Excision of 5-Halogenated Uracils by Human Thymine DNA Glycosylase

Abstract

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...

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.…”

“…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.…”

“…These enzymes face the formidable task of removing a normal base, thymine, from G·T mispairs but not from A·T base pairs. Previous studies show that TDG activity is weak for G·T mispairs compared with most in vitro substrates (e.g., G·U), even though G·T mispairs are an important biological substrate for TDG (8,9,(16)(17)(18)(19). This might reflect a mechanism used by TDG to strike a balance between the requirement for efficient repair of mutagenic G·T lesions and the need for avoiding aberrant removal of T from A·T pairs, which can be mutagenic and cytotoxic (13,20,21).…”

“…Notably, such a compromise in G·T repair activity could account in part for the high frequency of C→T transitions at CpG sites in cancer and genetic disease (20,(22)(23)(24). 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.…”

“…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.…”