phi29 DNA聚合酶中的一个特定亚结构域赋予了持续性和链置换能力。
A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity.
作者信息
Rodríguez Irene, Lázaro José M, Blanco Luis, Kamtekar Satwik, Berman Andrea J, Wang Jimin, Steitz Thomas A, Salas Margarita, de Vega Miguel
机构信息
Instituto de Biología Molecular Eladio Viñuela, Consejo Superior de Investigaciones Científicas, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Canto Blanco, 28049 Madrid, Spain.
出版信息
Proc Natl Acad Sci U S A. 2005 May 3;102(18):6407-12. doi: 10.1073/pnas.0500597102. Epub 2005 Apr 21.
Recent crystallographic studies of phi29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a phi29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of phi29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.
近期对phi29 DNA聚合酶的晶体学研究为其链置换和持续合成能力提供了结构上的见解。一个名为末端蛋白区域2（TPR2）的特定插入序列，仅存在于蛋白引发的DNA聚合酶中，与核酸外切酶、拇指和手掌亚结构域一起，形成了两个能够与DNA相互作用的环面。为了分析该插入序列的功能作用，我们构建了一个缺失TPR2氨基酸残基Asp-398至Glu-420的phi29 DNA聚合酶缺失突变体。对该突变体DNA聚合酶的生化分析表明，其DNA结合能力减弱，显著降低了其持续合成能力。此外，去除TPR2插入序列消除了phi29 DNA聚合酶进行与DNA合成偶联的链置换的内在能力。因此，此处描述的生化结果直接证明TPR2在链置换和持续合成能力中起关键作用。
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