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"Role of DNA ligase E in DNA damage following chemical or radiation stress"

par Panagiotou, Vassilios
Président du jury Roisin, Yves
Promoteur Van Doninck, Karine
Co-Promoteur Guérineau, Marc
Publication Non publié, 2024-06-26

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Résumé :

Background. All organisms face external and internal factors causing DNA damage, triggeringcrucial repair mechanisms to preserve genomic integrity. Bdelloid rotifers are recognized fortheir ability to withstand challenging environmental conditions. To understand and decipherthese extraordinary repair abilities, A. vaga constitutes an outstanding model. KVD lab showeda group of upregulated proteins following irradiation experiments. This ligase E has drawnattention due to its phylogenetic nature, being related to the class of prokaryotes. Indeed, it hasbeen found in several bdelloid eukaryotic organisms other than Adineta vaga, as well as in fungiwhere remarkably efficient and resistant responses to extreme environmental stress areobserved. Finally, Nicolas Emilien et al. 2023 demonstrated an increase in the survival rate ofhuman cells (HEKT 293 cell line) transfected with the constructs AvLigE_A and AvLigE_Bafter irradiation with potentially lethal doses. For these reasons, ligase E is strongly suspectedto be involved in this resistance capability.Aim. The aim is to decipher the mechanisms of ligase E, specifically to determine if it indeedplays a significant role in the efficiency of molecular repair.Materials and methods. The aim is first to test ligase E through heterologous expression in themodel prokaryotic organism E. coli, also test the effects of a radiomimetic agent (bleomycin)on Adineta vaga to determine if it is possible to substitute the irradiation technique withbleomycin. Finally, trials were conducted on human HEK293 cells transfected with Ligase E tomeasure accumulation of double-strand break markers (γH2AX) after exposure tobleomycin/Ionizing irradiation. This allowed to detect any effect of Ligase E upon efficacityDNA repair mechanisms compared to non-transfected cells.Results. Although the overexpression of Ligase E did occur in bacteria induced by IPTG, thephenotype showed higher mortality in induced bacteria than in non-induced ones. This is likelythe result of a non-functional protein or too high IPTG concentration, leading to toxicity.Experiments using bleomycin upon Adineta vaga have shown overexpression of PNKP andPolß in addition to AvLigE-B. No double strand breaks have been highlighted on Pulsed FieldElectrophoresis Gel (PFGE). The accumulation of γH2AX does not appear to be greater in nontransfectedcells compared to transfected cells.Conclusion. Escherichia coli does not appear to be a reliable model for further experimentsaiming to decipher the function of AvLigE. Concerning bleomycin tests, additional experimentsare needed to confirm the presence of single-strand breaks. Finally, the similar accumulationobserved in non-transfected cells following bleomycin/irradiation treatment does not suggestenhanced DNA repair efficiency conferred by Ligase E.