D3109: The role of 53BP1 in DSB DNA repair in Aedes aegypti

Vector-borne diseases (VBD) are estimated to account for more than 17% of all infectious diseases, with over 80% of the world’s population at risk of one or more VBD. With increasing insecticide and drug resistance in vectors, improvements in solutions for vector control are imperative, with genetic approaches such as gene drive holding great promise. Gene drive approaches which rely on the cell’s machinery to repair a double-strand break (DSB) require homology-directed repair (HDR) as the repair pathway choice over non-homologous end joining (NHEJ), a choice that in vertebrates is partially regulated by the chromatin-binding protein 53BP1. If 53BP1, which is a positive regulator of NHEJ, is inhibited from binding to DSB-specific histone marks via an inhibitor or a dominant negative, HDR rates will increase and NHEJ rates will decrease in Aedes aegypti cell lines, A20 and Aag2. Using 10 chemicals which are cell cycle arresting and induce DSBs, A20 and Aag2 cells were incubated with 10 concentrations per chemical for 48 hours. After 48 hours, the chemical dilutions were removed and cell viability (RLU) was measured via Spectramax using luciferase reporter protocol and ATPlite 1step cell cytotoxicity kit. Initial results show Camptothecin is significantly more toxic to both cell lines compared to all other chemicals tested, with Etoposide showing the second highest cytotoxicity. Its decrease in cell viability appears inversely correlated to its increase in concentration and is a promising candidate for use in chemical transfections in conjunction with an inhibitor of NHEJ to measure possible HDR promotion.