Arabidopsis thaliana DNA Damage Response Mutants Challenged with Genotoxic Agents-A Different Experimental Approach to Investigate the TDP1α and TDP1β Genes.

Abstract

Background/Objectives: DNA damage response (DDR) is a highly conserved and complex signal transduction network required for preserving genome integrity. DNA repair pathways downstream of DDR include the tyrosyl-DNA phosphodiesterase1 (TDP1) enzyme that hydrolyses the phosphodiester bond between the tyrosine residue of topoisomerase I (TopI) and 3'-phosphate end of DNA. A small TDP1 subfamily, composed of TDP1α and TDP1β, is present in plants. The aim of this work was to investigate the role of the two TDP1 genes in the DDR context. Methods: A series of Arabidopsis thaliana DDR single and double mutants defective in the sog1, e2fb, pol2A, atm, and atr genes, treated with the genotoxic agents camptothecin (CPT, inhibitor of TopI) and NSC120686 (NSC, inhibitor of TDP1), were used. These compounds were specifically used due to their known impact on the TDP1 function. The effect of the treatments was assessed via phenotypic analyses that included germination percentage, speed, and seedling growth. Subsequently, the expression of the TDP1α and TDP1β genes was monitored through qRT-PCR. Results: Overall, the gathered data indicate that the atm mutant was highly sensitive to NSC120686, both phenotypically and concerning the TDP1α gene expression profiles. Alternatively, the upregulation of TDP1β in e2fb, pol2a, and atr supports its implication in the replication stress response. Conclusions: The current study demonstrates that genotoxic stress induced by CPT and NSC has a genotype-dependent effect reflected by a differential expression of TDP1 genes and early phenotypic development.