PHOSPHORYLATION-DEPENDENT ASSOCIATION OF WRN WITH RPA IS REQUIRED FOR RECOVERY OF REPLICATION FORKS STALLED AT SECONDARY DNA STRUCTURES

Overview

Journal bioRxiv

Date 2023 Aug 23

PMID 37609214

Authors

Alessandro Noto

Pasquale Valenzisi

Federica Fratini

Tomasz Kulikowicz

Joshua A Sommers

Flavia Di Feo

Valentina Palermo

Maurizio Semproni

Marco Crescenzi

Robert M Brosh Jr

Annapaola Franchitto

Pietro Pichierri

Affiliations

Soon will be listed here.

Abstract

The WRN protein mutated in the hereditary premature aging disorder Werner syndrome plays a vital role in handling, processing, and restoring perturbed replication forks. One of its most abundant partners, Replication Protein A (RPA), has been shown to robustly enhance WRN helicase activity in specific cases when tested . However, the significance of RPA-binding to WRN at replication forks in vivo has remained largely unexplored. In this study, we have identified several conserved phosphorylation sites in the acidic domain of WRN that are targeted by Casein Kinase 2 (CK2). Surprisingly, these phosphorylation sites are essential for the interaction between WRN and RPA, both and in human cells. By characterizing a CK2-unphosphorylatable WRN mutant that lacks the ability to bind RPA, we have determined that the WRN-RPA complex plays a critical role in fork recovery after replication stress whereas the WRN-RPA interaction is not necessary for the processing of replication forks or preventing DNA damage when forks stall or collapse. When WRN fails to bind RPA, fork recovery is impaired, leading to the accumulation of single-stranded DNA gaps in the parental strands, which are further enlarged by the structure-specific nuclease MRE11. Notably, RPA-binding by WRN and its helicase activity are crucial for countering the persistence of G4 structures after fork stalling. Therefore, our findings reveal for the first time a novel role for the WRN-RPA interaction to facilitate fork restart, thereby minimizing G4 accumulation at single-stranded DNA gaps and suppressing accumulation of unreplicated regions that may lead to MUS81-dependent double-strand breaks requiring efficient repair by RAD51 to prevent excessive DNA damage.

References

Brosh Jr R, Waheed J, Sommers J . Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase. J Biol Chem. 2002; 277(26):23236-45. DOI: 10.1074/jbc.M111446200. View

Basile G, Leuzzi G, Pichierri P, Franchitto A . Checkpoint-dependent and independent roles of the Werner syndrome protein in preserving genome integrity in response to mild replication stress. Nucleic Acids Res. 2014; 42(20):12628-39. PMC: 4227752. DOI: 10.1093/nar/gku1022. View

Hashimoto Y, Ray Chaudhuri A, Lopes M, Costanzo V . Rad51 protects nascent DNA from Mre11-dependent degradation and promotes continuous DNA synthesis. Nat Struct Mol Biol. 2010; 17(11):1305-11. PMC: 4306207. DOI: 10.1038/nsmb.1927. View

Chen R, Wold M . Replication protein A: single-stranded DNA's first responder: dynamic DNA-interactions allow replication protein A to direct single-strand DNA intermediates into different pathways for synthesis or repair. Bioessays. 2014; 36(12):1156-61. PMC: 4629251. DOI: 10.1002/bies.201400107. View

van Wietmarschen N, Sridharan S, Nathan W, Tubbs A, Chan E, Callen E . Repeat expansions confer WRN dependence in microsatellite-unstable cancers. Nature. 2020; 586(7828):292-298. PMC: 8916167. DOI: 10.1038/s41586-020-2769-8. View

Cantor S . Revisiting the BRCA-pathway through the lens of replication gap suppression: "Gaps determine therapy response in BRCA mutant cancer". DNA Repair (Amst). 2021; 107:103209. PMC: 9049047. DOI: 10.1016/j.dnarep.2021.103209. View

Rossi M, Ghosh A, Bohr V . Roles of Werner syndrome protein in protection of genome integrity. DNA Repair (Amst). 2010; 9(3):331-44. PMC: 2827637. DOI: 10.1016/j.dnarep.2009.12.011. View

Sturzenegger A, Burdova K, Kanagaraj R, Levikova M, Pinto C, Cejka P . DNA2 cooperates with the WRN and BLM RecQ helicases to mediate long-range DNA end resection in human cells. J Biol Chem. 2014; 289(39):27314-27326. PMC: 4175362. DOI: 10.1074/jbc.M114.578823. View

Lebel M, Spillare E, Harris C, Leder P . The Werner syndrome gene product co-purifies with the DNA replication complex and interacts with PCNA and topoisomerase I. J Biol Chem. 1999; 274(53):37795-9. DOI: 10.1074/jbc.274.53.37795. View

10.

Pirzio L, Pichierri P, Bignami M, Franchitto A . Werner syndrome helicase activity is essential in maintaining fragile site stability. J Cell Biol. 2008; 180(2):305-14. PMC: 2213598. DOI: 10.1083/jcb.200705126. View

11.

Shorrocks A, Jones S, Tsukada K, Morrow C, Belblidia Z, Shen J . The Bloom syndrome complex senses RPA-coated single-stranded DNA to restart stalled replication forks. Nat Commun. 2021; 12(1):585. PMC: 7838300. DOI: 10.1038/s41467-020-20818-5. View

12.

Oshima J, Sidorova J, Monnat Jr R . Werner syndrome: Clinical features, pathogenesis and potential therapeutic interventions. Ageing Res Rev. 2016; 33:105-114. PMC: 5025328. DOI: 10.1016/j.arr.2016.03.002. View

13.

Doherty K, Sommers J, Gray M, Lee J, von Kobbe C, Thoma N . Physical and functional mapping of the replication protein a interaction domain of the werner and bloom syndrome helicases. J Biol Chem. 2005; 280(33):29494-505. DOI: 10.1074/jbc.M500653200. View

14.

Constantinou A, Tarsounas M, Karow J, Brosh R, Bohr V, Hickson I . Werner's syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO Rep. 2001; 1(1):80-4. PMC: 1083680. DOI: 10.1093/embo-reports/kvd004. View

15.

Sommers J, Sharma S, Doherty K, Karmakar P, Yang Q, Kenny M . p53 modulates RPA-dependent and RPA-independent WRN helicase activity. Cancer Res. 2005; 65(4):1223-33. DOI: 10.1158/0008-5472.CAN-03-0231. View

16.

Brosh Jr R, Orren D, Nehlin J, Ravn P, Kenny M, Machwe A . Functional and physical interaction between WRN helicase and human replication protein A. J Biol Chem. 1999; 274(26):18341-50. DOI: 10.1074/jbc.274.26.18341. View

17.

Behan F, Iorio F, Picco G, Goncalves E, Beaver C, Migliardi G . Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens. Nature. 2019; 568(7753):511-516. DOI: 10.1038/s41586-019-1103-9. View

18.

Lee M, Shin S, Uhm H, Hong H, Kirk J, Hyun K . Multiple RPAs make WRN syndrome protein a superhelicase. Nucleic Acids Res. 2018; 46(9):4689-4698. PMC: 5961295. DOI: 10.1093/nar/gky272. View

19.

Brosh Jr R, Bohr V . Human premature aging, DNA repair and RecQ helicases. Nucleic Acids Res. 2007; 35(22):7527-44. PMC: 2190726. DOI: 10.1093/nar/gkm1008. View

20.

Croteau D, Popuri V, Opresko P, Bohr V . Human RecQ helicases in DNA repair, recombination, and replication. Annu Rev Biochem. 2014; 83:519-52. PMC: 4586249. DOI: 10.1146/annurev-biochem-060713-035428. View