RESUMO
Using a monoclonal antibody that specifically recognizes thymine glycol (Tg) in DNA, we measured the kinetics of the removal of Tg from the genomes of wild-type and repair gene mutant strains of Escherichia coli treated with hydrogen peroxide. Tg is rapidly and efficiently removed from the total genomes of repair-proficient cells in vivo and the removal of Tg is completely dependent on the nth gene that encodes the endonuclease III glycosylase. Hence, it appears that little redundancy in the repair of Tg occurs in vivo, at least under the conditions used here. Moreover, previous studies have found that nth mutants are not sensitive to killing by hydrogen peroxide but xth mutant strains (deficient in the major AP endonuclease, exonuclease III) are sensitive. We find that cell death correlates with the persistence of single-strand breaks rather than the persistence of Tg. We attempted to measure transcription-coupled removal of Tg in the lactose operon using the Tg-specific monoclonal antibody in an immunoprecipitation approach but were not successful in achieving reproducible results. Furthermore, the analysis of transcription-coupled repair in the lactose operon is complicated by potent inhibition of beta-galactosidase expression by hydrogen peroxide.
Assuntos
Reparo do DNA , Desoxirribonuclease (Dímero de Pirimidina) , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano , Peróxido de Hidrogênio/farmacologia , Timina/análogos & derivados , Timina/metabolismo , Morte Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Cinética , TemperaturaRESUMO
We previously described a sensitive assay for measuring thymine glycol in the DNA of irradiated cells. The assay combines immunorecognition of the DNA lesion with capillary electrophoresis and laser-fluorescence detection to achieve an absolute detection level in the zeptomole (10(-21) mol) range. This article describes modifications to the protocol that overcome certain technical problems seen with the original methodology. In particular, the capillary electrophoresis is carried out at pH 8.3 rather than pH 10.5. The new protocol was used to examine removal of thymine glycol from the DNA of A549 lung adenocarcinoma cells and resting lymphocytes after exposure to 2 Gy gamma rays. Both cell types displayed similar repair kinetics. Removal of thymine glycol is almost complete at 4 hours postirradiation.
