To maintain genomic integrity, DNA repair enzymes continually remove damaged 
bases and lesions resulting from endogenous and exogenous processes. These 
repair enzymes must distinguish damaged bases from normal bases to prevent the 
inadvertent removal of normal bases, which would promote genomic instability. 
The mechanisms by which this high level of specificity is accomplished are as 
yet unresolved. One member of the uracil-DNA glycosylase family of repair 
enzymes, Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug), is 
reported to distinguish U:G mispairs from U:A base pairs based upon specific 
contacts with the mispaired guanine after flipping the target uracil out of the 
duplex. However, recent studies suggest other mechanisms for base selection, 
including local duplex stability. In this study, we used the modified base 
N6-methyladenine to probe the effect of local helix perturbation on Mug 
recognition of uracil. N6-Methyladenine is found in E. coli as part of both the 
mismatch repair and restriction-modification systems. In its cis isomer, 
N6-methyladenine destabilizes hydrogen bonding by interfering with 
pseudo-Watson-Crick base pairing. It is observed that the selection of uracil by 
Mug is sequence dependent and that uracil residues in sequences of reduced 
thermostability are preferentially removed. The replacement of adenine by 
N6-methyladenine increases the frequency of removal of the uracil residue paired 
opposite the modified adenine. These results are in accord with suggestions that 
local helix stability is an important determinant of base recognition by some 
DNA repair enzymes and provide a potential strategy for identifying the sequence 
location of modified bases in DNA.