Gemma Serrano-Heras

Protein p56 from the Bacillus subtilis phage ϕ29 inhibits DNA-binding ability of uracil-DNA glycosylase

Protein p56 (56 amino acids) from the Bacillus subtilis phage ϕ29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of β-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry.

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

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 ϕ29 is a linear double-stranded DNA with a terminal protein covalently linked at each 5′-end. Replication of ϕ29 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 ϕ29-infected cells were deficient in UDG activity. We suggested that inhibition of the cellular UDG is a defense mechanism developed by ϕ29 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 non-uracil-containing viral DNA.

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

Protein p56 encoded by the Bacillus subtilis phage φ29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage φ29 to prevent the action of UDG if uracilation occurs in DNA either from deamination of cytosine or the incorporation of dUMP during viral DNA replication. In this work, we analyzed the ability of φ29 DNA polymerase to insert dUMP into DNA. Primer extension analysis showed that viral DNA polymerase incorporates dU opposite dA with a catalytic efficiency only 2-fold lower than that for dT. Using the φ29 DNA amplification system, we found that φ29 DNA polymerase is also able to carry out the extension of the dA:dUMP pair and replicate past uracil. Additionally, UDG and apurinic-apyrimidinic endonuclease treatment of viral DNA isolated from φ29-infected cells revealed that uracil residues arise in φ29 DNA during replication, probably as a result of misincorporation of dUMP by the φ29 DNA polymerase. On the other hand, the action of UDG on uracil-containing φ29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing φ29 DNA was significantly higher in cells that constitutively synthesized p56 than in cells lacking this protein. Thus, our data support a model in which protein p56 ensures an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracil residues present in the φ29 genome.

In Vivo Assembly of Phage ϕ29 Replication Protein p1 into Membrane-associated Multimeric Structures

The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown. In the case of the Bacillus subtilis phage ϕ29, the viral protein p1 enhances the rate of in vivo viral DNA replication. Previous work showed that p1 generates highly ordered structures in vitro. We now show that protein p1, like integral membrane proteins, has an amphiphilic nature. Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location. By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane. These structures exist both during viral DNA replication and when ϕ29 DNA synthesis is blocked due to the lack of viral replisome components. In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional. We propose that a phage structure assembled on the cell membrane provides a specific site for ϕ29 DNA replication.

Crystal structure and functional insights into uracil-DNA glycosylase inhibition by phage ϕ29 DNA mimic protein p56

Uracil-DNA glycosylase (UDG) is a key repair enzyme responsible for removing uracil residues from DNA. Interestingly, UDG is the only enzyme known to be inhibited by two different DNA mimic proteins: p56 encoded by the Bacillus subtilis phage ϕ29 and the well-characterized protein Ugi encoded by the B. subtilis phage PBS1/PBS2. Atomic-resolution crystal structures of the B. subtilis UDG both free and in complex with p56, combined with site-directed mutagenesis analysis, allowed us to identify the key amino acid residues required for enzyme activity, DNA binding and complex formation. An important requirement for complex formation is the recognition carried out by p56 of the protruding Phe191 residue from B. subtilis UDG, whose side-chain is inserted into the DNA minor groove to replace the flipped-out uracil. A comparative analysis of both p56 and Ugi inhibitors enabled us to identify their common and distinctive features. Thereby, our results provide an insight into how two DNA mimic proteins with different structural and biochemical properties are able to specifically block the DNA-binding domain of the same enzyme.

Novel dimeric structure of phage ϕ29-encoded protein p56: insights into uracil-DNA glycosylase inhibition

Protein p56 encoded by the Bacillus subtilis phage ϕ29 inhibits the host uracil-DNA glycosylase (UDG) activity. To get insights into the structural basis for this inhibition, the NMR solution structure of p56 has been determined. The inhibitor defines a novel dimeric fold, stabilized by a combination of polar and extensive hydrophobic interactions. Each polypeptide chain contains three stretches of anti-parallel β-sheets and a helical region linked by three short loops. In addition, microcalorimetry titration experiments showed that it forms a tight 2:1 complex with UDG, strongly suggesting that the dimer represents the functional form of the inhibitor. This was further confirmed by the functional analysis of p56 mutants unable to assemble into dimers. We have also shown that the highly anionic region of the inhibitor plays a significant role in the inhibition of UDG. Thus, based on these findings and taking into account previous results that revealed similarities between the association mode of p56 and the phage PBS-1/PBS-2-encoded inhibitor Ugi with UDG, we propose that protein p56 might inhibit the enzyme by mimicking its DNA substrate.

Characterization of Bacillus subtilis uracil-DNA glycosylase and its inhibition by phage φ29 protein p56

Uracil‐DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram‐positive organism. The purified enzyme removes uracil preferentially from single‐stranded DNA over double‐stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp‐65 and His‐187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe‐78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29‐related phage, GA‐1, encodes a p56‐like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe‐191 of B. subtilis UDG is critical for the interaction with φ29 and GA‐1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.

Role of host factors in bacteriophage φ29 DNA replication.

During the course of evolution, viruses have learned to take advantage of the natural resources of their hosts for their own benefit. Due to their small dimension and limited size of genomes, bacteriophages have optimized the exploitation of bacterial host factors to increase the efficiency of DNA replication and hence to produce vast progeny. The Bacillus subtilis phage φ29 genome consists of a linear double-stranded DNA molecule that is duplicated by means of a protein-primed mode of DNA replication. Its genome has been shown to be topologically constrained at the size of the bacterial nucleoid and, as to avoid generation of positive supercoiling ahead of the replication forks, the bacterial DNA gyrase is used by the phage. In addition, the B. subtilis actin-like MreB cytoskeleton plays a crucial role in the organization of φ29 DNA replication machinery in peripheral helix-like structures. Thus, in the absence of an intact MreB cytoskeleton, φ29 DNA replication is severely impaired. Importantly, MreB interacts directly with the phage membrane protein p16.7, responsible for attaching φ29 DNA at the cell membrane. Moreover, the φ29-encoded protein p56 inhibits host uracil-DNA glycosylase activity and has been proposed to be a defense mechanism developed by the phage to prevent the action of the base excision repair pathway if uracil residues arise in replicative intermediates. All of them constitute incoming examples on how viruses have profited from the cellular machinery of their hosts.