PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

Yang Yang; Deepika Jayaprakash; Satpal S. Jhujh; John J. Reynolds; Steve Chen; Yanzhe Gao; Jay Ramanlal Anand; Elizabeth Mutter-Rottmayer; Pablo Ariel; Jing An; Xing Cheng; Kenneth H. Pearce; Sophie-Anne Blanchet; Nandana Nandakumar; Pei Zhou; Amélie Fradet-Turcotte; Grant S. Stewart; Cyrus Vaziri

doi:10.1093/nar/gkae918

PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

Yang Yang
, Deepika Jayaprakash
, Satpal S. Jhujh
, John J. Reynolds
, Steve Chen
, Yanzhe Gao
, Jay Ramanlal Anand
, Elizabeth Mutter-Rottmayer
, Pablo Ariel
, Jing An
, Xing Cheng
, Kenneth H. Pearce
, Sophie-Anne Blanchet
, Nandana Nandakumar
, Pei Zhou
, Amélie Fradet-Turcotte
, Grant S. Stewart^*
, Cyrus Vaziri^*

^*Corresponding author for this work

University of North Carolina School of Medicine
University of North Carolina at Chapel Hill
University College Birmingham
Cytiva
Harbin Medical University
Chongqing Cancer Hospital
UNC Eshelman School of Pharmacy
CHU de Québec-Université Laval
Duke University

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

5 Citations (SciVal)

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Abstract

RNF168 orchestrates a ubiquitin-dependent DNA damage response to regulate the recruitment of repair factors, such as 53BP1 to DNA double-strand breaks (DSBs). In addition to its canonical functions in DSB signaling, RNF168 may facilitate DNA replication fork progression. However, the precise role of RNF168 in DNA replication remains unclear. Here, we demonstrate that RNF168 is recruited to DNA replication factories in a manner that is independent of the canonical DSB response pathway regulated by Ataxia-Telangiectasia Mutated (ATM) and RNF8. We identify a degenerate Proliferating Cell Nuclear Antigen (PCNA)-interacting peptide (DPIP) motif in the C-terminus of RNF168, which together with its Motif Interacting with Ubiquitin (MIU) domain mediates binding to mono-ubiquitylated PCNA at replication factories. An RNF168 mutant harboring inactivating substitutions in its DPIP box and MIU1 domain (termed RNF168 1DPIP/1MIU1) is not recruited to sites of DNA synthesis and fails to support ongoing DNA replication. Notably, the PCNA interaction-deficient RNF168 1DPIP/1MIU1 mutant fully rescues the ability of RNF168−/− cells to form 53BP1 foci in response to DNA DSBs. Therefore, RNF168 functions in DNA replication and DSB signaling are fully separable. Our results define a new mechanism by which RNF168 promotes DNA replication independently of its canonical functions in DSB signaling.

Original language	English
Number of pages	17
Journal	Nucleic Acids Research
Volume	52
Issue number	21
Early online date	24 Oct 2024
DOIs	https://doi.org/10.1093/nar/gkae918
Publication status	Published - 27 Nov 2024

Bibliographical note

Copyright © The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site-for further information please contact [email protected]

Data Access Statement

The data underlying this article are available in the article and in its online supplementary material.

Funding

National Institutes of Health (NIH) [S10OD030223; R01 ES029079, CA215347 to C.V.; R01CA279034 to P.Z.]; Cancer Research-UK Programme Grant [C17183/A23303 to G.S.S.]; University of Birmingham (to J.J.R.); Canadian Institutes of Health Research [152948 to A.F.-T.]; Research Development Fund Award from the University of Birmingham (to S.S.J.); Department of Pathology and Laboratory Medicine [P30 CA016086]; UNC Lineberger Comprehensive Cancer Center. The open access publication charge for this paper has been waived by Oxford University Press – NAR Editorial Board members are entitled to one free paper per year in recognition of their work on behalf of the journal.

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10.1093/nar/gkae918Licence: CC BY-NC 4.0

PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling
Copyright © The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site-for further information please contact [email protected]
Final published version, 1.96 MBLicence: CC BY-NC 4.0

Cite this

Yang, Y., Jayaprakash, D., Jhujh, S. S., Reynolds, J. J., Chen, S., Gao, Y., Anand, J. R., Mutter-Rottmayer, E., Ariel, P., An, J., Cheng, X., Pearce, K. H., Blanchet, S.-A., Nandakumar, N., Zhou, P., Fradet-Turcotte, A., Stewart, G. S., & Vaziri, C. (2024). PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling. Nucleic Acids Research, 52(21). https://doi.org/10.1093/nar/gkae918

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title = "PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling",

abstract = "RNF168 orchestrates a ubiquitin-dependent DNA damage response to regulate the recruitment of repair factors, such as 53BP1 to DNA double-strand breaks (DSBs). In addition to its canonical functions in DSB signaling, RNF168 may facilitate DNA replication fork progression. However, the precise role of RNF168 in DNA replication remains unclear. Here, we demonstrate that RNF168 is recruited to DNA replication factories in a manner that is independent of the canonical DSB response pathway regulated by Ataxia-Telangiectasia Mutated (ATM) and RNF8. We identify a degenerate Proliferating Cell Nuclear Antigen (PCNA)-interacting peptide (DPIP) motif in the C-terminus of RNF168, which together with its Motif Interacting with Ubiquitin (MIU) domain mediates binding to mono-ubiquitylated PCNA at replication factories. An RNF168 mutant harboring inactivating substitutions in its DPIP box and MIU1 domain (termed RNF168 1DPIP/1MIU1) is not recruited to sites of DNA synthesis and fails to support ongoing DNA replication. Notably, the PCNA interaction-deficient RNF168 1DPIP/1MIU1 mutant fully rescues the ability of RNF168−/− cells to form 53BP1 foci in response to DNA DSBs. Therefore, RNF168 functions in DNA replication and DSB signaling are fully separable. Our results define a new mechanism by which RNF168 promotes DNA replication independently of its canonical functions in DSB signaling.",

author = "Yang Yang and Deepika Jayaprakash and Jhujh, \{Satpal S.\} and Reynolds, \{John J.\} and Steve Chen and Yanzhe Gao and Anand, \{Jay Ramanlal\} and Elizabeth Mutter-Rottmayer and Pablo Ariel and Jing An and Xing Cheng and Pearce, \{Kenneth H.\} and Sophie-Anne Blanchet and Nandana Nandakumar and Pei Zhou and Am{\'e}lie Fradet-Turcotte and Stewart, \{Grant S.\} and Cyrus Vaziri",

note = "Copyright {\textcopyright} The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site-for further information please contact [email protected]",

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Yang, Y, Jayaprakash, D, Jhujh, SS, Reynolds, JJ, Chen, S, Gao, Y, Anand, JR, Mutter-Rottmayer, E, Ariel, P, An, J, Cheng, X, Pearce, KH, Blanchet, S-A, Nandakumar, N, Zhou, P, Fradet-Turcotte, A, Stewart, GS & Vaziri, C 2024, 'PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling', Nucleic Acids Research, vol. 52, no. 21. https://doi.org/10.1093/nar/gkae918

TY - JOUR

T1 - PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

AU - Yang, Yang

AU - Jayaprakash, Deepika

AU - Jhujh, Satpal S.

AU - Reynolds, John J.

AU - Chen, Steve

AU - Gao, Yanzhe

AU - Anand, Jay Ramanlal

AU - Mutter-Rottmayer, Elizabeth

AU - Ariel, Pablo

AU - An, Jing

AU - Cheng, Xing

AU - Pearce, Kenneth H.

AU - Blanchet, Sophie-Anne

AU - Nandakumar, Nandana

AU - Zhou, Pei

AU - Fradet-Turcotte, Amélie

AU - Stewart, Grant S.

AU - Vaziri, Cyrus

N1 - Copyright © The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site-for further information please contact [email protected]

PY - 2024/11/27

Y1 - 2024/11/27

N2 - RNF168 orchestrates a ubiquitin-dependent DNA damage response to regulate the recruitment of repair factors, such as 53BP1 to DNA double-strand breaks (DSBs). In addition to its canonical functions in DSB signaling, RNF168 may facilitate DNA replication fork progression. However, the precise role of RNF168 in DNA replication remains unclear. Here, we demonstrate that RNF168 is recruited to DNA replication factories in a manner that is independent of the canonical DSB response pathway regulated by Ataxia-Telangiectasia Mutated (ATM) and RNF8. We identify a degenerate Proliferating Cell Nuclear Antigen (PCNA)-interacting peptide (DPIP) motif in the C-terminus of RNF168, which together with its Motif Interacting with Ubiquitin (MIU) domain mediates binding to mono-ubiquitylated PCNA at replication factories. An RNF168 mutant harboring inactivating substitutions in its DPIP box and MIU1 domain (termed RNF168 1DPIP/1MIU1) is not recruited to sites of DNA synthesis and fails to support ongoing DNA replication. Notably, the PCNA interaction-deficient RNF168 1DPIP/1MIU1 mutant fully rescues the ability of RNF168−/− cells to form 53BP1 foci in response to DNA DSBs. Therefore, RNF168 functions in DNA replication and DSB signaling are fully separable. Our results define a new mechanism by which RNF168 promotes DNA replication independently of its canonical functions in DSB signaling.

AB - RNF168 orchestrates a ubiquitin-dependent DNA damage response to regulate the recruitment of repair factors, such as 53BP1 to DNA double-strand breaks (DSBs). In addition to its canonical functions in DSB signaling, RNF168 may facilitate DNA replication fork progression. However, the precise role of RNF168 in DNA replication remains unclear. Here, we demonstrate that RNF168 is recruited to DNA replication factories in a manner that is independent of the canonical DSB response pathway regulated by Ataxia-Telangiectasia Mutated (ATM) and RNF8. We identify a degenerate Proliferating Cell Nuclear Antigen (PCNA)-interacting peptide (DPIP) motif in the C-terminus of RNF168, which together with its Motif Interacting with Ubiquitin (MIU) domain mediates binding to mono-ubiquitylated PCNA at replication factories. An RNF168 mutant harboring inactivating substitutions in its DPIP box and MIU1 domain (termed RNF168 1DPIP/1MIU1) is not recruited to sites of DNA synthesis and fails to support ongoing DNA replication. Notably, the PCNA interaction-deficient RNF168 1DPIP/1MIU1 mutant fully rescues the ability of RNF168−/− cells to form 53BP1 foci in response to DNA DSBs. Therefore, RNF168 functions in DNA replication and DSB signaling are fully separable. Our results define a new mechanism by which RNF168 promotes DNA replication independently of its canonical functions in DSB signaling.

UR - https://www.scopus.com/pages/publications/85211392561

UR - https://academic.oup.com/nar/article/52/21/13019/7833678

U2 - 10.1093/nar/gkae918

DO - 10.1093/nar/gkae918

M3 - Article

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AN - SCOPUS:85211392561

SN - 0305-1048

VL - 52

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 21

ER -

PCNA-binding activity separates RNF168 functions in DNA replication and DNA double-stranded break signaling

Abstract

Bibliographical note

Data Access Statement

Funding

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