Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

Vlad-Stefan Raducanu; Muhammad Tehseen; Amani Al-Amodi; Luay Joudeh; Alfredo De Biasio; Samir Hamdan

doi:10.1038/s41467-022-34751-2

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

Vlad-Stefan Raducanu, Muhammad Tehseen, Amani Al-Amodi, Luay Joudeh, Alfredo De Biasio, Samir Hamdan

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

8 Scopus citations

Abstract

The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

Original language	English (US)
Journal	Nature Communications
Volume	13
Issue number	1
DOIs	https://doi.org/10.1038/s41467-022-34751-2
State	Published - Nov 15 2022

Bibliographical note

KAUST Repository Item: Exported on 2022-11-17
Acknowledged KAUST grant number(s): CRG8 URF/1/4036-01-01
Acknowledgements: This work was supported by the King Abdullah University of Science and Technology under Competitive Research Award Grant CRG8 URF/1/4036-01-01 to S.M.H. and A.D.B. We thank Yujing Ouyang for the preparation of the functionalized coverslips. We also thank the members of Samir M. Hamdan’s lab for the helpful discussions. We thank Prof. Marc S. Wold for the generous gift of human RPA expression plasmid. We thank Prof. Petr Cejka for the generous gift of human DNA2 expression plasmid. We thank Prof. Andrew Jackson and Dr. Martin Reijns for the generous gift of human RNase H2 expression plasmid (pGEX6P1-hsRNASEH2BCA, Addgene plasmid #108692).

ASJC Scopus subject areas

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

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10.1038/s41467-022-34751-2

https://repository.kaust.edu.sa/bitstream/10754/685776/1/s41467-022-34751-2.pdf

Cite this

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title = "Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics",

abstract = "The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.",

author = "Vlad-Stefan Raducanu and Muhammad Tehseen and Amani Al-Amodi and Luay Joudeh and {De Biasio}, Alfredo and Samir Hamdan",

note = "KAUST Repository Item: Exported on 2022-11-17 Acknowledged KAUST grant number(s): CRG8 URF/1/4036-01-01 Acknowledgements: This work was supported by the King Abdullah University of Science and Technology under Competitive Research Award Grant CRG8 URF/1/4036-01-01 to S.M.H. and A.D.B. We thank Yujing Ouyang for the preparation of the functionalized coverslips. We also thank the members of Samir M. Hamdan{\textquoteright}s lab for the helpful discussions. We thank Prof. Marc S. Wold for the generous gift of human RPA expression plasmid. We thank Prof. Petr Cejka for the generous gift of human DNA2 expression plasmid. We thank Prof. Andrew Jackson and Dr. Martin Reijns for the generous gift of human RNase H2 expression plasmid (pGEX6P1-hsRNASEH2BCA, Addgene plasmid #108692).",

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doi = "10.1038/s41467-022-34751-2",

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AU - Raducanu, Vlad-Stefan

AU - Tehseen, Muhammad

AU - Al-Amodi, Amani

AU - Joudeh, Luay

AU - De Biasio, Alfredo

AU - Hamdan, Samir

N1 - KAUST Repository Item: Exported on 2022-11-17 Acknowledged KAUST grant number(s): CRG8 URF/1/4036-01-01 Acknowledgements: This work was supported by the King Abdullah University of Science and Technology under Competitive Research Award Grant CRG8 URF/1/4036-01-01 to S.M.H. and A.D.B. We thank Yujing Ouyang for the preparation of the functionalized coverslips. We also thank the members of Samir M. Hamdan’s lab for the helpful discussions. We thank Prof. Marc S. Wold for the generous gift of human RPA expression plasmid. We thank Prof. Petr Cejka for the generous gift of human DNA2 expression plasmid. We thank Prof. Andrew Jackson and Dr. Martin Reijns for the generous gift of human RNase H2 expression plasmid (pGEX6P1-hsRNASEH2BCA, Addgene plasmid #108692).

PY - 2022/11/15

Y1 - 2022/11/15

N2 - The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

AB - The final steps of lagging strand synthesis induce maturation of Okazaki fragments via removal of the RNA primers and ligation. Iterative cycles between Polymerase δ (Polδ) and Flap endonuclease-1 (FEN1) remove the primer, with an intermediary nick structure generated for each cycle. Here, we show that human Polδ is inefficient in releasing the nick product from FEN1, resulting in non-processive and remarkably slow RNA removal. Ligase 1 (Lig1) can release the nick from FEN1 and actively drive the reaction toward ligation. These mechanisms are coordinated by PCNA, which encircles DNA, and dynamically recruits Polδ, FEN1, and Lig1 to compete for their substrates. Our findings call for investigating additional pathways that may accelerate RNA removal in human cells, such as RNA pre-removal by RNase Hs, which, as demonstrated herein, enhances the maturation rate ~10-fold. They also suggest that FEN1 may attenuate the various activities of Polδ during DNA repair and recombination.

UR - http://hdl.handle.net/10754/685776

UR - https://www.nature.com/articles/s41467-022-34751-2

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DO - 10.1038/s41467-022-34751-2

M3 - Article

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SN - 2041-1723

VL - 13

JO - Nature Communications

JF - Nature Communications

IS - 1

ER -

Mechanistic investigation of human maturation of Okazaki fragments reveals slow kinetics

Abstract

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