Cytosine deamination and the misincorporation of 2-dUrd into DNA during replication result in the presence of uracil in DNA. Uracil-DNA glycosylases (UDGs) initiate the excision repair of this aberrant base by catalyzing the hydrolysis of the N-glycosidic bond. UDGs are expressed by nearly all known organisms, including some viruses, in which the functional role of the UDG protein remains unresolved. This issue could in principle be addressed by the availability of designed synthetic inhibitors that target the viral UDG without affecting the endogenous human UDG. Here, we report that double-stranded and single-stranded oligonucleotides incorporating either of two dUrd analogs tightly bind and inhibit the activity of herpes simplex virus type-1 (HSV-1) UDG. Both inhibitors are exquisitely specific for the HSV-1 UDG over the human UDG. These inhibitors should prove useful in structural studies aimed at understanding substrate recognition and catalysis by UDGs, as well as in elucidating the biologic role of UDGs in the life cycle of herpesviruses.Uracil-DNA glycosylases (UDGs) 1 are a highly conserved class of DNA repair enzymes that initiates excision repair of uracil in DNA by hydrolyzing the N-glycosidic bond (Fig. 1a). Uracil in DNA arises from the misincorporation of deoxyuridine triphosphate (dUTP) during replication and from the hydrolytic deamination of cytosine (1, 2). Cytosine deamination generates highly mutagenic G:U mismatches that lead to G:C to A:T transition mutations. Characterized by a high substrate specificity, UDGs are able to distinguish between such structurally similar bases as uracil and thymine. Unlike other DNA glycosylases, UDGs remove uracil from both single-stranded and double-stranded DNA, often with higher efficiency for the single-stranded substrates (1, 3).DNA glycosylases accomplish the formidable task of scanning a large excess of normal DNA bases, recognizing and then catalyzing the excision of damaged bases. A molecular-level description of how DNA glycosylases achieve recognition and repair requires the formation of stable protein-substrate complexes that can be studied by x-ray crystallography or NMR. Such studies are not ordinarily possible because of the transient nature of the enzyme-substrate interaction. One way to circumvent this problem involves mutating the enzyme so as to obtain a catalytically inactive form that retains the ability to recognize its substrate (4,5). An alternative approach is in effect to "mutate" the substrate by preparing synthetic analogs that bind the enzyme specifically but cannot be processed by it (6 -8). Both approaches have proven useful in structural and biochemical analysis of base excision DNA repair (4 -12). Powerful features of the substrate modification approach are that it requires no prior knowledge of the enzyme active site and that it can be structurally very conservative, entailing changes of as little as a single atom.UDGs have an unusually broad phylogenetic distribution, being present in organisms from the simplest free-stand...
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