Tatsuo Nunoshiba

Involvement of Y-Family DNA Polymerases in Mutagenesis Caused by Oxidized Nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

Mutagenic target for hydroxyl radicals generated in Escherichia coli mutant deficient in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron-uptake systems

We previously reported that mutations in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron uptake systems, simulated generation of hydroxyl radicals, and caused hypermutability in Escherichia coli. The predominant type of spontaneous mutation was GC --> TA, followed by AT --> CG, suggesting the involvement of 7,8-dihydro-8-oxoguanine (8-oxoG) and 1,2-dihydro-2-oxoadenine (2-oxoA) in DNA as well as 7,8-dihydro-8-oxodeoxyguanosine triphosphate (8-oxodGTP) and 1,2-dihydro-2-oxodeoxyadenosine triphosphate (2-oxodATP) in the nucleotide pool. To determine the targets contributing to oxidative mutagenesis, DNA or nucleotides, we characterized spontaneous mutations and compared the distribution to those in mutMY and mutT strains, in which GC --> TA and AT --> CG were predominantly induced, respectively. The hotspots and sequence contexts where AT --> CG occurred frequently in sodAB fur strain were almost identical to those in mutT strain,whereas, those where GC --> TA occurred frequently in sodAB fur strain were quite different from those in mutMY strain. These observations suggested that AT --> CG is due to 8-oxodGTP, while GC --> TA is produced by some other lesion(s). The 2-oxodATP is also a major oxidative lesion in nucleotides, and strongly induces GC --> TA. The expression of cDNA for MTH1, which can hydrolyze 2-oxodATP as well as 8-oxodGTP, partially but significantly, suppressed the GC --> TA mutator phenotype of the sodAB fur strain, whereas, it did not for the mutMY strain. Additionally, the sequence contextby 2-oxodATP in E. coli was similar to that in sodAB fur strain. These results suggested that the targets contributing to oxidative mutagenesis in sodAB fur strain are nucleotides such as dGTP and dATP, rather than DNA.

Role of glutathione on acrolein-induced cytotoxicity and mutagenicity in Escherichia coli.

The reduced form of glutathione (GSH) is a well-known antioxidant, while there have been few reports indicating the contribution of glutathione to protection of Escherichia coli cells from the lethal effect of oxidative damage. Here, we report that depletion of glutathione causes hypersensitivity of E. coli to acrolein, the structurally simplest alpha, beta-unsaturated aldehyde derived from lipid peroxide-degradation, and that GSH can chemically react with acrolein in vitro thus reducing its toxicity. We further demonstrated that acrolein inactivates glutathione oxidoreductase followed by depletion of glutathione, probably as a part of the toxic effect of acrolein. These results suggested that GSH contributes to cellular defense against the toxic effects.

Miscoding and misincorporation of 8-oxo-guanine during leading and lagging strand synthesis in Escherichia coli

Abstract. We examined whether strand identity with respect to DNA replication influences strand bias for 8-oxo-7,8-dihydroguanine (8-oxoG) mutagenesis. The specificity of 8-oxoG mutagenesis was determined in a mutM mutY or a mutT strain carrying the supF gene on one of two vectors that differed only in the orientation of supF with respect to a unique origin of replication. Most of the supF mutations in the mutM mutY strain were base substitutions (67%), predominantly G:C→T:A transversions (>64%), while the majority in the mutT strain were base substitutions (>92%), predominantly A:T→C:G transversions (>91%). The distributions of frequently mutated sites of G:C→T:A and A:T→C:G transversions in the supF gene in the mutM mutY and mutT strains, respectively, did not differ markedly between the two vectors. These results suggest that gene orientation is not an important determinant of the strand bias of 8-oxoG mutagenesis.

Phenyl hydroquinone, an Ames test-negative carcinogen, induces Hog1-dependent stress response signaling.

Recently, we have shown that phenyl hydroquinone, a hepatic metabolite of the Ames test‐negative carcinogen o‐phenylphenol, efficiently induced aneuploidy in Saccharomyces cerevisiae. We further found that phenyl hydroquinone arrested the cell cycle at G1 and G2/M. In this study, we demonstrate that phenyl hydroquinone can arrest the cell cycle at the G2/M transition as a result of stabilization of Swe1 (a Wee1 homolog), probably leading to inactivation of Cdc28 (a Cdk1/Cdc2 homolog). Furthermore, Hog1 (a p38 MAPK homolog) was robustly phosphorylated by phenyl hydroquinone, which can stabilize Swe1. On the other hand, Chk1 and Rad53 were not phosphorylated by phenyl hydroquinone, indicating that the Mec1/Tel1 DNA‐damage checkpoint was not functional. Mutations of swe1 and hog1 abolished phenyl hydroquinone‐induced arrest at the G2/M transition and the cells became resistant to phenyl hydroquinone lethality and aneuploidy development. These data suggest that a phenyl hydroqionone‐induced G2/M transition checkpoint that is activated by the Hog1–Swe1 pathway plays a role in the development of aneuploidy.

Hydrogen peroxide-induced microsatellite instability in the Escherichia coli K-12 endogenous tonB gene.

Damage to DNA by reactive oxygen species may be a significant source of endogenous mutagenesis in aerobic organisms. Using an endogenous tonB gene as a mutation selective marker in Escherichia coli, we have examined whether endogenous oxidative mutagenesis can contribute to genetic instability. We have also used oxyR(+) and oxyR(-) strains to evaluate how hydrogen peroxide scavenging system can contribute to genetic instability. The highest mutation frequency induced by hydrogen peroxide was 3.8x10(-6) at 600 microM and 5.3 x 10(-6) at 40 microM in oxyR(+) and oxyR(-), respectively. Hydrogen peroxide induced minus frameshift mutations predominantly followed by transversions (G:C-->T:A, G:C-->C:G, and A:T-->T:A). The types and the nature of the mutations did not differ between strains. Frameshift mutations occurred at G:C and A:T sites equally, and in repeated and non-repeated sequences equally. It is evident that endogenous oxidative damage to DNA can increase the frequency of strand slippage intermediates occurring during DNA replication and contribute to genomic instability. Our results further indicate that oxyR regulon does not take part in the DNA-repair pathway against oxidative damage induced by hydrogen peroxide.