2.0 Å resolution crystal structure of human polκ reveals a new catalytic function of N-clasp in DNA replication

Sci Rep. 2018 Oct 11;8(1):15125. doi: 10.1038/s41598-018-33371-5.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain*
  • DNA / chemistry
  • DNA / metabolism
  • DNA Replication
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Humans
  • Molecular Docking Simulation
  • Mutation

Substances

  • DNA
  • DNA-Directed DNA Polymerase
  • POLK protein, human