A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity
A specific subdomain in φ29 DNA polymerase confers both processivity and strand-displacement capacity
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DOI：
10.1073/pnas.0500597102
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发表时间：
2005-05-03
影响因子：
11.1
通讯作者：
de Vega, M
中科院分区：
文献类型：
--
作者：
Rodríguez, I;Lázaro, JM;de Vega, M
最近对phi 29DNA聚合酶的结晶学研究提供了对其链移位和加工能力的结构洞察。一个称为末端蛋白区域2(TPR2)的特定插入，只存在于蛋白质启动的DNA聚合酶中，与核酸外切酶、拇指和手掌亚区一起形成两个能够与DNA相互作用的环状结构。为了分析这个插入的功能，我们构建了一个缺失TPR2氨基酸残基Asp-398到Glu-420的phi 29DNA聚合酶缺失突变体。对突变的DNA聚合酶的生化分析表明，其与DNA的结合能力减弱，大大降低了其加工能力。此外，TPR2插入的去除取消了phi 29DNA聚合酶执行与DNA合成耦合的链置换的固有能力。因此，这里描述的生化结果直接证明了TPR2在链转移和加工中起着关键作用。
Recent crystallographic studies of phi 29 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 phi 29 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 phi 29 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.