Assuntos
Dano ao DNA/genética , Enzimas Reparadoras do DNA , Mutação/genética , Precursores de Ácido Nucleico , 8-Hidroxi-2'-Desoxiguanosina , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Animais , Enzimas Reparadoras do DNA/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Nucleotídeos de Desoxiguanina/genética , Nucleotídeos de Desoxiguanina/metabolismo , Desoxirribonuclease (Dímero de Pirimidina)/fisiologia , Proteínas de Escherichia coli/fisiologia , GTP Cicloidrolase/fisiologia , Precursores de Ácido Nucleico/genética , Precursores de Ácido Nucleico/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Pirofosfatases/fisiologiaRESUMO
To examine whether base excision repair suppresses mutations induced by oxidized deoxyribonucleotide 5'-triphosphates in the nucleotide pool, 8-hydroxy-dGTP (8-OH-dGTP) was introduced into Escherichia coli strains deficient in endonucleases III (Nth) and VIII (Nei), and MutY, and mutations in the chromosomal rpoB gene were analyzed. The spontaneous rpoB mutant frequency was also examined in mutT/nth and mutT/nei strains, to assess the influence on the mutations induced by the endogenous 8-OH-dGTP accumulated in the mutT mutant. Exogenous 8-OH-dGTP increased the rpoB mutant frequency more efficiently in the nth strain than that in the wild-type strain. The spontaneous mutant frequency in the mutT/nth strain was 1.8-fold higher than that in the mutT strain. These results suggest that E. coli endonuclease III also acts as a defense against the mutations caused by 8-OH-dGTP in the nucleotide pool.
Assuntos
Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA , Nucleotídeos de Desoxiguanina/metabolismo , Escherichia coli/genética , Mutagênese , 8-Hidroxi-2'-Desoxiguanosina , DNA Glicosilases/genética , DNA Glicosilases/fisiologia , Desoxirribonuclease (Dímero de Pirimidina)/genética , Desoxirribonuclease (Dímero de Pirimidina)/fisiologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Genes BacterianosRESUMO
The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylase-deficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions.