Argonaute 2 regulates nuclear DNA damage, repair, and phenotypes in Arabidopsis under genotoxic stress.

Argonaute (AGO) proteins are involved in gene expression and genome integrity during biotic and abiotic stress responses. AGO2 mediates double-strand break (DSB) repair in DNA damage response (DDR) induced by genotoxic stress. However, beyond DSB repair, the involvement of AGO proteins in DDR remains unknown. To investigate the potential roles and functions of AGO2 in DDR, we exposed three different ago2 mutants, each harboring a T-DNA insertion in the promoter, the N-terminal domain of exon 2, or the P-element-induced wimpy testis (PIWI) domain of exon 3, to genotoxic stress, and examined their DDR phenotypes. DDR phenotypes, such as root cell death and growth inhibition following γ-irradiation and zeocin treatment, were significantly suppressed by defects in the promoter or N-terminal domain of AGO2 but not by defects in the PIWI domain, which is responsible for RNA silencing. The weak DDR phenotypes were rescued by AGO2 overexpression and were attributed to reduced nuclear DNA damage despite impaired DNA repair, including DSB repair, as shown in comet and γH2AX assays. These results suggest that AGO2 regulates overall nuclear DNA damage and DDR phenotypes beyond DSB repair through the N-terminal domain rather than the PIWI domain. The potential role of AGO2 in the DDR implies that DNA repair may not be the primary factor for determining susceptibility to genotoxic stress.