Abstract
Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, initiates the uracil excision repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits UDGs by serving as a remarkable substrate mimic. Structure determination of UDGs has identified regions important for the exquisite specificity in the detection and removal of uracils from DNA and in their interaction with Ugi. In this study, we carried out mutational analysis of the Escherichia coli UDG at Leu191 within the 187HPSPLS192 motif (DNA intercalation loop). We show that with the decrease in side chain length at position 191, the stability of the UDG-Ugi complexes regresses. Further, while the L191V and L191F mutants were as efficient as the wild type protein, the L191A and L191G mutants retained only 10 and 1% of the enzymatic activity, respectively. Importantly, however, substitution of Leu191 with smaller side chains had no effect on the relative efficiencies of uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of UDG, and it contributes to the formation of a physiologically irreversible complex with Ugi. We also envisage a role for Leu191 in stabilizing the productive enzyme-substrate complex.
Highlights
Icated to the indomitable task of freeing the DNA from uracil residues [5, 6]
Our results suggest that leucine within the HPSPLS motif is crucial for the uracil excision activity of Uracil DNA glycosylase (UDG), and it contributes to the formation of a physiologically irreversible complex with Ugi
The mechanism of uracil excision that has emerged from various structural studies and mutational analyses of UDGs is that the glycosidic bond between uracil and the sugar is cleaved by the attack of a hydroxyl nucleophile on the deoxyribose C1Ј atom
Summary
Icated to the indomitable task of freeing the DNA from uracil residues [5, 6]. UDGs have been identified from a large number of prokaryotic and eukaryotic organisms, and these enzymes show a high degree of conservation from bacteria to viruses to humans [7, 8]. The major interactions, which render the complex between UDG and Ugi physiologically irreversible, are defined by (i) hydrogen bonding and packing contacts derived from the complementarity between the conserved Leu loop (187HPSPLS192) of E. coli UDG and eight hydrophobic residues of Ugi (Met, Val, Val, Ile, Val, Met, Leu, and Val71) in a cavity burrowed between the ␣2 and the antiparallel  sheet of Ugi and (ii) the electrostatic interactions between the acidic residues of the 1 edge of Ugi with the key active site residues of E. coli UDG This structure showed that the interaction between UDG and Ugi results in the burial of about 2200 Å2 of the total accessible surface area. During these studies, we have developed a novel urea-polyacrylamide gel system, which has allowed us to monitor the real time dissociation of UDG-
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