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

Claudia Lancey, Muhammad Tehseen, Souvika Bakshi, Matthew Percival, Masateru Takahashi, Mohamed A. Sobhy, Vlad S. Raducanu, Kerry Blair, Frederick W. Muskett, Timothy J. Ragan, Ramon Crehuet, Samir M. Hamdan () and Alfredo De Biasio ()
Additional contact information
Claudia Lancey: University of Leicester
Muhammad Tehseen: Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology
Souvika Bakshi: University of Leicester
Matthew Percival: University of Leicester
Masateru Takahashi: Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology
Mohamed A. Sobhy: Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology
Vlad S. Raducanu: Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology
Kerry Blair: University of Leicester
Frederick W. Muskett: University of Leicester
Timothy J. Ragan: University of Leicester
Ramon Crehuet: CSIC-Institute for Advanced Chemistry of Catalonia (IQAC) C/ Jordi Girona 18-26
Samir M. Hamdan: Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology
Alfredo De Biasio: University of Leicester

Nature Communications, 2021, vol. 12, issue 1, 1-14

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-26251-6

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