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 Abdelmaboud Sobhy; Vlad-Stefan Raducanu; Kerry Blair; Frederick W. Muskett; Timothy J. Ragan; Ramon Crehuet; Samir Hamdan; Alfredo De Biasio

doi:10.1038/s41467-021-26251-6

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 Abdelmaboud Sobhy, Vlad-Stefan Raducanu, Kerry Blair, Frederick W. Muskett, Timothy J. Ragan, Ramon Crehuet, Samir Hamdan, Alfredo De Biasio

Research output: Contribution to journal › Article › peer-review

24 Scopus citations

Abstract

AbstractY-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.

Original language	English (US)
Journal	Nature Communications
Volume	12
Issue number	1
DOIs	https://doi.org/10.1038/s41467-021-26251-6
State	Published - Oct 19 2021

Bibliographical note

KAUST Repository Item: Exported on 2021-10-21
Acknowledged KAUST grant number(s): CRG8 URF/1/4036-01-01
Acknowledgements: This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H.) and the Competitive Research Award Grant CRG8 URF/1/4036-01-01 (to S.M.H. and A.D.B.), and by the Wellcome Trust (to A.D.B.). R.C. acknowledges funding from the MINECO (CTQ2016-78636-P) and to AGAUR, (2017 SGR 324). The MD project has been carried out using CSUC resources. We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva (LISCB, University of Leicester) for his help in cryo-EM data collection and advice on data processing

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology
General Chemistry
General Physics and Astronomy

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10.1038/s41467-021-26251-6

https://repository.kaust.edu.sa/bitstream/10754/672905/1/s41467-021-26251-6.pdf

Cite this

@article{cee88b9da1e242e995e2037316010a17,

title = "Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA",

abstract = "AbstractY-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.",

author = "Claudia Lancey and Muhammad Tehseen and Souvika Bakshi and Matthew Percival and Masateru Takahashi and Sobhy, {Mohamed Abdelmaboud} and Vlad-Stefan Raducanu and Kerry Blair and Muskett, {Frederick W.} and Ragan, {Timothy J.} and Ramon Crehuet and Samir Hamdan and {De Biasio}, Alfredo",

note = "KAUST Repository Item: Exported on 2021-10-21 Acknowledged KAUST grant number(s): CRG8 URF/1/4036-01-01 Acknowledgements: This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H.) and the Competitive Research Award Grant CRG8 URF/1/4036-01-01 (to S.M.H. and A.D.B.), and by the Wellcome Trust (to A.D.B.). R.C. acknowledges funding from the MINECO (CTQ2016-78636-P) and to AGAUR, (2017 SGR 324). The MD project has been carried out using CSUC resources. We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva (LISCB, University of Leicester) for his help in cryo-EM data collection and advice on data processing",

year = "2021",

month = oct,

day = "19",

doi = "10.1038/s41467-021-26251-6",

language = "English (US)",

volume = "12",

journal = "Nature Communications",

issn = "2041-1723",

publisher = "Nature Research",

number = "1",

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TY - JOUR

T1 - Cryo-EM structure of human Pol κ bound to DNA and mono-ubiquitylated PCNA

AU - Lancey, Claudia

AU - Tehseen, Muhammad

AU - Bakshi, Souvika

AU - Percival, Matthew

AU - Takahashi, Masateru

AU - Sobhy, Mohamed Abdelmaboud

AU - Raducanu, Vlad-Stefan

AU - Blair, Kerry

AU - Muskett, Frederick W.

AU - Ragan, Timothy J.

AU - Crehuet, Ramon

AU - Hamdan, Samir

AU - De Biasio, Alfredo

N1 - KAUST Repository Item: Exported on 2021-10-21 Acknowledged KAUST grant number(s): CRG8 URF/1/4036-01-01 Acknowledgements: This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H.) and the Competitive Research Award Grant CRG8 URF/1/4036-01-01 (to S.M.H. and A.D.B.), and by the Wellcome Trust (to A.D.B.). R.C. acknowledges funding from the MINECO (CTQ2016-78636-P) and to AGAUR, (2017 SGR 324). The MD project has been carried out using CSUC resources. We acknowledge The Midlands Regional Cryo-EM Facility at the Leicester Institute of Structural and Chemical Biology (LISCB), major funding from MRC (MC_PC_17136). We thank Christos Savva (LISCB, University of Leicester) for his help in cryo-EM data collection and advice on data processing

PY - 2021/10/19

Y1 - 2021/10/19

N2 - AbstractY-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.

AB - AbstractY-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.

UR - http://hdl.handle.net/10754/672905

UR - https://www.nature.com/articles/s41467-021-26251-6

U2 - 10.1038/s41467-021-26251-6

DO - 10.1038/s41467-021-26251-6

M3 - Article

C2 - 34667155

SN - 2041-1723

VL - 12

JO - Nature Communications

JF - Nature Communications

IS - 1

ER -

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

Abstract

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