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

Author

Listed:
  • 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
    Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology)

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.

Suggested Citation

  • 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, 2021. "Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-26251-6
    DOI: 10.1038/s41467-021-26251-6
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    References listed on IDEAS

    as
    1. Claudia Lancey & Muhammad Tehseen & Vlad-Stefan Raducanu & Fahad Rashid & Nekane Merino & Timothy J. Ragan & Christos G. Savva & Manal S. Zaher & Afnan Shirbini & Francisco J. Blanco & Samir M. Hamdan, 2020. "Structure of the processive human Pol δ holoenzyme," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    2. Clément Madru & Ghislaine Henneke & Pierre Raia & Inès Hugonneau-Beaufet & Gérard Pehau-Arnaudet & Patrick England & Erik Lindahl & Marc Delarue & Marta Carroni & Ludovic Sauguet, 2020. "Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
    3. Alfredo De Biasio & Alain Ibáñez de Opakua & Gulnahar B. Mortuza & Rafael Molina & Tiago N. Cordeiro & Francisco Castillo & Maider Villate & Nekane Merino & Sandra Delgado & David Gil-Cartón & Irene L, 2015. "Structure of p15PAF–PCNA complex and implications for clamp sliding during DNA replication and repair," Nature Communications, Nature, vol. 6(1), pages 1-12, May.
    4. Matteo Tiberti & Elena Papaleo & Tone Bengtsen & Wouter Boomsma & Kresten Lindorff-Larsen, 2015. "ENCORE: Software for Quantitative Ensemble Comparison," PLOS Computational Biology, Public Library of Science, vol. 11(10), pages 1-16, October.
    5. Matteo De March & Nekane Merino & Susana Barrera-Vilarmau & Ramon Crehuet & Silvia Onesti & Francisco J. Blanco & Alfredo De Biasio, 2017. "Structural basis of human PCNA sliding on DNA," Nature Communications, Nature, vol. 8(1), pages 1-7, April.
    6. Timothy D. Silverstein & Robert E. Johnson & Rinku Jain & Louise Prakash & Satya Prakash & Aneel K. Aggarwal, 2010. "Structural basis for the suppression of skin cancers by DNA polymerase η," Nature, Nature, vol. 465(7301), pages 1039-1043, June.
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    Cited by:

    1. Qing He & Feng Wang & Nina Y. Yao & Michael E. O’Donnell & Huilin Li, 2024. "Structures of the human leading strand Polε–PCNA holoenzyme," Nature Communications, Nature, vol. 15(1), pages 1-12, December.

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