Aedes aegypti transmits the viruses which cause yellow fever, dengue fever, and dengue hemorrhagic fever. Homing endonucleases are selfish genetic elements which introduce double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this study, we aimed to validate a somatic assay to detect recombinant homing endonuclease (rHE)-induced dsDNA breaks in both cultured cells and adult female Ae. aegypti. While the cell culture-based two plasmid assay used to test rHE ability to induce dsDNA breaks was inconclusive, assays used to test rHEs in Ae. aegypti were successful. Recognition sequences for various rHEs were introduced into Ae. aegypti through germline transformation, and imperfect repair at each of these exogenous sites was evaluated. In mosquitoes containing a single exogenous HE site, imperfect gap repair was detected in 40% and 21% of clones sequenced from mosquitoes exposed to I-PpoI and Iâ SceI, respectively. In mosquitoes containing two exogenous HE sites flanking a marker gene (EGFP), 100% of clones sequenced from mosquitoes exposed to I-PpoI, I-CreI, and I-AniI demonstrated excision of EGFP. No evidence of EGFP excision or imperfect repair at any HE recognition site was detected in mosquitoes not exposed to a rHE. In summary, a somatic genomic footprint assay was developed and validated to detect rHE or other meganuclease-induced site-specific dsDNA breaks in chromosomal DNA in Ae. aegypti. / Master of Science in Life Sciences

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder because of a LMNA gene mutation that produces a mutant lamin A protein (progerin). Progerin also has been correlated to physiological aging and related diseases. However, how progerin causes the progeria remains unknown. Here, we report that the large subunit (RFC1) of replication factor C is cleaved in HGPS cells, leading to the production of a truncated RFC1 of ∼75kDa, which appears to be defective in loading proliferating cell nuclear antigen (PCNA) and pol δ onto DNA for replication. Interestingly, the cleavage can be inhibited by a serine protease inhibitor, suggesting that RFC1 is cleaved by a serine protease. Because of the crucial role of RFC in DNA replication, our findings provide a mechanistic interpretation for the observed early replicative arrest and premature aging phenotypes of HPGS and may lead to novel strategies in HGPS treatment. Furthermore, this unique truncated form of RFC1 may serve as a potential marker for HGPS.