Abstract
S. pneumoniae is an important pathogen causing pulmonary infection, acute otitis media and purulent meningitis in infants and children. Type II topoisomerases are enzymes that play essential roles in DNA replication, chromosome segregation and recombination throughout all living organisms. Topoisomerases IV can make a transient break in DNA strands in one chromosome. These enzymes are very important antibacterial as well as anticancer targets and potential anti-trypanosomal targets. Levofloxacin has shown efficient inhibition of Type II topoisomerases in S. pneumoniae. Its mechanism of action is to inhibit the activity of DNA topoisomerase, prevent bacterial DNA synthesis and replication leading to bacterial death. We focused on solving the key structures of topoisomerase IV-DNA-levofloxacin complexes by negative staining electron microscopy and the resulting model was obtained at 32 Å by 3D autorefine in Relion 3.0. This study was to try and obtain the structure of the whole complex with DNA bound in the G-gate and the T-gate in order to study DNA capture and transport in type II topoisomerases.
Highlights
MethodsPreparation and purification of the Topoisomerase IV-DNA-Levofloxacin complexes
The negative stain could not achieve the high resolution which leads to the final structure of Topo IV-DNA-levofloxacin is a lowresolution model
The expected complex is a 191pb DNA composed of G-DNA and T-DNA linked to a closed ATPase domain. 14-mer DNA passes through a 22 Å cavity made up of Pare44 ATPase domains
Summary
Preparation and purification of the Topoisomerase IV-DNA-Levofloxacin complexes. The preconceived DNA is 191bp has a molecular weight of 126,060 Da, which G-DNA and T-DNA binding domains have been engineered into this synthetic, and ParE as a MW of 72000 Da while ParC has a MW of 92000 Da. An equimolar complex of ParC, ParE and DNA was formed in the 10 micromolar range by mixing the correct combinations of ParC, ParE and DNA 5.38 uM, 71.8 uM, 58.3uM respectively. High performance liquid chromatography was used for the purification of complex. The topoisomerase IVDNA-drug was purified by high performance liquid chromatography (HPLC) using AKTA high performance liquid chromatography system. More purified samples can be selected by observing the trace map
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