Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 105, Issue 49, Pages 19044-19049Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0808797105
Keywords
phi 29 DNA polymerase; DNA repair; protein-primed replication; dUMP incorporation
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Funding
- Spanish Ministry of Education and Science [BFU2005-00733/BMC]
- Consolider-Ingenio [24717]
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Protein p56 encoded by the Bacillus subtilis phage phi 29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage phi 29 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 phi 29 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 phi 29 DNA amplification system, we found that phi 29 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 phi 29-infected cells revealed that uracil residues arise in phi 29 DNA during replication, probably as a result of misincorporation of dUMP by the phi 29 DNA polymerase. On the other hand, the action of UDG on uracil-containing phi 29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing phi 29 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 phi 29 genome.
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