A Yeast Homologue of the Human Phosphotyrosyl Phosphatase Activator PTPA Is Implicated in Protection against Oxidative DNA Damage Induced by the Model Carcinogen 4-Nitroquinoline 1-Oxide

Abstract

The model carcinogen 4-nitroquinoline 1-oxide (4-NQO) has historically been characterized as "UV-mimetic" with respect to its genotoxic properties. However, recent evidence indicates that 4-NQO, unlike 254-nm UV light, may exert significant cytotoxic and/or mutagenic potential via the generation of reactive oxygen species. To elucidate the response of eukaryotic cells to 4-NQOinduced oxidative stress, we isolated Saccharomyces cerevisiae mutants exhibiting hypersensitivity to the cytotoxic effects of this mutagen. One such mutant, EBY1, was cross-sensitive to the oxidative agents UVA and diamide while retaining parental sensitivities to 254-nm UV light, methyl methanesulfonate, and ionizing radiation. A complementing gene (designated yPTPA1), restoring full UVA and 4-NQO resistance to EBY1 and encoding a protein that shares 40% identity with the human phosphotyrosyl phosphatase activator hPTPA, has been isolated. Targeted deletion of yPTPA1 in wild type yeast engendered the identical pattern of mutagen hypersensitivity as that manifested by EBY1, in addition to a spontaneous mutator phenotype that was markedly enhanced upon exposure to either UVA or 4-NQO but not to 254-nm UV or methyl methanesulfonate. Moreover, the yptpa1 deletion mutant exhibited a marked deficiency in the recovery of high molecular weight DNA following 4-NQO exposure, revealing a defect at the level of DNA repair. These data (i) strongly support a role for active oxygen intermediates in determining the genotoxic outcome of 4-NQO exposure and (ii) suggest a novel mechanism in yeast involving yPtpa1p-mediated activation of a phosphatase that participates in the repair of oxidative DNA damage, implying that hPTPA may exert a similar function in humans.Treatment with the agent 4-nitroquinoline 1-oxide (4-NQO) 1 has been widely employed in mammalian systems as a paradigm for DNA damage-induced carcinogenesis. To exert its neoplastic effect, 4-NQO must first undergo metabolic activation to the proximate carcinogen 4-hydroxyaminoquinoline 1-oxide, which, following acylation, reacts with DNA to form stable quinoline-purine monoadducts, i.e. at the exocyclic N-2 and N-6 positions of guanine and adenine, respectively (1, 2). In bacteria, yeast, and mammalian cells, these genotoxic "bulky" DNA lesions are processed largely by the nucleotide excision repair (NER) pathway in a manner analogous to classical dipyrimidine photoproducts (viz. cyclobutane pyrimidine dimers and (6 -4) pyrimidine-pyrimidone photoproducts) generated by the model DNA-damaging agent 254-nm UV light (3, 4). As such, mutants that are deficient in NER are hypersensitive to the genotoxic effects of 254-nm UV light, as well as 4-NQO (3-6). The apparent strong similarity in modes of cellular processing for 254-nm UV light-and 4-NQO-induced DNA damage in diverse prokaryotic and eukaryotic systems has often resulted in categorization of the latter agent as "UV-mimetic" (7).However, this designation may be inappropriate, because several recent investigations have clearly demonstrated that ...

Genetic evidence for a role of Saccharomyces cerevisiae Mph1 in recombinational DNA repair under replicative stress

“…Similar phenotypic differences are reported in a recent study where mph1 srs2 strains showed a growth defect but not synthetic lethality (Banerjee et al, 2008). We found viable mph1 srs2 segregants synergistically sensitive to MMS and 4-NQO, thought to form DNA lesions potently blocking replisome progression (Pegg, 1984;Ramotar et al, 1998) and, in particular, sensitive to camptothecin ( Figure 4A). Camptothecin is thought to induce replication fork collapse and the formation of double-strand ends (Pommier et al, 1998;Strumberg et al, 2000;Lin et al, 2000).…”

UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae

“…Indeed, non-DNA damage is likely the main cause of cell death after UVA irradiation and hides the effect of cytotoxic DNA lesions. To the best of our knowledge, the only yeast mutants unusually sensitive to the lethal effect of UVA are not affected in DNA repair but in the phosphotyrosyl phosphatase activator protein (Ptpa1p) or in the threonine͞serine phosphatase (Sit4p) (37,38). These authors suggest that UVA radiation triggers Swe1p- The percentages represent the fraction of total mutational events.…”

Identification of biomarkers that distinguish human papillomavirus (HPV)-positive versus HPV-negative head and neck cancers in a mouse model

“…Because tobacco use is implicated in HNSCC, we monitored cancers in K14E6͞K14E7 doubly transgenic mice treated with the chemical carcinogen, 4-nitroquinoline 1-oxide (4NQO). 4NQO causes a spectrum of DNA damage similar to that caused by tobacco-associated carcinogens (26)(27)(28)(29)(30), and it induces cancers of the oral cavity in rodents when it is supplied in their drinking water (31). 4NQO-treated, HPV16-transgenic mice developed HNSCC at a much higher frequency than nontransgenic mice.…”