Research Projects

Human DNA Topoisomerase 1 (Top1) is crucial for DNA supercoils restoration and genetic transmission. It is selectively trapped by anticancer drugs like camptothecin (CPT) and its clinical analogs, Topotecan and Irinotecan, which form the backbone of chemotherapy for lung, ovarian, and breast cancer patients. Top1 breaks produce toxic protein-linked DNA breaks (Top1cc), which can cause genomic instability and cell death. Arginine methylation is a key posttranslational modification responsible for the addition of the methyl group on about 0.5% of arginine residues in humans and is involved in various cellular events, including DNA repair and genome maintenance. Protein Arginine Methyltransferase 5 (PRMT5) catalyzes the formation of symmetric di-methylation (SDMA) of arginine residues to acceptor proteins. PRMT5 inhibitors are emerging as potent anticancer agents. Preliminary data show novel binding of human Top1 with PRMT5 in co-immunoprecipitation experiments, independent of TDP1 or Top1cc repair. Top1-mediated DNA relaxation activity was markedly reduced in PRMT5 knockdown cells. The proposed study aims to dissect the biological significance of the Top1-PRMT5 association by generating PRMT5 knockout cells using CRISPR-Cas9 technology. The study will uncover novel arginine methylation sites of human Top1 and test the role of methylation in the catalytic cycle of Top1 in single turnover equilibrium cleavage/relegation assays and DNA binding assays. The study will also evaluate the preclinical rationale for the combination of PRMT5 and Top1 inhibitors in tumor regression as chemotherapeutic strategies using in vivo tumors generated as xenografts in mice.