MYCOBACTERIAL TYPE IA TOPOISOMERASE SUFFER ENERGETICALLY AFTER C-TERMINAL DELETION

Regulating DNA topology is essential for cellular processes. This is handled by DNA topoisomerases. The Type IA family resolves DNA supercoils via a strand passage mechanism. Type IA topoisomerases feature toroidally shaped N-terminal domains, highly conserved from the archetypal E. coli topoisomerase IA. These enzymes cleave a single strand of denatured duplex DNA, creating a protein-mediated DNA gate that aids the passage of the intact strand. Despite the similarity in N-terminal domains, there is considerable variation in the C-terminal domains among different organisms. When compared to E. Coli, the mycobacteria genus is underexplored. These enzymes possess unique DNA-binding C-terminal domains that are involved in passing the intact strand during activity. Making them potential targets for highly specific antibiotics. A particularly notable candidate is Mycobacterium tuberculosis (Mtb). An organism that displays high antibiotic resistance. Mycobacterium smegmatis topoisomerase IA (MsmTopIA) serves as a valuable, nonpathogenic model for MtbTopIA. Using Single Molecule Magnetic Tweezers (MT) and computational modeling, we investigated wild-type MsmTopIA and variants with specific C-terminal domains removed via mutagenesis. Our study reveals nuanced behavioral differences among these enzymes. Further validating results from ensemble assays. We also outline the force and energy dependencies governing their supercoil relaxation abilities. These insights provide a deeper understanding of mycobacterial topoisomerases.