The Role of 53BP1 in Double-Strand Break DNA Repair in Aedes aegypti

Abstract

Aedes aegypti is a mosquito species of high medical relevance as a vector of arboviruses such as dengue, yellow fever, Zika, and chikungunya. Vector control via drainage and insecticide use has been the main strategy to mitigate disease transmission for over a century, yet population shifts toward urbanization and increased insecticide resistance have favored vectors. Advancements in gene editing using the CRISPR/Cas9 endonuclease system has made genetic control mechanisms an attractive alternative. CRISPR/Cas9 utilizes an organism’s endogenous repair pathways, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ), to repair double-strand DNA break (DSB) induced via the Cas9 endonuclease. Repair pathway choice influences the resulting product and repair via the HDR pathway is necessary when the desired outcome is integration of genetic constructs, however NHEJ is the cell’s pathway of choice most often due to the lower energy costs and faster rates. The DSB DNA repair pathway choice in vertebrates is partially regulated by the chromatin-binding protein 53BP1, an NHEJ-promoting protein, which competes with BRCA1, an HDR-promoting protein. Factors influencing repair pathway choice in Ae. aegypti are uncertain as neither 53BP1 nor BRCA1 orthologs have been identified at present. Through a BLAST of Homo sapiens TP53BP1 and 53BP1 related domains against the Ae. aegypti genome, we identified an Ae. aegypti 53BP1 ortholog with a Tudor domain and tandem BRCT domains. To investigate the function of this 53BP1 ortholog, we designed five GFP-tagged dominant negative (DN) versions to inhibit endogenous 53BP1 and eliminate downstream recruitment of NHEJ factors. Chemotherapy agents were used as the DNA damaging agents for DSB induction and cell cycle arrest. Using a cytotoxicity assay, a dose-dependent relationship was identified between camptothecin and mortality of the Ae. aegypti cell lines, A20 and Aag2. These cytotoxicity assays were performed with transfections of each DN 53BP1 construct and a decrease in cell mortality was observed with multiple constructs. Additionally, nuclear localization of 53BP1 mutants was confirmed via fluorescent microscopy, with four of the five DN constructs displaying nuclear localization, moreso than the control plasmid with a known nuclear localization signal. The nuclear localization, in addition to the decreased mortality observed with multiple DN 53BP1 mutants, demonstrates the potential of 53BP1 as a target for locally inhibiting NHEJ in Ae. aegypti, without the deleterious effects of global NHEJ inhibition.