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.