A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation
Manon Budzyk, Anthony Simon, Anne-Sophie Mace, Renata Basto
Abstract
Unscheduled whole genome duplication (WGD), also described as unscheduled or non-physiological polyploidy, can lead to genetic instability and is commonly observed in human cancers. WGD generates DNA damage due to scaling defects between replication factors and DNA content. As a result DNA damage repair mechanisms are thought to be critical for ensuring cell viability and proliferation under these conditions. In this study, we explored the role of homologous recombination and Holliday junction resolution in non-physiological polyploidy in vivo. Using Drosophila genetics and high-resolution imaging, we identified a key and surprising role for Gen/Gen1 nuclease.
Introduction
Polyploidy, resulting from WGD is defined as the presence of more than two copies of all chromosomes. Several cellular strategies can be used to obtain polyploid cells, such as endoreplication, cell fusion and endomitosis, which includes mitotic slippage and cytokinesis failure [1,2]. In mammals, certain organs contain polyploid cells, such as cardiomyocytes and hepatocytes in the heart and liver, respectively, contributing to essential physiological roles [1]. A protective role for polyploidy in the ageing Drosophila brain has recently been shown [3]. Further, polyploid neurons have been described in the mouse neocortex during embryonic development [4].
Materials and method
Fly husbandry
Flies were raised on cornmeal medium (0.75% agar, 3.5% organic wheat flour, 5.0% yeast, 5.5% sugar, 2.5% nipagin, 1.0% penicillin-streptomycin and 0.4% propionic acid). Fly stocks were maintained at 18°C. Crosses were carried out in plastic vials and maintained at 25°C unless differently specified. Stocks were maintained using balancer inverted chromosomes to prevent recombination. In all experiments, larvae were staged to obtain comparable stages of development. Egg collection was performed at 25 °C for 24h. After 5 days of development at 25 °C, third instar larvae were used for brain dissection.
Results
Rad51 and Gen impair polyploid cell proliferation
To identify DNA damage repair specific vulnerabilities of unscheduled polyploid proliferation, we focused on the homologous recombination (HR) pathway. We reasoned that HR should be involved in DNA damage repair since an essential role for this pathway in polyploid yeast has been previously shown [30]. In addition, in newly born unscheduled human tetraploid cells, DNA damage is generated during S-Phase [10], which relies on HR for repair before mitosis.
Discussion
In this study we explored the function of Gen nuclease in vivo using a variety of genetic conditions. Our results reveal three important aspects concerning Gen nuclease biology.
Acknowledgments
We thank the PiCT-IBiSA platform and Nikon Imaging Center at the Institut Curie and Vincent Fraisier for excellent and continuous support on image acquisition. We thank S. Gemble, V. Marthiens, A. Terrizzano and R. Salame for helpful discussions and comments on the manuscript, and L. Jawish for help with dissections and immunostaining. We thank J. Sekelsky (University of North Carolina, NC, USA) for reagents and G. Mazon (IGR, Villejuif, FR), S. West (CRICK Institute, London UK) and J. Matos (Vienna University, Vienna, AT) for insightful discussions.
Citation: Budzyk M, Simon A, Mace A-S, Basto R (2025) A novel DNA repair-independent role for Gen nuclease in promoting unscheduled polyploidy cell proliferation. PLoS Genet 21(9): e1011605. https://doi.org/10.1371/journal.pgen.1011605
Editor: Louise Cheng, Peter MacCallum Cancer Institute Central Cancer Library: Peter MacCallum Cancer Centre, AUSTRALIA
Received: January 31, 2025; Accepted: August 11, 2025; Published: September 8, 2025
Copyright: © 2025 Budzyk et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are in the manuscript and/or Supporting information files.
Funding: This work was supported by the European Research Council Consolidator Grant (ChromoNumber-LS3, ERC-2016-COG) to R.B. and the French government for the ScalingRules project from ANR chair excellence through the Agence Nationale de la Recherche from France 2030 (ANR-23-CHBS-0012) to R.B, the InCA (www.e-cancer.fr) (2021-1- PREV-Bio grant to R.B.), the Institut Curie and the Centre National de la Recherche Scientifique. M.B.’s salary was funded by the ministry of Science and the ARC foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
