Type IA topoisomerases are ubiquitous in the three kingdoms of life, and play critically important roles in maintaining proper DNA topology during the vital cellular processes of replication, transcription, recombination, and repair. The PI?s research activities have provided seminal biochemical and structural findings for this class of essential genome regulator, and continue to address key questions on the catalytic mechanism of type IA topoisomerases and provide new insights into their functional and regulatory interactions. This information is needed to utilize type IA topoisomerases present in every bacterial pathogen as a novel therapeutic target for finding new antibiotics to help face our serious global health challenge of antibiotic resistance. Type IA topoisomerases catalyze the relaxation of negatively supercoiled DNA by cleaving a single DNA strand in the underwound duplex DNA and passing the complementary DNA single strand through the break before religation of the cleaved strand to change the DNA topology. The molecular mechanism of the large enzyme conformational changes that are required for the coordinated movement of the passing DNA in and out of the DNA gate is the critical barrier for elucidating how bacterial TOP1 can relax negatively supercoiled DNA with high efficiency to prevent hypernegative DNA supercoiling and R-loop stabilization that can arise during transcription. This important function of bacterial TOP1 is facilitated by the direct TOP1 interaction with RNA polymerase that we have characterized and found to be targeted by endogenous toxin in mycobacteria. For future studies, we will create new TOP1 mutants perturbed in interdomain interactions at a distance from the active site and investigate the effect on the in vivo relaxation activity and in vitro interactions with DNA substrate. Mutants with reduced catalytic efficiency will be further studied to determine if the mutations affected the gate opening-closing dynamics and DNA strand passage. We will capture new structural conformations of the TOP1-DNA complex that may represent different stages of the catalytic cycle with X-ray crystallography and measure the gate opening-closing dynamics with single molecule assays. Structural studies will also incorporate other ligands including RNA. Type IA topoisomerases have evolved to include TOP1 and TOP3 enzymes in all three kingdoms of life that possess dual activities on both DNA and RNA substrates. The RNA topoisomerase activity of human TOP3B has been shown to be required for neurodevelopment and the enzyme is also involved in R-loop suppression and genome stability. We are modeling the RNA interaction of type IA topoisomerases with molecular dynamics simulations to determine how the DNA and RNA substrate may be accommodated differentially by change in enzyme conformation and interacting residues. We have initiated studies to identify a separation of function mutation or small molecule probe that can be used to distinguish between the DNA and RNA topoisomerase activity in vivo. Such research tools for study of cellular RNA topoisomerase activity and regulation will have an important and lasting impact on the field.
The goal of this project is to advance our knowledge on the structure, mechanism, interactions and regulation of type IA topoisomerases found in all life forms. The results will facilitate the discovery of much needed new antibiotics leads against bacterial topoisomerase I, a validated drug target for treatment of drug resistant infections. The investigation will also develop tools that can be used in vivo to differentiate the dual DNA and RNA topoisomerase activity of human topoisomerase 3B implicated in neurodevelopment and genome stability.