Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair

Ronak Shah; Muhammad Assad Aslam; Aldo Spanjaard; Daniel de Groot; Lisa M. Zurcher; Maarten Altelaar; Liesbeth Hoekman; Colin E. J. Pritchard; Bas Pilzecker; Paul C. M. van den Berk; Heinz Jacobs

doi:10.1093/pnasnexus/pgae242

Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair

Ronak Shah, Muhammad Assad Aslam, Aldo Spanjaard, Daniel de Groot, Lisa M. Zurcher, Maarten Altelaar, Liesbeth Hoekman, Colin E. J. Pritchard, Bas Pilzecker, Paul C. M. van den Berk, Heinz Jacobs^*

^*Corresponding author for this work

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

Abstract

The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/-) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.

Original language	English
Article number	pgae242
Number of pages	14
Journal	PNAS Nexus
Volume	3
Issue number	7
DOIs	https://doi.org/10.1093/pnasnexus/pgae242
Publication status	Published - 2 Jul 2024

Bibliographical note

Publisher Copyright:
© The Author(s) 2024.

Funding

This work was supported and made possible by two grants from the Dutch Cancer Society: KWF NKI-2017-10032 and NKI-2017-10796 to H.J. This research was supported by an institutional grant of the Dutch Cancer Society and of the Dutch Ministry of Health, Welfare and Sport. The authors would like to thank the biotechnical staff of the NKI-AVL for support, especially Anita Pfauth, Frank van Diepen, and Martijn van Baalen, for their expertise and help with flow cytometry experiments and cell sorting. The authors moreover thank the Animal Pathology Facility for providing H&E stains of mouse organs. The authors thank Dr. Ron van Kerkhoven, Marja Nieuwland, and Roel Kluin for their help with the RNA sequencing. The authors finally thank Prof. Dr. Titia Sixma for critical reading of the manuscript. This work was supported and made possible by two grants from the Dutch Cancer Society: KWF NKI-2017-10032 and NKI-2017-10796 to H.J. This research was supported by an institutional grant of the Dutch Cancer Society and of the Dutch Ministry of Health, Welfare and Sport.

Funders	Funder number
Martijn van Baalen
Ministerie van Volksgezondheid, Welzijn en Sport
KWF Kankerbestrijding	KWF NKI-2017-10032, NKI-2017-10796

Keywords

Fanconi anemia
Interstrand crosslink
Mismatch repair
PCNA monoubiquitination
Replication stress

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Shah, R., Aslam, M. A., Spanjaard, A., de Groot, D., Zurcher, L. M., Altelaar, M., Hoekman, L., Pritchard, C. E. J., Pilzecker, B., van den Berk, P. C. M., & Jacobs, H. (2024). Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair. PNAS Nexus, 3(7), Article pgae242. https://doi.org/10.1093/pnasnexus/pgae242

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abstract = "The Fanconi anemia (FA) repair pathway governs repair of highly genotoxic DNA interstrand crosslinks (ICLs) and relies on translesion synthesis (TLS). TLS is facilitated by REV1 or site-specific monoubiquitination of proliferating cell nuclear antigen (PCNA) (PCNA-Ub) at lysine 164 (K164). A PcnaK164R/K164R but not Rev1-/- mutation renders mammals hypersensitive to ICLs. Besides the FA pathway, alternative pathways have been associated with ICL repair (1, 2), though the decision making between those remains elusive. To study the dependence and relevance of PCNA-Ub in FA repair, we intercrossed PcnaK164R/+; Fancg-/+ mice. A combined mutation (PcnaK164R/K164R; Fancg-/-) was found embryonically lethal. RNA-seq of primary double-mutant (DM) mouse embryonic fibroblasts (MEFs) revealed elevated levels of replication stress-induced checkpoints. To exclude stress-induced confounders, we utilized a Trp53 knock-down to obtain a model to study ICL repair in depth. Regarding ICL-induced cell toxicity, cell cycle arrest, and replication fork progression, single-mutant and DM MEFs were found equally sensitive, establishing PCNA-Ub to be critical for FA-ICL repair. Immunoprecipitation and spectrometry-based analysis revealed an unknown role of PCNA-Ub in excluding mismatch recognition complex MSH2/MSH6 from being recruited to ICLs. In conclusion, our results uncovered a dual function of PCNA-Ub in ICL repair, i.e. exclude MSH2/MSH6 recruitment to channel the ICL toward canonical FA repair, in addition to its established role in coordinating TLS opposite the unhooked ICL.",

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author = "Ronak Shah and Aslam, {Muhammad Assad} and Aldo Spanjaard and {de Groot}, Daniel and Zurcher, {Lisa M.} and Maarten Altelaar and Liesbeth Hoekman and Pritchard, {Colin E. J.} and Bas Pilzecker and {van den Berk}, {Paul C. M.} and Heinz Jacobs",

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AU - de Groot, Daniel

AU - Zurcher, Lisa M.

AU - Altelaar, Maarten

AU - Hoekman, Liesbeth

AU - Pritchard, Colin E. J.

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Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair

Abstract

Bibliographical note

Funding

Keywords

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