Uracil-DNA glycosylase

Uracil-DNA glycosylase, also known as UNG or UDG, is a human is_associated_with::gene though is_associated_with::orthologs exist ubiquitously among prokaryotes and eukaryotes and even in some DNA viruses. The first uracil DNA-glycosylase was isolated from Escherichia coli.

Structure
UDG is made of a four-stranded parallel is_associated_with::β-sheet surrounded by eight is_associated_with::α-helices. The is_associated_with::active site comprises five highly conserved motifs that collectively catalyze is_associated_with::glycosidic bond cleavage:

1) Water-activating loop: 63-QDPYH-67

2) Pro-rich loop: 165-PPPPS-169

3) Uracil-binding motif: 199-GVLLLN-204

4) Gly-Ser loop: 246-GS-247

5) Minor groove intercalation loop: 268-HPSPLS-273

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 is_associated_with::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 is_associated_with::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. is_associated_with::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 is_associated_with::thymine C5 is_associated_with::methyl group, while a specific is_associated_with::hydrogen bond between the uracil O2 is_associated_with::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 is_associated_with::nucleophile and protonate the uracil is_associated_with::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 is_associated_with::aspartic acid and is_associated_with::histidine residues are consistent across catalytic studies.

Interactions
Uracil-DNA glycosylase has been shown to interact with is_associated_with::RPA2.