Abstract
BackgroundMycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. It shares a common transesterification domain with other type IA DNA topoisomerases. There is, however, no homology between the C-terminal DNA binding domains of Escherichia coli and M. tuberculosis DNA topoisomerase I proteins.ResultsA new protocol for expression and purification of recombinant M. tuberculosis DNA topoisomerase I (MtTOP) has been developed to produce enzyme of much higher specific activity than previously characterized recombinant enzyme. MtTOP was found to be less efficient than E. coli DNA topoisomerase I (EcTOP) in removal of remaining negative supercoils from partially relaxed DNA. DNA cleavage by MtTOP was characterized for the first time. Comparison of DNA cleavage site selectivity with EcTOP showed differences in cleavage site preferences, but the preferred sites of both enzymes have a C nucleotide in the -4 position.ConclusionRecombinant M. tuberculosis DNA topoisomerase I can be expressed as a soluble protein and purified in high yield from E. coli host with a new protocol. Analysis of DNA cleavage with M. tuberculosis DNA substrate showed that the preferred DNA cleavage sites have a C nucleotide in the -4 position.
Highlights
Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product
Expression and purification of M. tuberculosis topoisomerase I Genome sequencing of M. tuberculosis H37Rv strain has revealed the presence of topA gene Rv3646c which encodes a DNA topoisomerase I (MtTOP) comprising of 934 amino acids with an estimated molecular weight of 102.3 kDa [11]
Our efforts to express and purify recombinant M. tuberculosis DNA topoisomerase I (MtTOP) in E. coli BL21 (DE3) by induction of the T7 promoter were frustrated by the insolubility of the expressed protein
Summary
Mycobacterium tuberculosis DNA topoisomerase I is an attractive target for discovery of novel TB drugs that act by enhancing the accumulation of the topoisomerase-DNA cleavage product. The major classes of topoisomerases, type IA and type IIA, modify DNA topology by transiently cleaving and rejoining one or two strands of DNA respectively [1,2]. Both of these classes form a 5'-phosphotyrosyl enzyme-DNA linkage during the catalytic cycle of DNA cleavage and religation [1]. Bacterial DNA gyrase and topoisomerase IV from the type IIA class are targets of antibiotics such as quinolones and fluoroquinolones These antibiotics exhibit their bactericidal properties by trapping the covalent protein-DNA complexes formed by (page number not for citation purposes)
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