Abstract
We report the development of a rapid method to detect binding of supercoiled DNA to Escherichia coli topoisomerase I using the scintillation proximity assay (SPA). Streptavidin-SPA beads were coated with biotinylated topoisomerase I produced in vivo as a chimeric fusion protein. The hybrid biotinyl-fusion protein was overproduced in E. coli and purified in a single step by monomeric avidin affinity chromatography. The assay signal originates from both covalent and noncovalent binding of [3H]DNA to the SPA bead-immobilized enzyme. About 20-30% of the total [3H]DNA bound to the bead-immobilized enzyme remains associated with the bead in the presence of 0.5% SDS. The residual signal arises from the trapping of covalent [3H]DNA-enzyme complexes. The assay was employed in a high throughput screen that identified two general classes of topoisomerase inhibitors: agents that (1) inhibit DNA binding or (2) stabilize a covalent enzyme-DNA intermediate.
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