The RAD52 double-ring remodels replication forks restricting fork reversal

Masayoshi Honda; Mortezaali Razzaghi; Paras Gaur; Eva Malacaria; Giorgia Marozzi; Ludovica Di Biagi; Francesca Antonella Aiello; Emeleeta A Paintsil; Andrew J Stanfield; Bailey J Deppe; Lokesh Gakhar; Nicholas J Schnicker; M Ashley Spies; Pietro Pichierri; Maria Spies

doi:10.1038/s41586-025-08753-1

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The RAD52 double-ring remodels replication forks restricting fork reversal

Journal article

Peer reviewed

The RAD52 double-ring remodels replication forks restricting fork reversal

Masayoshi Honda, Mortezaali Razzaghi, Paras Gaur, Eva Malacaria, Giorgia Marozzi, Ludovica Di Biagi, Francesca Antonella Aiello, Emeleeta A Paintsil, Andrew J Stanfield, Bailey J Deppe, …

Nature (London), Vol.641(8062), pp.512-519

05/08/2025

DOI: 10.1038/s41586-025-08753-1

PMCID: PMC12360664

PMID: 40175552

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https://pmc.ncbi.nlm.nih.gov/articles/PMC12360664/View

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Abstract

Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability, including protection of stalled DNA replication forks from excessive degradation . In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress, protecting them from reversal by SMARCAL1 motor . The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses, we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the single-stranded DNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.

Details

Title: Subtitle: The RAD52 double-ring remodels replication forks restricting fork reversal
Creators: Masayoshi Honda - Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Mortezaali Razzaghi - University of Iowa
Paras Gaur - University of Iowa
Eva Malacaria - Istituto Superiore di Sanità
Giorgia Marozzi - Istituto Superiore di Sanità
Ludovica Di Biagi - Istituto Superiore di Sanità
Francesca Antonella Aiello - Istituto Superiore di Sanità
Emeleeta A Paintsil - Medical College of Wisconsin
Andrew J Stanfield - Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Bailey J Deppe - Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Lokesh Gakhar - PAQ Therapeutics, Burlington, MA, USA
Nicholas J Schnicker - University of Iowa
M Ashley Spies - University of Iowa, Medicinal and Natural Products Chemistry
Pietro Pichierri - Istituto Superiore di Sanità
Maria Spies - Department of Biochemistry and Molecular Biology, University of Iowa Carver College of Medicine, Iowa City, IA, USA. maria-spies@uiowa.edu
Resource Type: Journal article
Publication Details: Nature (London), Vol.641(8062), pp.512-519
DOI: 10.1038/s41586-025-08753-1
PMID: 40175552
PMCID: PMC12360664
NLM abbreviation: Nature
ISSN: 1476-4687
eISSN: 1476-4687
Publisher: NATURE PORTFOLIO
Grant note: National Institutes of Health (NIH): R01 CA232425 NIH NCI T32 in Free Radicals and Radiation Biology training programme: CA078586 University of Iowa Healthcare Distinguished Scholar Program awardNIH NCI: P30 CA086862 University of Iowa Holden Comprehensive Cancer CenterNIH Common Fund Transformative High Resolution Cryo-Electron Microscopy Program: U24 GM129539 Simons FoundationNY State AssemblyNIH: U24GM129547 DOE Office of Science User FacilityOffice of Biological and Environmental ResearchUniversity of Iowa Carver College of Medicine
This work was supported by grants from the National Institutes of Health (NIH), grants no. R01 CA232425 to M.S., P.P. and M.A.S. M.R. was supported by a postdoctoral fellowship from the NIH NCI T32 in Free Radicals and Radiation Biology training programme CA078586. The work was also supported in part by the University of Iowa Healthcare Distinguished Scholar Program award to M.S and the NIH NCI P30 CA086862 in support of the University of Iowa Holden Comprehensive Cancer Center. Some of this work was performed at the National Center for CryoEM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy Program (grant no. U24 GM129539), and by grants from the Simons Foundation (grant no. SF349247) and NY State Assembly. A portion of this research was supported by NIH grant no. U24GM129547 and performed at the Pacific Northwest Center for Cryo-EM (PNCC) at OHSU and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We would like to acknowledgement use of Protein & Crystallography Facility resources that are supported through funding from the University of Iowa Carver College of Medicine.
Language: English
Electronic publication date: 04/02/2025
Date published: 05/08/2025
Academic Unit: Molecular Physiology and Biophysics; Pharmaceutical Sciences and Experimental Therapeutics; Radiation Oncology; Biochemistry and Molecular Biology; Medicine Administration; Medicinal and Natural Products Chemistry
Record Identifier: 9984805010502771

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