Uracil-DNA Glycosylase - Mechanism

Mechanism

Glycosidic bond cleavage follows a “pinch-push-pull” mechanism using the five conserved motifs.

Pinch: UDG scans DNA for uracil by nonspecifically binding to the strand and creating a kink in the backbone, thereby positioning the selected base for detection. The Pro-rich and Gly-Ser loops form polar contacts with the 3’ and 5’ phosphates flanking the examined base. This compression of the DNA backbone, or “pinch,” allows for close contact between UDG and base of interest.

Push: To fully assess the nucleotide identity, the intercalation loop penetrates, or pushes into, the DNA minor groove and induces a conformational change to flip the nucleotide out of the helix. Backbone compression favors eversion of the now extrahelical nucleotide, which is positioned for recognition by the uracil-binding motif. The coupling of intercalation and eversion helps compensate for the disruption of favorable base stacking interactions within the DNA helix. Leu272 fills the void left by the flipped nucleotide to create dispersion interactions with neighboring bases and restore stacking stability.

Pull: Now accessible to the active site, the nucleotide interacts with the uracil binding motif. The active site shape complements the everted uracil structure, allowing for high substrate specificity. Purines are too large to fit in the active site, while unfavorable interactions with other pyrimidines discourage binding alternative substrates. The side chain of Tyr147 interferes sterically with the thymine C5 methyl group, while a specific hydrogen bond between the uracil O2 carbonyl and Gln144 discriminates against a cytosine substrate, which lacks the necessary carbonyl. Once uracil is recognized, cleavage of the glycosidic bond proceeds according to the mechanism below.

The position of the residues that activate the water nucleophile and protonate the uracil leaving group are widely debated, though the most commonly followed mechanism employs the water activating loop detailed in the enzyme structure. Regardless of position, the identities of the aspartic acid and histidine residues are consistent across catalytic studies.