Resistance of tardigrades to genotoxic stress : Importance of genome protection and DNA repair systems

• Main Author: Anoud, Marwan
• Other Authors: Structure et Instabilité des Génomes (STRING), Muséum national d'Histoire naturelle (MNHN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie - CNRS Chimie (INC-CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université Paris-Saclay, Carine Giovannangeli, Jean-Paul Concordet, Anne De Cian
• Format: Doctoral or Postdoctoral Thesis
• Language: French
• Published: HAL CCSD 2023
• Subjects: Tardigrade; Radiotolerance; DNA repair; Proteomics; Transcriptomics; Dsup; Radiotolérance; Réparation de l'ADN; Proméomique; Transcriptomique; [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry; Molecular Biology/Molecular biology; [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry; Molecular Biology/Genomics [q-bio.GN]; Acutuncus antarcticus; Antarc*; antarcticus
• Online Access: https://theses.hal.science/tel-04476478; https://theses.hal.science/tel-04476478/document; https://theses.hal.science/tel-04476478/file/104448_ANOUD_2023_archivage.pdf

Tardigrades are small aquatic metazoans capable of withstanding a wide variety of extreme abiotic conditions; in particular, desiccation and ionizing radiation which can induce extensive DNA damage and threaten genome integrity. In the tardigrade Ramazzottius varieornatus, a protein called RvDSup (for R. varieornatus DNA Damage Suppressor), is thought to protect the genome from X-rays. In order to better understand the resistance to DNA damage induced by ionizing radiation, we chose to focus on three tardigrade species: Hypsibius exemplaris, whose genome has been sequenced, Acutuncus antarcticus and Paramacrobiotus fairbanksi, which are still little studied and whose genomes are being sequenced. DNA single-strand breaks induced by doses of 100 or 1000 Gy of γ- radiation were analyzed in H exemplaris by migration of genomic DNA on denaturing agarose gel and double-strand breaks by Western blot with an antibody specific for the phosphorylated form of a histone H2AX of H. exemplaris. These analyses confirmed that DNA damage is dose dependent and is mostly repaired 72 h after irradiation. To identify mechanisms involved in tardigrade resistance, we measured changes in gene expression in response to γ-radiation and bleomycin, a radiomimetic antibiotic, by RNA-seq. These analyses allowed us to identify a set of genes highly overexpressed in response to both treatments in all 3 species analyzed. We completed this transcriptome analysis with a proteomic analysis in H. exemplaris, which confirmed that a large number of genes induced at the mRNA level are also induced at the protein level. These genes include many genes from the classical DNA double-strand break repair pathways (NHEJ, HR, MMEJ) but also genes of unknown function that appear to be unique to tardigrades. These genes are candidate genes for γ-radiation resistance in tardigrades whose function we chose to study. To this end, we performed gain-of- function experiments by heterologous expression in human U2OS cells, which allowed us to identify their ...