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

<div class='caption-title'>Cryo-EM structure of the Pol κ−DNA−wt-PCNA complex.</div>
<div class='caption-title'>Details of the cryo-EM structure of the Pol κ−DNA−wt-PCNA complex and comparison with Pol δ processive holoenzyme structure.</div>
<div class='caption-title'>MD simulations of the Pol κ−PCNA complex.</div>
Lancey C, Tehseen M, Bakshi S, Percival M, Takahashi M, Sobhy MA, Raducanu VS, Blair K, Muskett FW, Ragan TJ, Crehuet R, Hamdan SM, De Biasio A
OpenAccess logo Nat Commun 12 6095 (2021)
Cited: 13 times
EuropePMC logo PMID: 34667155

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.

Reviews citing this publication (5)

  1. Contribution of Microhomology to Genome Instability: Connection between DNA Repair and Replication Stress. Jiang Y. Int J Mol Sci 23 12937 (2022)
  2. Recent Advances in Understanding the Structures of Translesion Synthesis DNA Polymerases. Ling JA, Frevert Z, Washington MT. Genes (Basel) 13 915 (2022)
  3. Repair and tolerance of DNA damage at the replication fork: A structural perspective. Eichman BF. Curr Opin Struct Biol 81 102618 (2023)
  4. Observing protein dynamics during DNA-lesion bypass by the replisome. Wilkinson EM, Spenkelink LM, van Oijen AM. Front Mol Biosci 9 968424 (2022)
  5. Embracing Heterogeneity: Challenging the Paradigm of Replisomes as Deterministic Machines. Lewis JS, van Oijen AM, Spenkelink LM. Chem Rev 123 13419-13440 (2023)

Articles citing this publication (8)

  1. Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing. Blair K, Tehseen M, Raducanu VS, Shahid T, Lancey C, Rashid F, Crehuet R, Hamdan SM, De Biasio A. Nat Commun 13 7833 (2022)
  2. Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics. Raducanu VS, Tehseen M, Al-Amodi A, Joudeh LI, De Biasio A, Hamdan SM. Nat Commun 13 6973 (2022)
  3. Interaction of human HelQ with DNA polymerase delta halts DNA synthesis and stimulates DNA single-strand annealing. He L, Lever R, Cubbon A, Tehseen M, Jenkins T, Nottingham AO, Horton A, Betts H, Fisher M, Hamdan SM, Soultanas P, Bolt EL. Nucleic Acids Res 51 1740-1749 (2023)
  4. Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids. Huntington B, Zhao L, Bron P, Shahul Hameed UF, Arold ST, Qureshi BM. Front Mol Biosci 9 890390 (2022)
  5. Efficient tagging of endogenous proteins in human cell lines for structural studies by single-particle cryo-EM. Choi W, Wu H, Yserentant K, Huang B, Cheng Y. Proc Natl Acad Sci U S A 120 e2302471120 (2023)
  6. Implications of Translesion DNA Synthesis Polymerases on Genomic Stability and Human Health. Venkadakrishnan J, Lahane G, Dhar A, Xiao W, Bhat KM, Pandita TK, Bhat A. Mol Cell Biol 43 401-425 (2023)
  7. Structure of the PCNA unloader Elg1-RFC. Zheng F, Yao NY, Georgescu RE, Li H, O'Donnell ME. Sci Adv 10 eadl1739 (2024)
  8. Canonical binding of Chaetomium thermophilum DNA polymerase δ/ζ subunit PolD3 and flap endonuclease Fen1 to PCNA. Alphey MS, Wolford CB, MacNeill SA. Front Mol Biosci 10 1320648 (2023)


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