Measuring the effects of C-terminal deletion in mycobacterial type IA topoisomerases using magnetic tweezers.

Abstract

In the cell, protein replication and transcription are processes that introduce supercoils to DNA. Regulation of this DNA topology is handled by enzymes called DNA topoisomerases. Type IA topoisomerases resolve supercoils in discrete steps by cleaving a single strand of duplex DNA, creating a protein mediated DNA gate, and passing the opposing strand through the opening. The mycobacteria genus of topoisomerases has been less well-studied than the prototypical type IA topoisomerases of E. coli. Two enzymes of interest are Mycobacterium tuberculosis topoisomerase IA (mtbTopIA) and Mycobacterium smegmatis topoisomerase IA (msmTopIA). Mycobacteria type IA topoisomerases have DNA-binding C-terminal domains that are involved in passing the second DNA strand of a melted duplex through the DNA gate. Because these C-terminal domains are unique, they are potential targets for highly specific antibiotics. Using single-molecule magnetic tweezers (MT), we have characterized the relaxation behavior of wild type msmTopIA as well as variants that have had domains from their C-termini removed by mutagenesis. We report clear differences in behavior between the enzymes studied, as well as provide further exploration regarding the purpose and mechanism of the C-termini of these enzymes. In addition, we report that our results agree with those done previously in ensemble assays.