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

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.

Mammalian DNA Polymerase Kappa Activity and Specificity

“…Until recently, the crystal structures of pol κ lacked sufficient resolution to identify interactions between the N-clasp residues and other parts of the complex. However, a 2.0-Å resolution crystal structure was reported in 2018 that shows structural characteristics and interactions crucial for our understanding of the role of the N-clasp [23]. This crystal structure shows that the N-terminal residues are well-ordered and interact with the N-clasp, fingers, and thumb domains.…”

“…This crystal structure shows that the N-terminal residues are well-ordered and interact with the N-clasp, fingers, and thumb domains. The electron density around R18 and K25 is weaker than other residues, which the authors argue demonstrates high flexibility [23]. The active site contains many ordered water molecules engaged in an extensive network of hydrogen bonds, especially with the highly conserved residues R18, R149, and K25.…”

“…K25 was identified as having a key role in activity through primer extension assays and kinetics assays. Pol κ K25 is at an analogous position and proposed to function similarly to a conserved lysine residue in the O-helix of pol I that makes contacts with phosphates on the incoming nucleotide [23]; pol I K758 is involved in dNTP binding through a conformational change in the fingers domain. However, while Y-family pols do not undergo a similar conformational change in the fingers domain as A-family DNA pols [24,25,26,27,28], this lysine residue K25 still acts as a proton donor to facilitate the formation of pyrophosphate and the reaction to form the phosphodiester bond [23].…”

“…Other TLS polymerases bypass this lesion by misincorporation of dA [85,86,87,88]. The open catalytic core of pol κ easily accommodates the bulky BPDE- N 2 -dG adduct oriented towards the minor groove, which pairs with dC in a Watson–Crick base pair with an extra H-bond between O 2 -dC and O 7 from the BPDE- N 2 -dG to allow accurate insertion [23,89,90]. Comparable conformations of ternary complexes with and without adducted DNA templates have revealed unique interactions between the core and little finger domains mediated by the N-clasp domain, resulting in a partial coverage of the DNA duplex minor groove [89].…”

“…However, the subsequent insertion and extension steps at dG-5′ are catalyzed with increased efficiency comparable to normal DNA replication. Pol κ is the most accurate relative to polymerases η and ι and predominantly incorporates correct nucleotides in insertion and extension steps [23]. Furthermore, pol κ can carry out strand displacement synthesis and can fully extend the primer having inserted a dNTP opposite cisplatin-interstrand crosslinked guanine [106].…”