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

Abstract

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.

Lesion processing by a repair enzyme is severely curtailed by residues needed to prevent aberrant activity on undamaged DNA

“…The MD results are also consistent with our previous findings that nucleotide flipping is destabilized for G·T relative to G·U substrates (17,19,25,26). The MD simulations suggest a transient flipping intermediate that is substantially populated for TDG with G·T but not G·U substrates.…”

“…The results indicate that Ala145 destabilizes nucleotide flipping for dT (but not dU) via steric hindrance, as suggested by previous studies (17,19,25,26). In addition, our findings suggest that His151 forms a repulsive electrostatic interaction with thymine (or uracil) that slows the chemical step of the reaction.…”

“…Previous studies indicate that these backbone interactions could be important for retaining the flipped nucleotide in the active site, and they may contribute to the chemical step by stabilizing negative charge that develops on O2 and O4 upon cleavage of the basesugar bond (39,40). Notably, the interactions formed with O2 could potentially stabilize the flipping and/or excision of fC, caC, and other cytosine analogs (6,25). As shown in Fig.…”

“…Previous studies indicate that steric hindrance limits the active-site access of thymine and other bases with a bulky C-5 substituent (i.e., 5-BrUra), and that steric effects largely account for the much weaker binding and base-excision activity for G·T relative to G·U mispairs (11,17,19,25). However, residue(s) that could potentially impose this steric hindrance were unknown.…”

“…A mechanism for limiting thymine excision could be needed if the capacity of TDG to discriminate between G·T and A·T pairs, which is approximately 10 4.3 -fold (17), is not sufficient to allow for highly efficient G·T repair in the absence of aberrant A·T activity. Consistent with this possibility, our previous studies strongly suggest that steric hindrance involving the methyl of thymine weakens substrate binding and slows base excision for G·T mispairs (11,17,19,25,26), but direct structural evidence of such a mechanism was lacking. We address these and other questions regarding the basis of TDG specificity and catalysis here, using a combination of structural, biochemical, and computational approaches.…”