Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China; University of Chinese Academy of Sciences, Beijing, 100049, China.
Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, 201800, China.
Biochem Biophys Res Commun. 2019 Mar 19;510(4):573-579. doi: 10.1016/j.bbrc.2019.01.144. Epub 2019 Feb 7.
A novel DNA polymerase found in the deep-sea vent phage NrS-1, was confirmed to have both DNA polymerase and primase activities. In this polymerase, the N-terminal residues 1-300 (referred to as N300) are the core region required for polymerizing DNA and catalyzing de novo DNA synthesis. Here, the crystal structure of N300 was solved at a resolution of 1.80 Å. The overall structure consists of a prim/pol domain and a helix bundle domain, which are separated by a 14-residue-long flexible tether (residues 177-190). Both the prim/pol domain of N300 and other primase-polymerases (prim-pol) encompass an analogous fold with conserved catalytic residues. Mutagenesis and enzymatic activity assays show that the acidic active-site residue E139 is required for both polymerase and primase activities. Functional assays confirm the essentiality of the helix bundle domain for primase activity. Furthermore, we identified a mutant (N300-Y261A) of the helix bundle domain, which probably plays an indispensable role in the primer initiation and recognition of template DNA.
在深海喷口噬菌体 NrS-1 中发现的一种新型 DNA 聚合酶，被证实具有 DNA 聚合酶和引物酶活性。在该聚合酶中，N 端残基 1-300（称为 N300）是聚合 DNA 和催化从头 DNA 合成所必需的核心区域。在这里，N300 的晶体结构在 1.80 Å 的分辨率下得到解决。整体结构由一个引物/聚合酶域和一个螺旋束域组成，它们由一个 14 个残基长的柔性连接物（残基 177-190）隔开。N300 的 prim/pol 结构域和其他引物聚合酶（prim-pol）都包含类似的折叠结构，具有保守的催化残基。诱变和酶活性测定表明，酸性活性位点残基 E139 对于聚合酶和引物酶活性都是必需的。功能测定证实了螺旋束域对于引物酶活性的重要性。此外，我们还鉴定了螺旋束域的突变体（N300-Y261A），它可能在引物起始和模板 DNA 的识别中发挥不可或缺的作用。
