RFWD3/FANCW orchestrates post-replicative filling of ssDNA gaps to promote UV tolerance

DNA lesions can stall replication, generating ssDNA gaps that require filling to maintain genome stability. The E3 ubiquitin ligase RFWD3/FANCW targets RPA and PCNA to regulate replication dynamics and damage tolerance, but its roles in these processes and influence on cell mutability remain incompletely understood. Here, we show that RFWD3 prevents the accumulation of ssDNA gaps in UV-irradiated cells through its E3 ligase activity and interaction with RPA. Mutational signature analysis implicates RFWD3 in the spontaneous mutability of human cells and reveals its involvement in 6-4 photoproducts tolerance. Accordingly, photorepair of 6-4 photoproducts, but not of cyclobutane pyrimidine dimers, alleviates gap accumulation in RFWD3-depleted cells. RFWD3 KO has limited impact on PCNA ubiquitylation, but its depletion sharply decreases UV-induced RPA ubiquitylation. Moreover, cells expressing an RPA mutant that cannot be ubiquitylated accumulate ssDNA gaps epistatically with RFWD3 loss. We propose that RFWD3-dependent RPA ubiquitylation regulates post-replicative ssDNA gaps to promote helix-distorting lesion tolerance. DNA lesions caused by UV light can block replication and generate single-stranded DNA gaps. Here, the authors reveal that the E3 ubiquitin ligase RFWD3 ubiquitylates RPA to prevent gap accumulation and promote DNA damage tolerance. This process helps maintain genome stability and regulates cell mutability.

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Journal
Nature Communications
Published
2026-10-05
DOI
https://doi.org/10.1038/s41467-026-78252-y
Primary Topic
DNA Repair Mechanisms
Type
article
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article

RFWD3/FANCW orchestrates post-replicative filling of ssDNA gaps to promote UV tolerance

Elliot A. Drobetsky, Isabelle Marois, Pierre‐Étienne Jacques, Luc R. Gaudreau et al.
Nature Communications
DNA Repair Mechanisms
article

RFWD3/FANCW orchestrates post-replicative filling of ssDNA gaps to promote UV tolerance

Elliot A. Drobetsky, Isabelle Marois, Pierre‐Étienne Jacques, Luc R. Gaudreau, Andréanne Blondeau, Alexandre Maréchal, Maïlyn Yates, Hugo Würtele, Amélie Fradet‐Turcotte, Edlie St-Hilaire, Anja‐Katrin Bielinsky, Ryan M. Baxley, Billel Djerir, Félix Racine-Brassard, Stéphane Coulon, Laurent Cappadocia, Samuel Zimmer, Lisa Casimir, Félix Goudreau, Yosra Mehrjoo, François Bélanger, Rima Kochman, Wendy Leung, Nadia Côté, Jean-Christophe Dubois, Gaëlle Lescalet, Vincent Mimee, Alexandra Octaviana Szenti
article en

Abstract

DNA lesions can stall replication, generating ssDNA gaps that require filling to maintain genome stability. The E3 ubiquitin ligase RFWD3/FANCW targets RPA and PCNA to regulate replication dynamics and damage tolerance, but its roles in these processes and influence on cell mutability remain incompletely understood. Here, we show that RFWD3 prevents the accumulation of ssDNA gaps in UV-irradiated cells through its E3 ligase activity and interaction with RPA. Mutational signature analysis implicates RFWD3 in the spontaneous mutability of human cells and reveals its involvement in 6-4 photoproducts tolerance. Accordingly, photorepair of 6-4 photoproducts, but not of cyclobutane pyrimidine dimers, alleviates gap accumulation in RFWD3-depleted cells. RFWD3 KO has limited impact on PCNA ubiquitylation, but its depletion sharply decreases UV-induced RPA ubiquitylation. Moreover, cells expressing an RPA mutant that cannot be ubiquitylated accumulate ssDNA gaps epistatically with RFWD3 loss. We propose that RFWD3-dependent RPA ubiquitylation regulates post-replicative ssDNA gaps to promote helix-distorting lesion tolerance. DNA lesions caused by UV light can block replication and generate single-stranded DNA gaps. Here, the authors reveal that the E3 ubiquitin ligase RFWD3 ubiquitylates RPA to prevent gap accumulation and promote DNA damage tolerance. This process helps maintain genome stability and regulates cell mutability.

Nature Communications
Centre National de la Recherche Scientifique (FR), University of Minnesota (US), Université de Sherbrooke (CA), Inserm (FR), Université du Québec à Montréal (CA), Hôpital Maisonneuve-Rosemont (CA), Centre de Recherche en Cancérologie de Marseille (FR), Institut Paoli-Calmettes (FR), Université Laval (CA), Masonic Cancer Center, University of Virginia (US), Université de Montréal (CA)
Openalex Percentile: Top 21%
DNA Repair Mechanisms
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