title: Slow Replication Fork Velocity of Homologous Recombination-Defective Cells Results from Endogenous Oxidative Stress.
abstract: Replications forks are routinely hindered by different endogenous stresses. Because homologous recombination plays a pivotal role in the reactivation of arrested replication forks, defects in homologous recombination reveal the initial endogenous stress(es). Homologous recombination-defective cells consistently exhibit a spontaneously reduced replication speed, leading to mitotic extra centrosomes. Here, we identify oxidative stress as a major endogenous source of replication speed deceleration in homologous recombination-defective cells. The treatment of homologous recombination-defective cells with the antioxidant N-acetyl-cysteine or the maintenance of the cells at low O2 levels (3%) rescues both the replication fork speed, as monitored by single-molecule analysis (molecular combing), and the associated mitotic extra centrosome frequency. Reciprocally, the exposure of wild-type cells to H2O2 reduces the replication fork speed and generates mitotic extra centrosomes. Supplying deoxynucleotide precursors to H2O2-exposed cells rescued the replication speed. Remarkably, treatment with N-acetyl-cysteine strongly expanded the nucleotide pool, accounting for the replication speed rescue. Remarkably, homologous recombination-defective cells exhibit a high level of endogenous reactive oxygen species. Consistently, homologous recombination-defective cells accumulate spontaneous γH2AX or XRCC1 foci that are abolished by treatment with N-acetyl-cysteine or maintenance at 3% O2. Finally, oxidative stress stimulated homologous recombination, which is suppressed by supplying deoxynucleotide precursors. Therefore, the cellular redox status strongly impacts genome duplication and transmission. Oxidative stress should generate replication stress through different mechanisms, including DNA damage and nucleotide pool imbalance. These data highlight the intricacy of endogenous replication and oxidative stresses, which are both evoked during tumorigenesis and senescence initiation, and emphasize the importance of homologous recombination as a barrier against spontaneous genetic instability triggered by the endogenous oxidative/replication stress axis.
authors.affiliation: CNRS UMR 8200, Gustave Roussy Cancer Institute, Université Paris-Saclay, Team labeled "Ligue 2014", Villejuif, France.
authors.forename: Therese
authors.initials: T
authors.lastname: Wilhelm
authors.affiliation: CNRS UMR 8200, Gustave Roussy Cancer Institute, Université Paris-Saclay, Team labeled "Ligue 2014", Villejuif, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Ragu
authors.affiliation: CNRS UMR 8200, Gustave Roussy Cancer Institute, Université Paris-Saclay, Team labeled "Ligue 2014", Villejuif, France.
authors.forename: Indiana
authors.initials: I
authors.lastname: Magdalou
authors.affiliation: Laboratoire de Biochimie et Toxicologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Pierre-Bénite, France.|Laboratoire de Chimie Analytique, Université de Lyon, Université Lyon 1, ISPB Faculté de Pharmacie, Lyon, France.
authors.forename: Christelle
authors.initials: C
authors.lastname: Machon
authors.affiliation: CNRS UMR 8200, Gustave Roussy Cancer Institute, Université Paris-Saclay, Team labeled "Ligue 2014", Villejuif, France.
authors.forename: Elodie
authors.initials: E
authors.lastname: Dardillac
authors.affiliation: Institut Curie, Centre de Recherche, Paris, France, UPMC Université Paris 06, Paris, France, CNRS UMR 3244, Paris, France.
authors.forename: Hervé
authors.initials: H
authors.lastname: Técher
authors.affiliation: Laboratoire de Biochimie et Toxicologie, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Pierre-Bénite, France.|Laboratoire de Toxicologie, Université Lyon 1, ISPB, Faculté de Pharmacie, Lyon, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Guitton
authors.affiliation: Institut Curie, Centre de Recherche, Paris, France, UPMC Université Paris 06, Paris, France, CNRS UMR 3244, Paris, France.
authors.forename: Michelle
authors.initials: M
authors.lastname: Debatisse
authors.affiliation: CNRS UMR 8200, Gustave Roussy Cancer Institute, Université Paris-Saclay, Team labeled "Ligue 2014", Villejuif, France.
authors.forename: Bernard S
authors.initials: BS
authors.lastname: Lopez
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells; D018385:Centrosome / Q000187:drug effects; D003412:Cricetulus; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D053263:Gene Regulatory Networks / Q000235:genetics; D006657:Histones / Q000235:genetics; D059765:Homologous Recombination / Q000235:genetics*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008938:Mitosis / Q000235:genetics*; D018384:Oxidative Stress / Q000187:drug effects / Q000235:genetics*; D000072760:Single Molecule Imaging; D000076105:X-ray Repair Cross Complementing Protein 1
chemical_list: D004268:DNA-Binding Proteins; D006657:Histones; D000076105:X-ray Repair Cross Complementing Protein 1; D006861:Hydrogen Peroxide; D000111:Acetylcysteine