Oxidative DNA Damage and Repair in the Radioresistant Archaeon Thermococcus gammatolerans.
The hyperthermophilic archaeon Thermococcus gammatolerans can resist huge doses of γ-irradiation, up to 5.0 kGy, without loss of viability. The potential to withstand such harsh conditions is probably due to complementary passive and active mechanisms, including repair of damaged chromosomes. In this work, we documented the formation and repair of oxidative DNA lesions in T. gammatolerans. The basal level of the oxidized nucleoside, 8-oxo-2'-deoxyguanosine (8-oxo-dGuo), was established at 9.2 (± 0.9) 8-oxo-dGuo per 106nucleosides, a higher level than those usually measured in eukaryotic cells or bacteria. A significant increase in oxidative damage, i.e., up to 24.2 (± 8.0) 8-oxo-dGuo/106nucleosides, was measured for T. gammatolerans exposed to a 5.0 kGy dose of γ-rays. Surprisingly, the yield of radiation-induced modifications was lower than those previously observed for human cells exposed to doses corresponding to a few grays. One hour after irradiation, 8-oxo-dGuo levels were significantly reduced, indicating an efficient repair. Two putative base excision repair (BER) enzymes, TGAM_1277 and TGAM_1653, were demonstrated both by proteomics and transcriptomics to be present in the cells without exposure to ionizing radiation. Their transcripts were moderately upregulated after gamma irradiation. After heterologous production and purification of these enzymes, biochemical assays based on electrophoresis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectrometry indicated that both have a β-elimination cleavage activity. TGAM_1653 repairs 8-oxo-dGuo, whereas TGAM_1277 is also able to remove lesions affecting pyrimidines (1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd)). This work showed that in normal growth conditions or in the presence of a strong oxidative stress, T. gammatolerans has the potential to rapidly reduce the extent of DNA oxidation, with at least these two BER enzymes as bodyguards with distinct substrate ranges.
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Ewa
E
Barbier
University of Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.|University of Perpignan, IHPE - UMR 5244 CNRS/IFREMER/Univ. Montpellier, Montpellier, F-34095, France.
Arnaud
A
Lagorce
University of Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.
Amine
A
Hachemi
University of Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.
Murielle
M
Dutertre
University of Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.
Aurore
A
Gorlas
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Lucie
L
Morand
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Christine
C
Saint-Pierre
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Jean-Luc
JL
Ravanat
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Thierry
T
Douki
CEA, DSV-Li2D, Laboratory "Innovative Technologies for Detection and Diagnostics", BP 17171, Bagnols-sur-Cèze, F-30207, France.
Jean
J
Armengaud
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Didier
D
Gasparutto
University of Paris-Sud, Institute for Integrative Biology of the Cell (I2BC), Université Paris Saclay, CEA, CNRS, Orsay, France.
Fabrice
F
Confalonieri
University of Grenoble Alpes, INAC, LCIB , F-38000 Grenoble, France.|CEA, INAC, SyMMES, F-38000 Grenoble, France.
Jean
J
Breton
D004249:DNA Damage; D004260:DNA Repair; D019641:DNA, Archaeal / Q000737:chemistry*; D010084:Oxidation-Reduction; D040901:Proteomics; D011836:Radiation Tolerance; D019712:Thermococcus / Q000235:genetics*; D059467:Transcriptome
D019641:DNA, Archaeal