EFFECT OF MG(II) BINDING ON THE STRUCTURE AND ACTIVITY OF ESCHERICHIA-COLI DNA TOPOISOMERASE-I

Escherichia coli DNA topoisomerase I requires Mg(II) as a cofactor for the relaxation of negatively supercoiled DNA. Mg(II) binding to the e nzyme was shown by fluorescence spectroscopy to affect the tertiary st ructure of the enzyme. Addition of 2 mM MgCl2 resulted in a 30% decrea se in the maximum emission of tryptophan fluorescence of the enzyme. T hese Mg(II)-induced changes in fluorescence properties were reversible by the addition of EDTA and not obtained with other divalent cations. After incubation with Mg(II) and dialysis, inductively coupled plasma (ICP) analysis showed that each enzyme molecule could form a complex with 1-2 Mg(II) bound to each enzyme molecule. Such Mg(II) enzyme comp lexes were found to be active in the relaxation of negatively supercoi led DNA in the absence of additional Mg(II). Results from ICP analysis after equilibrium dialysis and relaxation assays with limiting Mg(II) concentrations indicated that both Mg(ZI) binding sites had to be occ upied for the enzyme to catalyze relaxation of negatively supercoiled DNA.