Analysis of DNA relaxation and cleavage activities of recombinant Mycobacterium tuberculosis DNA topoisomerase I from a new expression and purification protocol

Abstract

Background: 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. 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.

Results: A 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.

Conclusion: Recombinant 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.

Figure 1

Expression of recombinant MtTOP in E. coli Arctic express (DE3)RP strain. SDS PAGE analysis of total cell lysate (lanes 1,3) and soluble cell lysate (lane 2,4) of Arctic express (DE3)RP cells transformed with pLIC-MTOP and induced with 0 mM (lanes 1,2) and 1 mM IPTG (lanes 3,4) at the end of 24 hours of induction in LB at 12°C. M: molecular weight standards.

Figure 2

SDS PAGE at different stages of MtTOP purification. A. Wash (lanes 1–4) and elution (lanes 5–9) fractions of fusion protein passed through a Ni-NTA agarose column. Lanes M represents molecular marker and lanes L and F represent the initial load and flow through of the Ni-NTA agarose column before wash and elution. B. Wash fractions (lanes 1–4) and potassium chloride gradient elution fractions (lanes 5–14) of TEV digested fusion protein loaded onto single-strand DNA cellulose column.

Figure 3

Relaxation of negatively supercoiled DNA to determine unit activity. A. 500 ng of negatively supercoiled plasmid DNA was subjected to relaxation as per the conditions in "materials and methods" with the indicated amount of EcTOP and MtTOP. B. DNA relaxation by different amounts of EcTOP and MtTOP was quantitated as percent relaxation for comparison. The percent relaxation was determined by dividing the distance between the negatively supercoiled band (SC); and the weighted center of the partially relaxed band (PR); by the distance between the supercoiled band (SC); and the fully relaxed band (FR). In simple terms, percent relaxation = (SC-PR)/(SC-FR) [18]. The percent relaxation values reported are averages of at least three independent experiments. Error bars denote the standard error of mean.

Figure 4

Time course of DNA relaxation. A. 50 ng of EcTOP (lanes 1–10) and MtTOP (lanes 11–20) was utilized in a relaxation assay as described in the "materials and methods" section over time course of 0, 10, 20, 30, 45, 60, 75, 90, 120, 180 sec respectively for each enzyme. B. Quantitation of the relaxation time course. The percent relaxation values reported are averages of at least three independent experiments. Error bars denote the standard error of mean.

Figure 5

Effect of Mg²⁺ ion concentration on DNA relaxation. Effect of different concentrations of Mg²⁺ ion concentration ranging from 0–20 mM in DNA relaxation assays containing (A) high level, 1 enzyme unit corresponding to 100 ng of EcTOP and 500 ng of MtTOP; or (B) low level (50 ng) of EcTOP or MtTOP. C: control with no enzyme.

Figure 6

Mapping cleavage sites on single-stranded DNA substrate. A. Single-stranded DNA substrate Mtop (216 bases) amplified from topA gene of M. tuberculosis was utilized to map cleavage sites of EcTOP and MtTOP. Lanes 1–4: cleavage reactions containing 400, 300, 200, 100 ng of EcTOP respectively. Similarly lanes 5–8 contain 400, 300, 200, 100 ng of MtTOP respectively. B. Single-stranded DNA substrate PBAD (556 bases) amplified from pBAD/thio plasmid was utilized to map cleavage sites of EcTOP and MtTOP. Lanes 1–2: cleavage reactions containing 400, 300 ng of EcTOP respectively. Similarly lanes 3–4 contain 400, 300 ng of MtTOP respectively. Lanes A, G, C, T containing the sequencing reactions with the corresponding nucleotide termination mixes were electrophoresed along with EcTOP and MtTOP cleavage reactions to determine the cleavage sites.