The gene for the mismatch-specific uracil DNA glycosylase (MUG) was identified 
in the Escherichia coli genome as a sequence homolog of the human thymine DNA 
glycosylase with activity against mismatched uracil base pairs. Examination of 
cell extracts led us to detect a previously unknown xanthine DNA glycosylase 
(XDG) activity in E. coli. DNA glycosylase assays with purified enzymes 
indicated the novel XDG activity is attributable to MUG. Here, we report a 
biochemical characterization of xanthine DNA glycosylase activity in MUG. The 
wild type MUG possesses more robust activity against xanthine than uracil and is 
active against all xanthine-containing DNA (C/X, T/X, G/X, A/X and 
single-stranded X). Analysis of potentials of mean force indicates that the 
double-stranded xanthine base pairs have a relatively narrow energetic 
difference in base flipping, whereas the tendency for uracil base flipping 
follows the order of C/U > G/U > T/U > A/U. Site-directed mutagenesis performed 
on conserved motifs revealed that Asn-140 and Ser-23 are important determinants 
for XDG activity in E. coli MUG. Molecular modeling and molecular dynamics 
simulations reveal distinct hydrogen-bonding patterns in the active site of E. 
coli MUG that account for the specificity differences between E. coli MUG and 
human thymine DNA glycosylase as well as that between the wild type MUG and the 
Asn-140 and Ser-23 mutants. This study underscores the role of the favorable 
binding interactions in modulating the specificity of DNA glycosylases.