Uracil-DNA glycosylase inhibitor (Ugi) is a B. subtilis bacteriophage protein that protects the uracil-containing phage DNA by irreversibly inhibiting the key DNA repair enzyme uracil-DNA glycosylase (UDG). The 1.9 A crystal structure of Ugi complexed to human UDG reveals that the Ugi structure, consisting of a twisted five-stranded antiparallel beta sheet and two alpha helices, binds by inserting a beta strand into the conserved DNA-binding groove of the enzyme without contacting the uracil specificity pocket. The resulting interface, which buries over 1200 A2 on Ugi and involves the entire beta sheet and an alpha helix, is polar and contains 22 water molecules. Ugi binds the sequence-conserved DNA-binding groove of UDG via shape and electrostatic complementarity, specific charged hydrogen bonds, and hydrophobic packing enveloping Leu-272 from a protruding UDG loop. The apparent mimicry by Ugi of DNA interactions with UDG provides both a structural mechanism for UDG binding to DNA, including the enzyme-assisted expulsion of uracil from the DNA helix, and a crystallographic basis for the design of inhibitors with scientific and therapeutic applications.

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Escherichia coli uracil-DNA glycosylase was shown to catalyze the hydrolysis of a site-specific uracil residue from a defined single-stranded oligonucleotide (25-mer). With duplex 25-mer, the rate of uracil removal from double-stranded DNA containing a U.G mispair was approximately 2-fold greater than a U.A base pair. The mechanism by which E. coli and rat liver mitochondrial uracil-DNA glycosylase located sequential uracil residues within double-stranded DNA was investigated. Two concatemeric polynucleotide substrates were constructed by ligation of homologous 5'-end 32P-labeled 25-mer double-stranded oligonucleotides that contained either a site-specific U.G or U.A target site at intervals of 25 nucleotides along one strand of the DNA. Reaction of uracil-DNA glycosylase with these concatemeric DNAs, followed by alkaline hydrolysis of the resultant AP-sites, would produce predominantly [32P]25-mer products, if a processive mechanism was used to locate successive uracil residues, or oligomeric multiples of [32P]25-mer, if a distributive mode was exhibited. Both the bacterial and the mitochondrial enzymes were found to act processively on U.A- and U.G-containing DNA in the absence of NaCl, based on the initial rate of 25-mer produced relative to the total amount of uracil excised. Approximately 50% of the total uracil excised resulted in the release of 25-mer product. The addition of NaCl (> or = 50 mM) caused reduced processivity on both U.A- and U.G-containing DNA substrates. The mode of action of uracil-DNA glycosylase was very similar to that observed for the EcoRI endonuclease cleavage of restriction sites contained in the same DNA substrate which was used as a positive control.

Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pullpush", as opposed to a "push-pull", mechanism for nucleotide flipping.