Separating Substrate Recognition from Base Hydrolysis in Human Thymine DNA Glycosylase by Mutational Analysis

Abstract

Human thymine DNA glycosylase (TDG) was discovered as an enzyme that can initiate base excision repair at sites of 5-methylcytosine-or cytosine deamination in DNA by its ability to release thymine or uracil from G⅐T and G⅐U mismatches. Crystal structure analysis of an Escherichia coli homologue identified conserved amino acid residues that are critical for its substrate recognition/interaction and base hydrolysis functions. Guided by this revelation, we performed a mutational study of structure function relationships with the human TDG. Substitution of the postulated catalytic site asparagine with alanine (N140A) resulted in an enzyme that bound mismatched substrates but was unable to catalyze base removal. Mutation of Met-269 in a motif with a postulated role in protein-substrate interaction selectively inactivated stable binding of the enzyme to mismatched substrates but not so its glycosylase activity. These results establish that the structure function model postulated for the E. coli enzyme is largely applicable to the human TDG. We further provide evidence for G⅐U being the preferred substrate of TDG, not only at the mismatch recognition step of the reaction but also in base hydrolysis, and for the importance of stable complementary strand interactions by TDG to compensate for its comparably poor hydrolytic potential.DNA of all organisms is susceptible to modification and damage through the action of a variety of exogenous and endogenous reagents. A prominent form of spontaneous damage arises through hydrolytic deamination of bases carrying exocyclic amino groups such as cytosine and 5-methylcytosine. Deamination of cytosine in double-stranded DNA (dsDNA) 1 generates a uracil⅐guanine mispair and, similarly, deamination of 5-methycytosine generates a thymine⅐guanine mispair. In vitro, both events occur at appreciable rates, with 5-methylcytosine deamination being slightly faster than that of cytosine (1) and, in vivo, both deamination products are mutagenic and will produce C3 T transitions upon DNA replication, if left unrepaired. Whereas accurate repair of G⅐U mispairs is mediated by enzymes that specifically recognize and process uracil in DNA, e.g. uracil DNA glycosylase (UDG) (2), correction of G⅐T mispairs to G⅐C base pairs requires a repair function that is able to discriminate between a mutagenic thymine in a G⅐T mismatch and a normal thymine base-paired with adenine.A G⅐T mismatch-specific thymine glycosylase activity was discovered in HeLa cell extracts (3). It was purified to apparent homogeneity (4), and the encoding cDNA was cloned (5). The biochemical properties of this thymine DNA glycosylase (TDG) are compatible with a function of the enzyme in cellular defense against mutagenesis by cytosine and 5-methylcytosine deamination. It is capable of recognizing G⅐T and G⅐U mismatches in DNA and initiating their restoration to G⅐C base pairs through a base excision repair process involving DNA polymerase ␤ (6 -8). Two bacterial open reading frames with significant homology to the central part of hum...

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

“…A key question regarding the catalytic mechanism of DNA glycosylases is how they bind and position the nucleophilic water molecule. Previous structural studies indicated that Asn18 of Escherichia coli MUG coordinates the nucleophile (33,34), and biochemical studies showed that the corresponding residue in TDG, Asn140, is essential for G·T glycosylase activity, consistent with a major role in catalysis (19,35). Although a mechanism of nucleophile binding involving the catalytic Asn was proposed for MUG and TDG enzymes (33), it has remained unsubstantiated because a putative nucleophilic water molecule has not been directly observed in any of the existing DNA-bound structures of TDG or MUG enzymes (29,33,34).…”

Uracil incorporation into genomic DNA does not predict toxicity caused by chemotherapeutic inhibition of thymidylate synthase

“…The influence of UDG activity on sensitivity to thymidylate deprivation in S. cerevisiae appeared to be transient [24,26]. S. cerevisiae lack SMUG1, MBD4, and TDG homologues [27], all of which are reported to remove uracil and 5-FU at least in vitro [8,10,11]. The absence of APN1, the major AP endonuclease in S. cerevisiae, leads to heightened sensitivity to 5-FU or antifolates [24,25].…”

“…It was shown that the SMUG1 DNA glycosylase can remove 5-FU from DNA and that this activity protects MEFs from 5-FU toxicity [8]. Interpreting the causes of 5-FU toxicity is complicated by the fact that 5-FU incorporated into DNA can be recognized by mismatch repair [9], and two additional DNA glycosylases of BER, namely TDG and MBD4 [10,11]. Thus, the precise role of BER during thymidylate deprivation remains unclear.…”

Uracil in DNA – occurrence, consequences and repair

“…The rate-limiting step in the mechanism in vitro is the release of the product (e.g. G : AP-site) due to rigid hydrogen bonding interactions between the enzyme and the Watson-Crick face of the guanine opposite the AP-site (Hardeland et al, 2000). This shields the cytotoxic APsite until the next enzyme in the pathway, APendonuclease APE1 (also called HAP1) enters.…”