Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

Sandhya Balasubramanian; Matteo Andreani; Júlia Goncalves Andrade; Tannishtha Saha; Devakumar Sundaravinayagam; Javier Garzón; Wenzhu Zhang; Oliver Popp; Shin-Ichiro Hiraga; Ali Rahjouei; Daniel B Rosen; Philipp Mertins; Brian T Chait; Anne D Donaldson; Michela Di Virgilio

doi:10.7554/eLife.75047

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

Sandhya Balasubramanian, Matteo Andreani, Júlia Goncalves Andrade, Tannishtha Saha, Devakumar Sundaravinayagam, Javier Garzón, Wenzhu Zhang, Oliver Popp, Shin-Ichiro Hiraga, Ali Rahjouei, Daniel B Rosen, Philipp Mertins, Brian T Chait, Anne D Donaldson, Michela Di Virgilio^* (Corresponding Author)

^*Corresponding author for this work

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Abstract

RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

Original language	English
Article number	e75047
Number of pages	30
Journal	eLife
Volume	11
DOIs	https://doi.org/10.7554/eLife.75047
Publication status	Published - 13 Apr 2022

Bibliographical note

Acknowledgements
We thank all members of the Di Virgilio lab for their feedback and discussion; V Delgado-Benito (Di Virgilio lab, MDC, Berlin) for her contribution to the project development; L Keller (Di Virgilio lab, MDC, Berlin) for support with cloning, mutagenesis, and mice genotyping; C Brischetto (Scheidereit Lab, MDC, Berlin) for assistance with confocal microscopy; Aberdeen Proteomics facility (University of Aberdeen) for the mass spec analysis of Aph-induced hRIF1 phosphorylation; and the MDC FACS Core Facility and Dr. HP Rahn for support with cell sorting. Aliquots of ATRi and ATMi were gener- ously provided by AG Henssen (MDC and ECRC, Berlin). Figures 1B and D, 2A, and 4C contain items created with BioRender.com. This work was supported by ERC grant 638897 (to MDV), the Helmholtz- Gemeinschaft Zukunftsthema 'Immunology and Inflammation' ZT-0027 (to MDV), P41 GM109824 and P41 GM103314 (to BTC), and Cancer Research UK awards C1445/A19059 and DRCPGM\100,013 (to ADD and SH).

Keywords

Animals
DNA/metabolism
DNA Breaks, Double-Stranded
DNA Repair
DNA Replication
Mice
Phosphorylation
Telomere-Binding Proteins/genetics

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Balasubramanian, S., Andreani, M., Andrade, J. G., Saha, T., Sundaravinayagam, D., Garzón, J., Zhang, W., Popp, O., Hiraga, S.-I., Rahjouei, A., Rosen, D. B., Mertins, P., Chait, B. T., Donaldson, A. D., & Di Virgilio, M. (2022). Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region. eLife, 11, Article e75047. https://doi.org/10.7554/eLife.75047

Balasubramanian, S, Andreani, M, Andrade, JG, Saha, T, Sundaravinayagam, D, Garzón, J, Zhang, W, Popp, O, Hiraga, S-I, Rahjouei, A, Rosen, DB, Mertins, P, Chait, BT, Donaldson, AD & Di Virgilio, M 2022, 'Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region', eLife, vol. 11, e75047. https://doi.org/10.7554/eLife.75047

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title = "Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region",

abstract = "RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.",

keywords = "Animals, DNA/metabolism, DNA Breaks, Double-Stranded, DNA Repair, DNA Replication, Mice, Phosphorylation, Telomere-Binding Proteins/genetics",

author = "Sandhya Balasubramanian and Matteo Andreani and Andrade, {J{\'u}lia Goncalves} and Tannishtha Saha and Devakumar Sundaravinayagam and Javier Garz{\'o}n and Wenzhu Zhang and Oliver Popp and Shin-Ichiro Hiraga and Ali Rahjouei and Rosen, {Daniel B} and Philipp Mertins and Chait, {Brian T} and Donaldson, {Anne D} and {Di Virgilio}, Michela",

note = "Acknowledgements We thank all members of the Di Virgilio lab for their feedback and discussion; V Delgado-Benito (Di Virgilio lab, MDC, Berlin) for her contribution to the project development; L Keller (Di Virgilio lab, MDC, Berlin) for support with cloning, mutagenesis, and mice genotyping; C Brischetto (Scheidereit Lab, MDC, Berlin) for assistance with confocal microscopy; Aberdeen Proteomics facility (University of Aberdeen) for the mass spec analysis of Aph-induced hRIF1 phosphorylation; and the MDC FACS Core Facility and Dr. HP Rahn for support with cell sorting. Aliquots of ATRi and ATMi were gener- ously provided by AG Henssen (MDC and ECRC, Berlin). Figures 1B and D, 2A, and 4C contain items created with BioRender.com. This work was supported by ERC grant 638897 (to MDV), the Helmholtz- Gemeinschaft Zukunftsthema 'Immunology and Inflammation' ZT-0027 (to MDV), P41 GM109824 and P41 GM103314 (to BTC), and Cancer Research UK awards C1445/A19059 and DRCPGM\100,013 (to ADD and SH).",

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doi = "10.7554/eLife.75047",

language = "English",

volume = "11",

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T1 - Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

AU - Balasubramanian, Sandhya

AU - Andreani, Matteo

AU - Andrade, Júlia Goncalves

AU - Saha, Tannishtha

AU - Sundaravinayagam, Devakumar

AU - Garzón, Javier

AU - Zhang, Wenzhu

AU - Popp, Oliver

AU - Hiraga, Shin-Ichiro

AU - Rahjouei, Ali

AU - Rosen, Daniel B

AU - Mertins, Philipp

AU - Chait, Brian T

AU - Donaldson, Anne D

AU - Di Virgilio, Michela

N1 - Acknowledgements We thank all members of the Di Virgilio lab for their feedback and discussion; V Delgado-Benito (Di Virgilio lab, MDC, Berlin) for her contribution to the project development; L Keller (Di Virgilio lab, MDC, Berlin) for support with cloning, mutagenesis, and mice genotyping; C Brischetto (Scheidereit Lab, MDC, Berlin) for assistance with confocal microscopy; Aberdeen Proteomics facility (University of Aberdeen) for the mass spec analysis of Aph-induced hRIF1 phosphorylation; and the MDC FACS Core Facility and Dr. HP Rahn for support with cell sorting. Aliquots of ATRi and ATMi were gener- ously provided by AG Henssen (MDC and ECRC, Berlin). Figures 1B and D, 2A, and 4C contain items created with BioRender.com. This work was supported by ERC grant 638897 (to MDV), the Helmholtz- Gemeinschaft Zukunftsthema 'Immunology and Inflammation' ZT-0027 (to MDV), P41 GM109824 and P41 GM103314 (to BTC), and Cancer Research UK awards C1445/A19059 and DRCPGM\100,013 (to ADD and SH).

PY - 2022/4/13

Y1 - 2022/4/13

N2 - RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

AB - RIF1 is a multifunctional protein that plays key roles in the regulation of DNA processing. During repair of DNA double-strand breaks (DSBs), RIF1 functions in the 53BP1-Shieldin pathway that inhibits resection of DNA ends to modulate the cellular decision on which repair pathway to engage. Under conditions of replication stress, RIF1 protects nascent DNA at stalled replication forks from degradation by the DNA2 nuclease. How these RIF1 activities are regulated at the post-translational level has not yet been elucidated. Here, we identified a cluster of conserved ATM/ATR consensus SQ motifs within the intrinsically disordered region (IDR) of mouse RIF1 that are phosphorylated in proliferating B lymphocytes. We found that phosphorylation of the conserved IDR SQ cluster is dispensable for the inhibition of DSB resection by RIF1, but is essential to counteract DNA2-dependent degradation of nascent DNA at stalled replication forks. Therefore, our study identifies a key molecular feature that enables the genome-protective function of RIF1 during DNA replication stress.

KW - Animals

KW - DNA/metabolism

KW - DNA Breaks, Double-Stranded

KW - DNA Repair

KW - DNA Replication

KW - Mice

KW - Phosphorylation

KW - Telomere-Binding Proteins/genetics

U2 - 10.7554/eLife.75047

DO - 10.7554/eLife.75047

M3 - Article

C2 - 35416772

VL - 11

JO - eLife

JF - eLife

M1 - e75047

ER -

Protection of nascent DNA at stalled replication forks is mediated by phosphorylation of RIF1 intrinsically disordered region

Abstract

Bibliographical note

Keywords

UN SDGs

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