Type IA topoisomerase inhibition by clamp closure

Abstract

Bacterial DNA topoisomerase I (topoI) catalyzes relaxation of negatively supercoiled DNA. The enzyme alters DNA topology through protein-operated DNA gate, switching between open and closed conformations during its reaction. We describe the mechanism of inhibition of Mycobacterium smegmatis and Mycobacterium tuberculosis topoI by monoclonal antibodies (mAbs) that bind with high affinity and inhibit at 10-50 nM concentration. Unlike other inhibitors of topoisomerases, the mAbs inhibited several steps of relaxation reaction, namely DNA binding, cleavage, strand passage, and enzyme-DNA dissociation. The enhanced religation of the cleaved DNA in presence of the mAb indicated closing of the enzyme DNA gate. The formation of enzyme-DNA heterocatenane in the presence of the mAbs as a result of closing the gate could be inferred by the salt resistance of the complex, visualized by atomic force microscopy and confirmed by fluorescence measurements. Locking the enzyme-DNA complex as a closed clamp restricted the movements of the DNA gate, affecting all of the major steps of the relaxation reaction. Enzyme trapped on DNA in closed clamp conformation formed roadblock for the elongating DNA polymerase. The unusual multistep inhibition of mycobacterial topoisomerases may facilitate lead molecule development, and the mAbs would also serve as valuable tools to probe the enzyme mechanism.

Direct regulation of topoisomerase activity by a nucleoid-associated protein

“…The relaxation reaction proceeds in sequential steps–binding of TopoI to DNA, cleavage, strand passage and religation (38). The effect of MtHU in each step of the reaction was examined using the assays described earlier and in Materials and Methods (18,29,33,37). In the first step of the process, MtHU did not affect the binding of MtTopoI to single-stranded 32 mer STS DNA at lower concentrations.…”

“…Next, to determine the effect of MtHU on the strand passage activity of MtTopoI, the enzyme was incubated with a radiolabeled relaxed circular form of pUC18 DNA (described in Materials and Methods) so that the DNA occupied the enzyme cavity (36). The occupancy of pUC18 DNA was ensured by using an anti-TopoI clamp-closing monoclonal antibody, which is shown to lock the TopoI clamp (37), thus trapping the plasmid DNA in the cavity of the enzyme. The TopoI–DNA complexes were incubated with MtHU or MtHUD17A and STS oligonucleotides were added to form an enzyme-DNA gate (schematic in Figure 7A).…”

“…The nick-translated radiolabeled pUC18 plasmid DNA was incubated with 500 nM MtTopoI in an assay buffer [40 mM Tris-HCl (pH 8.0), 20 mM NaCl and 1 mM EDTA, 10% glycerol] for 15 min on ice. To determine the initial counts in the control reaction, an anti-topoI clamp closing antibody 1E4F5 (37) was added to trap the plasmid DNA in the enzyme cavity. Two times molar excess of MtHU or MtHUD17A mutant were added followed by the addition of 5 pmoles of 32-mer STS oligonucleotides.…”

Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase

“…Cell debris was removed by centrifugation and the supernatant was used as input sample in subsequent IP experiments. The samples were incubated overnight at 4°C on a rotating wheel with antibodies (polyclonal anti-GyrA or monoclonal anti-Topo I [51]. An IP experiment without antibody served as negative control (Mock IP).…”

Targeting Bacterial Topoisomerase I to Meet the Challenge of Finding New Antibiotics

“…It is plausible that small molecules could be effective antibiotics by mimicking the action of protein inhibitors of bacterial topoisomerase I. Monoclonal antibodies that can inhibit the relaxation activity of M. tuberculosis and M. smegmatis topoisomerase I have been described [86,87]. These antibodies can act with different mechanisms, including stimulation of DNA cleavage [86] as well as enhancing the closing of the DNA gate after DNA cleavage [87].…”