RESUMO
Electronic excited molecular oxygen (singlet oxygen, (1)O(2)) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by (1)O(2) in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a (1)O(2)-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G-->T and G-->C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with (1)O(2)-induced DNA damage are linked to the context of the damaged sequence.
Assuntos
DNA Glicosilases , Reparo do DNA/genética , Endodesoxirribonucleases , Proteínas de Escherichia coli , Escherichia coli/genética , Mutagênese/efeitos dos fármacos , Mutação/genética , Oxigênio/farmacologia , Proteínas de Bactérias/genética , Sequência de Bases , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Replicação do DNA , DNA-Formamidopirimidina Glicosilase , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Vetores Genéticos/efeitos dos fármacos , Vetores Genéticos/genética , Mutagênese/genética , Mutação/efeitos dos fármacos , N-Glicosil Hidrolases/genética , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Oxigênio Singlete , Transformação BacterianaRESUMO
The repair of singlet oxygen (1O2)-induced DNA lesions requires several enzymes of the nucleotide and base excision repair pathways, including exonuclease III and endonuclease IV that are known apurinic/apyrimidinic-endonucleases in Escherichia coli. In order to better understand the relevance of exonuclease III on the repair of these lesions, we investigated the mutagenic events that result from the replication of a 1O2-damaged plasmid in an exonuclease-deficient host (xth). The mutation spectrum in the tRNA supF gene target indicated that the absence of exonuclease III does not change the types of mutations induced by 1O2 (mostly of G:C-->T:A and G:C-->C:G transversions). However, the spectrum shows that the mutations are scattered in the supF gene, which is significatively different from the one obtained in wild-type bacteria. Thus, exonuclease III may act on the repair of 1O2-induced lesions altering the DNA repair sequence specificity.