Uracil-DNA glycosylase (UDG) is an enzyme involved in the base excision repair pathway. It specifically removes uracil from both single-stranded and double-stranded DNA. The genome of the Bacillus subtilis phage 29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5-end. Replication of 29 DNA starts by a protein-priming mechanism and generates intermediates that have long stretches of single-stranded DNA. By using in vivo chemical cross-linking and affinity chromatography techniques, we found that UDG is a cellular target for the early viral protein p56. Addition of purified protein p56 to B. subtilis extracts inhibited the endogenous UDG activity. Moreover, extracts from 29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by 29 to prevent the action of the base excision repair pathway if uracil residues arise in their replicative intermediates. Protein p56 is the first example of a UDG inhibitor encoded by a nonuracil-containing viral DNA.Uracil in DNA may arise from spontaneous deamination of cytosine and from the occasional use of dUTP instead of dTTP during DNA replication. Hydrolytic deamination of cytosine generates G:U mismatches that cause G:C to A:T transition mutations. To maintain the genome integrity, most prokaryotic and eukaryotic cells rapidly eliminate uracil from DNA by the base excision repair (BER) 2 pathway, which is initiated by the uracil-DNA glycosylase (UDG) enzyme. UDGs have an unusually broad phylogenetic distribution. Some DNA viruses, such as herpesviruses and poxviruses, also encode a UDG activity, whereas the human immunodeficiency virus, type 1, packages cellular UDG (UNG2 enzyme) into virus particles. In these instances, the UDG activity appears to have an important role in virus replication (1).The UDG enzyme hydrolyzes the N-glycosidic bond between the uracil residue and the deoxyribose sugar of the DNA backbone, generating an apurinic-apyrimidinic (AP) site. The first UDG activity reported was purified from Escherichia coli cells (2). Since then, enzymes highly homologous to the archetypal E. coli UDG have been identified in numerous organisms, including human cells (Family-1 UDGs) (3). Studies on substrate specificity showed that Family-1 UDGs efficiently remove uracil residues from both single-stranded and double-stranded DNAs, often with preference for the single-stranded substrates (4, 5). The AP site generated by the UDG enzyme is further recognized by an AP endonuclease, which cleaves the phosphodiester bond of the DNA backbone 5Ј to the AP site. Several AP endonucleases are active not only on double-stranded DNAs but also on singlestranded DNAs (6 -8). Further repair can be accomplished via two pathways that involve different subsets of enzymes and result in replacement of one (short patch pathway) or more (long patch pathway) nucleotides (9).Over the course of evolution, bacteriophages have developed unique proteins that bind to and inactivate critical cellula...