Specificity of Human Thymine DNA Glycosylase Depends on N-Glycosidic Bond Stability

Initiating the DNA base excision repair pathway, DNA glycosylases find and hydrolytically excise damaged bases from DNA. While some DNA glycosylases exhibit narrow specificity, others remove multiple forms of damage. Human thymine DNA glycosylase (hTDG) cleaves thymine from mutagenic G·T mispairs, recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the million-fold excess of normal G·C pairs over G·T mispairs. The mechanism of this remarkable and essential specificity has remained obscure. Here, we examine the possibility that hTDG specificity depends on the stability of the scissile base−sugar bond by determining the maximal activity (kmax) against a series of nucleobases with varying leaving-group ability. We find that hTDG removes 5-fluorouracil 78-fold faster than uracil, and 5-chlorouracil, 572-fold faster than thymine, differences that can be attributed predominantly to leaving-group ability. More...