title: Oxidative stress in mammalian cells impinges on the cysteines redox state of human XRCC3 protein and on its cellular localization.
abstract: In vertebrates, XRCC3 is one of the five Rad51 paralogs that plays a central role in homologous recombination (HR), a key pathway for maintaining genomic stability. While investigating the potential role of human XRCC3 (hXRCC3) in the inhibition of DNA replication induced by UVA radiation, we discovered that hXRCC3 cysteine residues are oxidized following photosensitization by UVA. Our in silico prediction of the hXRCC3 structure suggests that 6 out of 8 cysteines are potentially accessible to the solvent and therefore potentially exposed to ROS attack. By non-reducing SDS-PAGE we show that many different oxidants induce hXRCC3 oxidation that is monitored in Chinese hamster ovarian (CHO) cells by increased electrophoretic mobility of the protein and in human cells by a slight decrease of its immunodetection. In both cell types, hXRCC3 oxidation was reversed in few minutes by cellular reducing systems. Depletion of intracellular glutathione prevents hXRCC3 oxidation only after UVA exposure though depending on the type of photosensitizer. In addition, we show that hXRCC3 expressed in CHO cells localizes both in the cytoplasm and in the nucleus. Mutating all hXRCC3 cysteines to serines (XR3/S protein) does not affect the subcellular localization of the protein even after exposure to camptothecin (CPT), which typically induces DNA damages that require HR to be repaired. However, cells expressing mutated XR3/S protein are sensitive to CPT, thus highlighting a defect of the mutant protein in HR. In marked contrast to CPT treatment, oxidative stress induces relocalization at the chromatin fraction of both wild-type and mutated protein, even though survival is not affected. Collectively, our results demonstrate that the DNA repair protein hXRCC3 is a target of ROS induced by environmental factors and raise the possibility that the redox environment might participate in regulating the HR pathway.
authors.affiliation: Institut Curie, Centre de Recherche, Orsay, France ; CNRS, UMR3348, Orsay, France.
authors.forename: Pierre-Marie
authors.initials: PM
authors.lastname: Girard
authors.forename: Dany
authors.initials: D
authors.lastname: Graindorge
authors.forename: Violetta
authors.initials: V
authors.lastname: Smirnova
authors.forename: Pascal
authors.initials: P
authors.lastname: Rigolet
authors.forename: Stefania
authors.initials: S
authors.lastname: Francesconi
authors.forename: Susan
authors.initials: S
authors.lastname: Scanlon
authors.forename: Evelyne
authors.initials: E
authors.lastname: Sage
mesh_terms: D000818:Animals; D016466:CHO Cells; D002166:Camptothecin / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D004260:DNA Repair / Q000187:drug effects; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D059765:Homologous Recombination / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D017382:Reactive Oxygen Species; C110901:X-ray repair cross complementing protein 3; D003545:Cysteine; D002166:Camptothecin