Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo.
The 3,N(4)-ethenocytosine (epsilon C) residue might have biological role in vivo since it is recognized and efficiently excised in vitro by the E. coli mismatch-specific uracil-DNA glycosylase (MUG) and the human thymine-DNA glycosylase (hTDG). In the present work we have generated mug defective mutant of E. coli by insertion of a kanamycin cassette to assess the role of MUG in vivo. We show that human TDG complements the enzymatic activity of MUG when expressed in a mug mutant. The epsilon C-DNA glycosylase defective strain did not exhibit spontaneous mutator phenotype and did not show unusual sensitivity to any of the following DNA damaging treatments: methylmethanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet light, H(2)O(2), paraquat. However, plasmid DNA damaged by 2-chloroacetaldehyde treatment in vitro was inactivated at a greater rate in a mug mutant than in wild-type host, suggesting that MUG is required for the in vivo processing of the ethenobases. In addition, 2-chloroacetaldehyde treatment induces preferentially G.C --> C.G and A.T --> T.A transversions in mug mutant. Comparison of the mutation frequencies induced by the site-specifically incorporated epsilon C residue in E. coli wild-type versus mug indicates that MUG repairs more than 80% of epsilon C residues in vivo. Furthermore, the results show that nucleotide excision repair and recombination are not involved in the processing of epsilon C in E. coli. Based on the mutagenesis data we suggest that epsilon C may be less toxic and less mutagenic than expected. The increased spontaneous mutation rate for G.C --> A.T transition in the ung mug double mutant as compared to the single ung mutant suggest that MUG may be a back-up repair enzyme to the classic uracil-DNA glycosylase.
Jurado
Juan
J
Groupe Réparation de l'AND, CNRS UMR 8113, LBPA-ENS Cachan, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France.
Maciejewska
Agnieszka
A
Krwawicz
Joanna
J
Laval
Jacques
J
Saparbaev
Murat K
MK
D000079:Acetaldehyde / Q000031:analogs & derivatives* / Q000494:pharmacology; D020137:Base Pair Mismatch / Q000235:genetics / Q000502:physiology*; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics / Q000502:physiology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D008826:Microbial Sensitivity Tests; D016254:Mutagenesis, Insertional / Q000235:genetics; D009153:Mutagens / Q000494:pharmacology; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000187:drug effects / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000235:genetics / Q000502:physiology*
C018289:3,N(4)-ethenocytosine; D009153:Mutagens; D003596:Cytosine; C004656:chloroacetaldehyde; D044502:Thymine DNA Glycosylase; C113921:mismatch-specific thymine uracil-DNA glycosylase; D000079:Acetaldehyde