The DNA relaxation activity and covalent complex accumulation of Mycobacterium tuberculosis topoisomerase I can be assayed in Escherichia coli:application for identification of potential FRET-dye labeling sites

摘要

Background: Mycobacterium tuberculosis topoisomerase I (MtTOP1) and Escherichia coli topoisomerase I havehighly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains.To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it isdesirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence fromaccumulation of its covalent complex.Results: We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topAfunction of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation thatinhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues inthe MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical orspectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for developmentof high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites inMtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteinesubstitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 wasutilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement withfluorophore-labeled oligonucleotide substrate.Conclusions: The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can bemeasured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapytargeting this essential enzyme