DNA N-glycosidases: properties of uracil-DNA glycosidase from Escherichia coli.

Abstract

Uracil-DNA glycosidase, an enzyme that catalyzes the release of free uracil from uracil-containing DNA, has been purified ll,OOO-fold from Escherichia coli cell extracts. The enzyme preparation was essentially homogenous, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of a molecular weight of 24,500 + 1,000. The enzyme efficiently releases uracil from single-stranded DNA and also from double-stranded DNA with uracil residues hydrogen-bonded to either adenine or guanine residues. On the other hand, DNA molecules containing 5-bromouracil residues, pyrimidine dimers, or deaminated purine residues are not substrates. Uracil-DNA glycosidase does not cleave free dUMP at a detectable rate and shows little activity with uracil-containing oligonucleotides. It has no cofactor dependence and apparently acts by hydrolytic cleavage of the base-sugar bonds in dUMP residues, as there is no incorporation of phosphate or pyrimidines in the DNA when uracil is released. When the enzyme was incubated with covalently closed circular DNA containing a small number of uracil residues introduced by deamination of cytosine residues with bisulfite, alkali-labile sites, but no chain breaks were introduced in the DNA molecules. Such DNA molecules could subsequently be cleaved by an endonuclease that specifically attacks DNA at apurinic and apyrimidinic sites, E. coli endonuclease IV. These data indicate that uracil-DNA glycosidase functions in the repair of DNA containing accidentally introduced uracil residues.