Inhibition of DNA Topoisomerase1 and Tyrosyl DNA phosphodiesterase1: a new approach to study the DNA damage response during Medicago truncatula seed germination and seedling development (Inibizione della DNA Topoisomersi1 e della Tirosil DNA Fosfodiesterasi1: un nuovo approccio per studiare la risposta al danno del DNA durante la germinazione dei semi di Medicago truncatula e lo sviluppo della piantina)

Environmental agents and endogenous processes steadily inflict DNA damage affecting genome stability, plant growth and productivity. Like animals, plants as well have developed the DNA damage response (DDR) pathway able to perceive DNA damage, and, through transducers and mediators, it triggers effective responses. Genomic protection and stability are fundamental to ensure cellular functions. The control of cell-cycle arrest and the repair of DNA damage are essential processes to avoid the propagation of possible mutations. Within the context of the seed germination process, characterized by active cell proliferation, DNA damage must be repaired before the start of cell division in order to ensure the development of robust plants. The purpose of this study was to investigate the relation between DNA topoisomerase 1 (TOP1) and tyrosyl DNA phosphodiesterase 1 (TDP1) in Medicago truncatula seedlings. This was done by using specific inhibitors, namely camptothecin (CPT), inhibitor of TOP1 enzyme, and NSC120686, reported as inhibitor of the human TDP1 enzyme. In human cells, TDP1 has been implicated in the resistance to various types of anticancer agents, including radiation and topoisomerase poisons, due to its ability to repair various types of DNA lesions. The inhibitors were delivered to M. truncatula seeds, alone or in combination, to exploit their function in altering the enzyme–DNA interactions (cleavage complexes). After collision with replication forks, both DNA and RNA synthesis convert cleavage complexes into DNA lesions causing double strand breaks (DBS). Phenotypic (germination percentage and speed, seedling growth) and molecular (DNA damage assay, gene expression profiling) analyses were then used to characterize the response to the treatments. Although no effect on seed germination were observed, the seedling growth was significantly inhibited especially by CPT and the combination CPT+NSC. The comet assay revealed accumulation of DNA damage in response to all treatments. Subsequently, the expression of plant TDPs, TOPs, as well as several genes with known roles in alternative DNA repair pathways and cell-cycle regulation were investigated by quantitative RealTime PCR (qRT-PCR). Interestingly, different patterns of gene expression were observed in response to the treatments, namely a contrasting response was noticed when CPT and NSC were provided alone, while a cumulative effect was detected in the case of the CPT+NSC combination. Thus, these results provide new and important findings in relation to the roles played by TDP1 and TOP1 genes in DDR during early seedling development of the M. truncatula model legume.