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 and recognizes many additional lesions, and has a strong preference for nucleobases paired with guanine rather than adenine. Yet, hTDG avoids cytosine, despite the millionfold 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 (k max ) 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. Moreover, hTDG readily excises cytosine analogues with improved leaving ability, including 5-fluorocytosine, 5-bromocytosine, and 5-hydroxycytosine, indicating that cytosine has access to the active site. A plot of log(k max ) versus leaving group pK a reveals a Brønsted-type linear free energy relationship with a large negative slope of β lg = −1.6 ± 0.2, consistent with a highly dissociative reaction mechanism. Further, we find that the hydrophobic active site of hTDG contributes to its specificity by enhancing the inherent differences in substrate reactivity. Thus, hTDG specificity depends on N-glycosidic bond stability, and the discrimination against cytosine is due largely to its very poor leaving ability rather than its exclusion from the active site.

Thymine DNA glycosylase (TDG) excises thymine from G⅐T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG⅐X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG⅐FU, CpG⅐ClU, and CpG⅐BrU than for CpG⅐T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k max ) for G⅐X substrates depends significantly on the 5 base pair. For example, k max decreases by 6-, 11-, and 82-fold for TpG⅐ClU, GpG⅐ClU, and ApG⅐ClU, respectively, as compared with CpG⅐ClU. For the other G⅐X substrates, the 5-neighbor effects have a similar trend but vary in magnitude. The activity for G⅐FU, G⅐ClU, and G⅐BrU, with any 5-flanking pair, meets and in most cases significantly exceeds the CpG⅐T activity. Strikingly, human TDG activity is reduced 10 2.3 -10 4.3 -fold for A⅐X relative to G⅐X pairs and reduced further for A⅐X pairs with a 5 pair other than C⅐G. The effect of altering the 5 pair and/or the opposing base (G⅐X versus A⅐X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.The nucleobases in DNA are subject to continuous chemical modification, generating a broad range of mutagenic and cytotoxic lesions that can lead to cancer and other diseases (1, 2). To counteract this inevitable damage, the cellular machinery includes systems for DNA repair (3). Damage occurring to the nucleobases is the purview of base excision repair, a pathway that is initiated by a damage-specific DNA glycosylase. These enzymes find damaged or mismatched bases within the vast expanse of normal DNA and catalyze the cleavage of the basesugar (N-glycosidic) bond, producing an abasic or apurinic/ apyrimidinic (AP) 2 site in the DNA. The repair process is continued by follow-on base excision repair enzymes.Human thymine DNA glycosylase (hTDG) was discovered as an enzyme that removes thymine from G⅐T and uracil from G⅐U mispairs in DNA (4, 5). In vertebrates, G⅐T mispairs arise from replication errors, which are handled by the mismatch repair pathway or from the deamination of 5-methylcytosine to T (6, 7). Because cytosine methylation occurs at CpG dinucleotides (8, 9), G⅐T mispairs caused by 5-methylcytosine deamination are found at CpG sites. It has been shown that hTDG is most active for G⅐T mispairs with a 5Ј C⅐G pair, suggesting that a predominant biological role of the enzyme is to initiate the repair of CpG⅐T lesions (10, 11). DNA methylation at CpG plays a fundamental role in many cellular processes, including transcriptional regulation and the silencing of repetitive genetic elements (8, 9). Sugg...

DNA Glycosylases hydrolytically excise damaged or mismatched bases from DNA. hTDG (human thymidine DNA glycosylase), is active against G·T mispairs and other lesions. We have shown that 5‐fluorouracil (FU), as well as its 5‐chlorouracil (ClU) and 5‐bromouracil (BrU) are excised much faster than the traditional G·T substrate (Bennett, M.T., et al JACS 128, –12519). Previous studies indicate that hTDG is specific for lesions paired with G and located at CpG sites. We investigated the contribution of the 5′‐base pair to hTDG activity using single turnover kinetics with substrates containing FU, ClU, and BrU lesions. For ClU, kmax was 10‐fold lower for GpG·ClU, 5‐fold lower for TpG·ClU, and 85‐fold lower for ApG·ClU as compared to CpG·ClU. Similar trends were observed for FU and BrU. Our findings indicate that hTDG is more active against FU·G, ClU·G, and BrU·G lesions in any DNA context than against CpG·T lesions. Thus, hTDG may offer general protection against ClU·G and BrU·G lesions, which may arise in DNA at sites of inflammation. However, the much slower activity for FU·A, ClU·A, and BrU·A pairs suggests a limited protective role for hTDG against these lesions.