Cryo-EM structure of human Pol \u03ba bound to DNA and mono-ubiquitylated PCNA

Claudia Lancey,Matthew Percival,Masateru Takahashi,Ramon Crehuet,Kerry Blair,Samir M Hamdan,Muhammad Tehseen,Frederick W Muskett,Vlad S Raducanu,Timothy J Ragan,Souvika Bakshi,Alfredo De Biasio,Mohamed A Sobhy

doi:10.1038/s41467-021-26251-6

Claudia Lancey, Matthew Percival + Show 11 more

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https://doi.org/10.1038/s41467-021-26251-6

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Abstract

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. Access of Pol κ to DNA damage sites is facilitated by its interaction with the processivity clamp PCNA and is regulated by PCNA mono-ubiquitylation. Here, we present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA). In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. The ubiquitin moieties are partly flexible and extend radially away from PCNA, with the ubiquitin at the Pol κ-bound protomer appearing more rigid. Activity assays suggest that, when the internal PIP-box interaction is lost, Pol κ is retained on DNA by a secondary interaction between the UBZs and the ubiquitins flexibly conjugated to PCNA. Our data provide a structural basis for the recruitment of a Y-family TLS polymerase to sites of DNA damage.

Highlights

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks
We present cryo-EM reconstructions of full-length human Pol κ bound to a primer/template (P/T) DNA substrate, an incoming nucleotide, and wild-type proliferating cell nuclear antigen (PCNA) or PCNA mono-ubiquitylated at K164 (Ub-PCNA), at resolutions between ~3.4 and ~6.4 Å
The structure has approximate dimensions of 127.7 × 88.0 × 89.1 Å, and displays the catalytic core of Pol κ sitting on top of the front face of PCNA in a remarkably angled orientation, with the axis of DNA in the catalytic cleft tilted by ~47° relative to the normal of the PCNA ring plane (Fig. 1f)

Summary

Introduction

Y-family DNA polymerase κ (Pol κ) can replicate damaged DNA templates to rescue stalled replication forks. We present cryo-EM reconstructions of human Pol κ bound to DNA, an incoming nucleotide, and wild type or mono-ubiquitylated PCNA (Ub-PCNA) In both reconstructions, the internal PIP-box adjacent to the Pol κ Polymerase-Associated Domain (PAD) docks the catalytic core to one PCNA protomer in an angled orientation, bending the DNA exiting the Pol κ active site through PCNA, while Pol κ C-terminal domain containing two Ubiquitin Binding Zinc Fingers (UBZs) is invisible, in agreement with disorder predictions. Eukaryotic TLS involves canonical high-fidelity as well as specialized error-prone TLS polymerases (e.g., Y-family Pol η, Pol ι, Pol κ and Rev1), which can synthesize DNA past a lesion due to their active sites being able to accommodate damaged templates[3,10,11] Both high-fidelity and TLS polymerases form complexes with the homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA), which encircles duplex DNA and tethers these polymerases to the template, enhancing their processivity[12,13,14]. The apo-enzyme was crystallized with the PAD positioned under the palm domain in two alternate positions, while in the DNA-bound structure the PAD is docked in the major groove; for the most divergent position, a movement requiring a 50 Å shift and a 143° rotation[30]

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