is not present in rich medium. Thus, ubiquitylation not only regulates Met4 by distinct degradation-dependent and -independent mechanisms, but also controls differential recruitment of a single transcription factor to distinct promoters, thereby diversifying transcriptional activator specificity.

Activity of the Met4 transcription factor is antagonized by the SCFMet30 ubiquitin ligase by degradation-dependent and degradation-independent mechanisms, in minimal and rich nutrient conditions, respectively. In this study, we show that the heavy metal Cd2+ over-rides both mechanisms to enable rapid Met4-dependent induction of metabolic networks needed for production of the antioxidant and Cd2+-chelating agent glutathione. Cd2+ inhibits SCFMet30 activity through rapid dissociation of the F-box protein Met30 from the holocomplex. In minimal medium, dissociation of SCFMet30 complex is sufficient to impair the methionine-induced degradation of Met4. In rich medium, dissociation of the SCFMet30 complex is accompanied by a deubiquitylation mechanism that rapidly removes inhibitory ubiquitin moieties from Met4. Post-translational control of SCFMet30 assembly by a physiological stress to allow rapid induction of a protective gene expression program represents a novel mode of regulation in the ubiquitin system