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Uracil-DNA glycosylase (UNG) is the key enzyme responsible for initiation of base excision repair. We have used both kinetic and binding assays for comparative analysis of UNG enzymes from humans and herpes simplex virus type 1 (HSV-1). Steady-state fluorescence assays showed that hUNG has a much higher specificity constant (k(cat)/K(m)) compared with the viral enzyme due to a lower K(m). The binding of UNG to DNA was also studied using a catalytically inactive mutant of UNG and non-cleavable substrate analogs (2'-deoxypseudouridine and 2'-alpha-fluoro-2'-deoxyuridine). Equilibrium DNA binding revealed that both human and HSV-1 UNG enzymes bind to abasic DNA and both substrate analogs more weakly than to uracil-containing DNA. Structure determination of HSV-1 D88N/H210N UNG in complex with uracil revealed detailed information on substrate binding. Together, these results suggest that a significant proportion of the binding energy is provided by specific interactions with the target uracil. The kinetic parameters for human UNG indicate that it is likely to have activity against both U.A and U.G mismatches in vivo. Weak binding to abasic DNA also suggests that UNG activity is unlikely to be coupled to the subsequent common steps of base excision repair.

Original publication




Journal article


J Biol Chem

Publication Date





4983 - 4992


Anisotropy, Binding Sites, Binding, Competitive, Biochemistry, Catalysis, Crystallography, X-Ray, DNA, Databases, Protein, Herpesvirus 1, Human, Humans, Kinetics, Mutation, Plasmids, Protein Binding, Recombinant Proteins, Sensitivity and Specificity, Species Specificity, Spectrometry, Fluorescence, Substrate Specificity, Thermodynamics, Uracil-DNA Glycosidase