Uncovering the functions of TDP1α and TDP1β genes in DNA damage response: a case study using Arabidopsis thaliana mutant collections

DNA Damage Response (DDR) is a complex signalling network required for preserving genome integrity. Exogenous (irradiation, ultraviolet light, pollutants, and chemical agents) and endogenous (errors introduced during DNA replication, DNA strand breaks, reactive oxygen species) factors represent a constant threat to genome stability. DDR is well conserved between plants and animals although it is far less understood in plants, due to the high genome complexity. Among the players implicated in DDR downstream pathways, the tyrosyl-DNA phosphodiesterase1 (TDP1) enzyme prevents the accumulation of DNA damage by removing the covalent intermediates formed between toposiomerase I (TopI) and DNA. A small TDP1 gene subfamily, composed of TDP1α and TDP1β, has been identified in plants. While TDP1α was demonstrated to be involved in DNA repair and maintenance of genome integrity, the functions of TDP1β are less clear. Arabidopsis thaliana was chosen as model plant in this study. Different approaches, including data mining, phenotypic studies, and molecular analyses, were employed to investigate the roles of TDP1α and TDP1β genes in plant DDR. An extensive data mining approach, using platforms where RNA-seq and microarray data are deposited, allowed to collect information regarding the TDP1α and TDP1β gene expression in different tissues, genetic backgrounds, and stress conditions. Both genes are required during seed germination, they are differentially expressed in response to hormone treatments, while TDP1β seems to have a more prominent role in root development. A time- and stress-dependent response of TDP1α and TDP1β to different biotic and abiotic stresses was also evidenced. An experimental system was set up to verify some of the mined data, using Col0 and A. thaliana sog1 mutant treated with gamma rays. The expression of TDP1α and TDP1β genes was evaluated at different time-points. The results proven that the lack of SOG1 gene is strongly correlated with defects in DNA repair activation and induction of programmed cell death. Moreover, the experimental TDP1α and TDP1β gene expression data presented similar patterns whit those retrieved from the data mining approach. To collect more information about the roles of TDP1α and TDP1β genes in plant DDR, an innovative experimental system was set up, using a collection of A. thaliana mutants defective in the function of TDP1 genes (tdp1α, tdp1β, tdp1α/β) and DDR players (sog1, pol2a, e2fb, pol2a/sog1, pol2a/e2fb, sog1/e2fb, atm, atr). These mutants were treated with five genotoxic compounds (camptothecin, NSC120686, zeocin, curcumin, cisplatin) inducing different types of DNA damage. The first two agents are inhibitors of TopI and hTDP1, while zeocin is an inducer of double strand breaks (DSBs). Curcumin was showed to inhibit different DNA repair pathways in mammals, while cisplatin causes the formation of DNA crosslinks and, together with camptothecin, is an inducer of replicative stress. Both overlapping and gene-specific functions for the TDP1α and TDP1β genes were evidenced. While both genes could be involved in DNA-protein crosslink repair, TDP1β may play additional functions related to DSBs and replication stress. This data adds novel and specific information on the activity of this gene, far less studied and unique to plants.