Global genome removal of thymine glycol in Escherichia coli requires endonuclease III but the persistence of processed repair intermediates rather than thymine glycol correlates with cellular sensitivity to high doses of hydrogen peroxide.
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.
Alanazi
Mohammed
M
Department of Biochemistry and Molecular Biology, University of Kentucky, Lexington, KY 40536, USA.
Leadon
Steven A
SA
Mellon
Isabel
I
D016923:Cell Death / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D016680:Genome, Bacterial; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007700:Kinetics; D013696:Temperature; D013941:Thymine / Q000031:analogs & derivatives* / Q000378:metabolism*
D029968:Escherichia coli Proteins; C029389:thymine glycol; D006861:Hydrogen Peroxide; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; D013941:Thymine