Magnetic tweezers investigations of the type IA topoisomerase of Mycobacterium smegmatis

Magnetic tweezers instruments use magnets to apply force and torque to paramagnetic beads attached to individual tethered molecules. This single molecule technique has proved useful for studying the intrinsic properties of DNA as well as the effect of various proteins on DNA structures. Topoisomerases are a class of enzyme that relax topologically strained DNA. They are essential for genome function and integrity, and as such are potential targets for antibiotics and chemotherapeutics. Type IA topoisomerases are a class of topoisomerase that relieve torsional strain in negatively supercoiled DNA. These enzymes work by cleaving the backbone of one strand of a DNA duplex, passing the other strand through the break, and re-ligating the DNA. Using magnetic tweezers, we have characterized the supercoil relaxation activity and conformational dynamics of Mycobacterium smegmatis topoisomerase I (MsmTOP1). In addition to a conserved core domain common to all type IA topoisomerases, Mycobacteria type IA topoisomerases have unique DNA-binding C-terminal domains that are involved in passage of the second DNA strand through a protein-mediated DNA gate. To better understand the role of these domains, we also measured the activity of a series of C-terminal deletion mutants. We found key differences in the relaxation rates and pausing behaviors of the mutants. Our results provide new information on the relationship between strand passage and relaxation activity for mycobacteria topoisomerases.