Mechanistic Studies Of A Novel AGO2-RAD51Interaction In Facilitating DSB Repair

DNA double strand breaks (DSB) are highly cytotoxic DNA damage and will severely threaten the physical and genetic integrity of genome if not properly repaired. Inappropriate repair of broken DNA ends may result in the generation of harmful genomic rearrangements. The accumulation of DSBs could lead to apoptosis or development of cancer.In order to minimize the catastrophic consequence, the eukaryotic cells are equipped with2major mechanisms to deal with DSB. One is nonhomologous DNA end-joining (NHEJ) and the other one is homologous recombination (HR). NHEJ can directly rejoin two DNA ends and functions mainly during phases of cell cycle, however, is often error-prone. On the contrary, HR is restricted in S and G2phases and can utilize the homologous strand as template to recover the damaged strand. Previously, protein enzymes are considered as the only component in DNA repair. Recently, a novel class of small RNA, called diRNA, is reported to play a role DSB repair. diRNA is21-nucleotide small RNAs are produced from the sequences in the vicinity of DSB sites. Its generation depends on nuclease Dicer and effect depends on AGO2. However, the mechanism of how diRNA facilitate DSB repair remains largely unknown.Here we report a novel protein-protein interaction between AGO2and RAD51through screening new AG02binding partners. We also show that AG02can bind to the vicinity of DSB site and facilitate the RAD51accumulation on DSB. Exogenous AG02IP identified RAD51and several DNA repair proteins as novel AG02partners. Among them, only the RAD51foci induced by IR treatment are affected by AGO2depletion, indicating diRNA and AGO2could influence RAD51recruitment. Then the AGO2-RAD51interaction was verified by exogenous and endogenous RAD51IP. Further results also find that IR treatment can dramatically enhance AGO2-RAD51interaction. However, AG02fragment IP failed to identify which domain is responsible for this interaction. diRNA binding and catalytic activity of AG02are dispensable for the interaction with RAD51. diRNA depletion by Dicer knockdown also didn’t affect AGO2-RAD51interaction, indicating the interaction is independent of small RNA. At last, ChIP assay identified the binding of AGO2near DSB sites.Taken together, these results suggest that AG02can facilitate RAD51recruitment through its bind to DSB site and direct interaction with RAD51. Thus, this project made a breakthrough in illustrating the molecular mechanism of how diRNA facilitate DSB repair, establish a bridge between small RNA pathway and DNA repair pathway and also broaden the understanding of the cellular function of both pathways. However, the detailed mechanism between diRNA and DSB repair requires further researches.