Department of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, N6A 5C1, Canada.
Sci Rep. 2018 Oct 11;8(1):15125. doi: 10.1038/s41598-018-33371-5.
Human polymerase kappa (polκ) is a distinct Y-family DNA polymerase with a unique N-terminal N-clasp domain. The N-clasp renders polκ's high efficiency and accuracy in DNA replication and lesion bypass. How N-clasp empowers polκ in replication remains unclear due to the disordering of N-clasp. Here, we present a 2.0-Å resolution crystal structure of a polκ ternary complex with DNA and an incoming nucleotide. The structure-function study reveals an ordered N-clasp domain that brings conserved and functionally important residues in contact with the replicating basepair in the active site and contributes to the nucleotidyl transfer reaction. Particularly, a fully ordered Lys25 from the N-clasp domain is in H-bonding with the α- and γ-phosphates of the incoming nucleotide. K25A mutation reduces the polymerase activity of polκ significantly. This lysine is structurally analogous to a conserved lysine in the A-family DNA polymerases in the closed form. In contrast, Lys25 in the previous structures of polκ does not have any contacts with the incoming nucleotide, resembling an open form of a DNA polymerase. Based on structural and functional similarity, we propose a local open/closed mechanism for polκ in DNA replication catalysis, which mimics the common mechanism for all DNA polymerases.
人类聚合酶 κ（polκ）是一种独特的 Y 家族 DNA 聚合酶，具有独特的 N 端 N 夹域。N 夹使 polκ 在 DNA 复制和损伤绕过中具有高效率和准确性。由于 N 夹的无序，N 夹如何赋予 polκ 在复制中的能力仍不清楚。在这里，我们展示了一个 2.0Å 分辨率的 polκ 三元复合物与 DNA 和一个进入的核苷酸的晶体结构。结构功能研究揭示了一个有序的 N 夹域，它使保守和功能重要的残基与活性位点中的复制碱基对接触，并有助于核苷酸转移反应。特别是，来自 N 夹域的完全有序的赖氨酸 25 与进入核苷酸的α-和γ-磷酸形成氢键。K25A 突变显著降低了 polκ 的聚合酶活性。该赖氨酸在封闭形式的 A 家族 DNA 聚合酶中与保守的赖氨酸结构类似。相比之下，polκ 先前结构中的赖氨酸 25 与进入的核苷酸没有任何接触，类似于 DNA 聚合酶的开放形式。基于结构和功能的相似性，我们提出了 polκ 在 DNA 复制催化中的局部开/闭机制，该机制模拟了所有 DNA 聚合酶的常见机制。
