Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

Kerry Blair; Muhammad Tehseen; Vlad Stefan Raducanu; Taha Shahid; Claudia Lancey; Fahad Rashid; Ramon Creuhet; Samir M. Hamdan; Alfredo De Biasio

doi:10.1038/s41467-022-35475-z

Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

Kerry Blair, Muhammad Tehseen, Vlad Stefan Raducanu, Taha Shahid, Claudia Lancey, Fahad Rashid, Ramon Creuhet, Samir M. Hamdan^*, Alfredo De Biasio^*

^*Corresponding author for this work

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

16 Scopus citations

Abstract

During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIP_N-term) and DNA binding domain (PIP_DBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIP_N-term is released from PCNA and only PIP_DBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

Original language	English (US)
Article number	7833
Journal	Nature Communications
Volume	13
Issue number	1
DOIs	https://doi.org/10.1038/s41467-022-35475-z
State	Published - Dec 2022

Bibliographical note

Funding Information:
This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H. and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.). 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 and T.J. Ragan (LISCB, University of Leicester) for their help in cryo-EM data collection and advice on data processing. This project has been carried out using the resources of CSUC.

Funding Information:
This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H. and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.). 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 and T.J. Ragan (LISCB, University of Leicester) for their help in cryo-EM data collection and advice on data processing. This project has been carried out using the resources of CSUC.

Publisher Copyright:
© 2022, The Author(s).

ASJC Scopus subject areas

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

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title = "Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing",

abstract = "During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.",

author = "Kerry Blair and Muhammad Tehseen and Raducanu, {Vlad Stefan} and Taha Shahid and Claudia Lancey and Fahad Rashid and Ramon Creuhet and Hamdan, {Samir M.} and {De Biasio}, Alfredo",

note = "Funding Information: This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H. and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.). 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 and T.J. Ragan (LISCB, University of Leicester) for their help in cryo-EM data collection and advice on data processing. This project has been carried out using the resources of CSUC. Funding Information: This research was supported by King Abdullah University of Science and Technology through core funding (to S.M.H. and A.D.B.) and the Competitive Research Award Grant CRG8 URF/1/4036‐01‐01 (to S.M.H. and A.D.B.). 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 and T.J. Ragan (LISCB, University of Leicester) for their help in cryo-EM data collection and advice on data processing. This project has been carried out using the resources of CSUC. Publisher Copyright: {\textcopyright} 2022, The Author(s).",

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AU - Blair, Kerry

AU - Tehseen, Muhammad

AU - Raducanu, Vlad Stefan

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AU - Rashid, Fahad

AU - Creuhet, Ramon

AU - Hamdan, Samir M.

AU - De Biasio, Alfredo

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PY - 2022/12

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N2 - During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

AB - During lagging strand synthesis, DNA Ligase 1 (Lig1) cooperates with the sliding clamp PCNA to seal the nicks between Okazaki fragments generated by Pol δ and Flap endonuclease 1 (FEN1). We present several cryo-EM structures combined with functional assays, showing that human Lig1 recruits PCNA to nicked DNA using two PCNA-interacting motifs (PIPs) located at its disordered N-terminus (PIPN-term) and DNA binding domain (PIPDBD). Once Lig1 and PCNA assemble as two-stack rings encircling DNA, PIPN-term is released from PCNA and only PIPDBD is required for ligation to facilitate the substrate handoff from FEN1. Consistently, we observed that PCNA forms a defined complex with FEN1 and nicked DNA, and it recruits Lig1 to an unoccupied monomer creating a toolbelt that drives the transfer of DNA to Lig1. Collectively, our results provide a structural model on how PCNA regulates FEN1 and Lig1 during Okazaki fragments maturation.

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Mechanism of human Lig1 regulation by PCNA in Okazaki fragment sealing

Abstract

Bibliographical note

ASJC Scopus subject areas

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