Evidence of Conformational Changes in the Structure of Y‐Family Human Polymerase Kappa

Abstract

Y‐family DNA polymerases, unlike replicative DNA polymerases, are enzymes characterized by their ability to bypass DNA lesions and continue extension of damaged DNA via Translesion Synthesis (TLS). One such polymerase, human polymerase kappa (Pol κ), can efficiently bypass minor groove adducts, most notably N2‐furfuryl‐deoxy‐guanosine (N2‐ffdG), but it is inhibited by major groove adducts such as O6‐methylated‐deoxy‐guanosine (O6‐Met‐dG). Pol k is composed of five domains including its novel N‐clasp domain, as well as the thumb, palm, fingers, and little finger (polymerase associated domain or PAD) domains which are found across Y‐family polymerases. The specificity that Pol κ demonstrates is thought to be related to Y‐family polymerases having smaller finger domains when compared to replicative DNA polymerases thus limiting major groove contact, while Pol κ’s PAD and catalytic core move to open the minor groove side. It is believed that Pol κ and its Escherichia colianalogue, DinB (DNA polymerase IV), undergo one of two conformational changes, open or closed, in their active site loops when in contact with undamaged DNA or minor groove adducts. Using molecular dynamics (MD) simulations, we examined conformational changes and utilized RMSD analysis to observe activity within Pol κ’s domains across three different Pol κ ternary structures in the presence of different templating lesions and their correct incoming nucleotides. These structures include, one with a minor groove N2‐furfuryl‐deoxy‐guanosine adduct (FDG for short), one with a major groove O6‐methylated‐deoxy‐guanosine adduct, and finally one with a mutation of a residue in Pol κ’s PAD (R507K) with an FDG lesion on the DNA templating strand. All systems were solvated using CHARMMgui, minimized, equilibrated, and run for 110 ns with NAMD, and observed in VMD. Our findings support that Pol κ exists in two conformations, which depend on the kind of lesion on the DNA and/or the incoming nucleotide. In particular, the movement is concentrated in the N‐clasp and fingers domains. Some residues which seem important for this transition are N52, M135, N464, and R507. Interestingly, kinetics studies have already identified some of these same residues as important for extension. Our MD studies give an atomistic explanation of why such residues are crucial for the correct functioning of this protein.