Objectives: To reconstitute purified subunits of DNA gyrase from the thermophilic archaebacterium Thermophilus thermus with a preferred DNA substrate, as a prelude to crystallographic analysis for structure-directed drug discovery.
Methods: Hexa-histidine tagged T. thermophilus DNA gyrase A and B subunits were separately cloned and expressed in Escherichia coli. The subunits were extensively purified by heat treatment of cell lysates at 65°C, nickel affinity chromatography and size exclusion chromatography. The DNA supercoiling activity of the reconstituted subunits was assayed by monitoring the conversion of relaxed plasmid pBR322 to its supercoiled form. The purified subunits were reconstituted with a suitable 130 bp DNA fragment of pBR322 (obtained by PCR) in the presence of Mg-ATP and the gyrase inhibitor novobiocin. The resultant complex was analysed using size exclusion chromatography and blue gel polyacrylamide electrophoresis.
Results: The supercoiling activity of DNA gyrase was optimal at the expected subunit molar ratio of 1:1 and was inhibited by the 130 bp fragment of pBR322. Reconstitution of the protein subunits with the DNA fragment gave a novobiocin-stabilised complex larger than either subunit A or B. Formation of the moderately stable gyrase-DNA complex appeared to require novobiocin but not Mg-ATP.
Conclusions: Preliminary evidence has been obtained for the reconstitution of a novobiocin-stabilised DNA-gyrase complex. Future studies will identify conditions that more effectively stabilise the gyrase-DNA complex. Crystallographic analysis may then help identify gyrase inhibitors with novel modes of action.