A Uracil-DNA Glycosylase Inhibitor Encoded by a Non-uracil Containing Viral DNA

Abstract

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...

Phage φ29 protein p56 prevents viral DNA replication impairment caused by uracil excision activity of uracil-DNA glycosylase

“…To study the in vivo role of protein p56, we used the same DNA preparations to transfect p56-producing cells to find out the effect of inhibiting the host UDG on the infectivity of uracilcontaining 29 DNA. These cells, which carry the recombinant plasmid pPR53.p56, constitutively synthesize protein p56 at levels only slightly lower than those detected at 40 min of phage infection (10). The results revealed that the infectivity of uracilcontaining DNAs increased substantially in B. subtilis cells whose UDG activity was inhibited because of the presence of p56, in comparison with p56 nonproducing cells (Fig.…”

“…We recently proposed that this inhibition is essential for an efficient infection, because the action of UDG on 29 ssDNA molecules that contain uracils would be deleterious for 29 DNA replication (10,11). To test this hypothesis, we have studied the consequences of uracil misincorporation on in vitro 29 DNA replication when UDG is present.…”

“…In earlier studies, we reported the identification of the first example of a UDG inhibitor (named protein p56) encoded by a nonuracil containing viral DNA (10,11). We proposed that inactivation of the host UDG by such inhibitor would ensure an efficient 29 DNA replication because it would avoid the deleterious effect produced by UDG on viral DNA synthesis if uracil residues arise in the single-stranded replicative intermediates.…”

“…Recently, we identified an inhibitor (protein p56) of the B. subtilis UDG encoded by bacteriophage 29 (10). In vitro experiments showed that protein p56 blocks the DNA-binding ability of UDG, probably by mimicking the structure of DNA (11).…”

“…Synthesis of the linear 29 DNA starts at both ends, where the replication origins are located, and proceeds by a strand-displacement mechanism, generating replicative intermediates with long stretches of ssDNA. We have suggested that inhibition of the host UDG by protein p56 is likely a defense mechanism developed by 29 and 29-related phages, which also encode p56-like proteins, to prevent the damaging action of the BER pathway if uracils arise in their replicative intermediates (10). The presence of these residues in the ssDNA intermediates, arising either by deamination of cytosine or misincorporation of dUMP during the previous replication round, could recruit components of the cellular BER pathway, such as UDGs and Apes.…”