Serrano-Heras Gemma, Bravo Alicia, Salas Margarita
Instituto de Biología Molecular Eladio Viñuela, Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma, Nicolás Cabrera 1, Canto Blanco, 28049 Madrid, Spain.
Proc Natl Acad Sci U S A. 2008 Dec 9;105(49):19044-9. doi: 10.1073/pnas.0808797105. Epub 2008 Oct 9.
Protein p56 encoded by the Bacillus subtilis phage phi29 inhibits host uracil-DNA glycosylase (UDG) activity. In previous studies, we suggested that this inhibition is likely a defense mechanism developed by phage phi29 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 phi29 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 phi29 DNA amplification system, we found that phi29 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 phi29-infected cells revealed that uracil residues arise in phi29 DNA during replication, probably as a result of misincorporation of dUMP by the phi29 DNA polymerase. On the other hand, the action of UDG on uracil-containing phi29 DNA impaired in vitro viral DNA replication, which was prevented by the presence of protein p56. Furthermore, transfection activity of uracil-containing phi29 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 phi29 genome.
枯草芽孢杆菌噬菌体 phi29 编码的蛋白质 p56 可抑制宿主尿嘧啶 - DNA 糖基化酶（UDG）的活性。在先前的研究中，我们提出这种抑制作用可能是噬菌体 phi29 形成的一种防御机制，以防在病毒 DNA 复制过程中，由于胞嘧啶脱氨基或 dUMP 掺入导致 DNA 中出现尿嘧啶化时 UDG 发挥作用。在这项工作中，我们分析了 phi29 DNA 聚合酶将 dUMP 插入 DNA 的能力。引物延伸分析表明，病毒 DNA 聚合酶在 dA 对面掺入 dU 的催化效率仅比掺入 dT 低 2 倍。使用 phi29 DNA 扩增系统，我们发现 phi29 DNA 聚合酶也能够延伸 dA:dUMP 碱基对并复制通过尿嘧啶。此外，对从 phi29 感染细胞中分离的病毒 DNA 进行 UDG 和脱嘌呤 - 脱嘧啶内切核酸酶处理后发现，在复制过程中 phi29 DNA 中会出现尿嘧啶残基，这可能是由于 phi29 DNA 聚合酶错误掺入 dUMP 所致。另一方面，UDG 对含尿嘧啶的 phi29 DNA 的作用会损害体外病毒 DNA 复制，而蛋白质 p56 的存在可防止这种情况发生。此外，含尿嘧啶的 phi29 DNA 在组成型合成 p56 的细胞中的转染活性明显高于缺乏该蛋白质的细胞。因此，我们的数据支持一种模型，即蛋白质 p56 确保了高效的病毒 DNA 复制，防止了 UDG 在消除 phi29 基因组中存在的尿嘧啶残基时所造成的有害影响。
