RESUMO
We previously demonstrated that reactive oxygen species (ROS) could be involved in ultraviolet-C (UVC)-induced DNA damage in Escherichia coli cells. In the present study, we evaluated the involvement of the GO system proteins in the repair of the 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxoG, GO) lesion, which is ROS-induced oxidative damage. We first found that the mutant strain Δfur, which produces an accumulation of iron, and the cells treated with 2,2'-dipyridyl, a iron chelator, were both as resistant to UVC-induced lethality as the wild strain. The 8-oxoG could be mediated by singlet oxygen ((1)O(2)). The Fpg protein repaired this lesion when it was linked to C (cytosine), whereas the MutY protein repaired 8-oxoG when it was linked to A (adenine). The survival assay showed that the Fpg protein, but not the MutY protein, was important to UVC-induced lethality and interacted with the UvrA protein, a nucleotide excision repair (NER) protein involved in UVC repair. The GC-TA reversion assay in the mutant strains from the '8-oxoG-repair' GO system showed that UVC-induced mutagenesis in the fpg mutants, but not in the MutY strain. The transformation assay demonstrated that the Fpg protein is important in UVC repair. These results suggest that UVC could also cause indirect ROS-mediated DNA damage and the Fpg protein plays a predominant role in repairing this indirect damage.
Assuntos
Quebras de DNA , Reparo do DNA , DNA-Formamidopirimidina Glicosilase/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Raios Ultravioleta/efeitos adversos , 8-Hidroxi-2'-Desoxiguanosina , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , DNA Bacteriano/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA-Formamidopirimidina Glicosilase/genética , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Mutagênese , Plasmídeos/genética , Plasmídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete/metabolismo , Transformação BacterianaRESUMO
We previously demonstrated that reactive oxygen species (ROS) could be involved in the DNA damage induced by ultraviolet-C (UVC). In this study, we evaluated singlet oxygen ((1)O(2)) involvement in UVC-induced mutagenesis in Escherichia coli cells. First, we found that treatment with sodium azide, an (1)O(2) chelator, protected cells against UVC-induced lethality. The survival assay showed that the fpg mutant was more resistant to UVC lethality than the wild-type strain. The rifampicin mutagenesis assay showed that UVC mutagenesis was inhibited five times more in cells treated with sodium azide, and stimulated 20% more fpg mutant. These results suggest that (1)O(2) plays a predominant role in UVC-induced mutagenesis. (1)O(2) generates a specific mutagenic lesion, 8-oxoG, which is repaired by Fpg protein. This lesion was measured by GC-TA reversion in the CC104 strain, its fpg mutant (BH540), and both CC104 and BH540 transformed with the plasmid pFPG (overexpression of Fpg protein). This assay showed that mutagenesis was induced 2.5-fold in the GC-TA strain and 7-fold in the fpg mutant, while the fpg mutant transformed with pFPG was similar to GC-TA strain. This suggests that UVC can also cause ROS-mediated mutagenesis and that the Fpg protein may be involved in this repair.
Assuntos
Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Mutagênese/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Raios Ultravioleta , Escherichia coli/genéticaRESUMO
The involvement of reactive oxygen species (ROS) in the induction of DNA damage to Escherichia coli cells caused by UVC (254 nm) irradiation was studied. We verified the expression of the soxS gene induced by UVC (254 nm) and its inhibition by sodium azide, a singlet oxygen (1O2) scavenger. Additional results showed that a water-soluble carotenoid (norbixin) protects against the lethal effects of UVC. These results suggest that UVC radiation can also cause ROS-mediated lethality.
