High throughput screen for inhibitors of bacterial DNA topoisomerase I using the scintillation proximity assay

Claude G. Lerner, Anne Y. Chiang Saiki, A. Craig Mackinnon, Xiaoling Xuei

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

We report the development of a rapid method to detect binding of supercoiled DNA to Escherichia colt 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.

Original languageEnglish (US)
Pages (from-to)135-143
Number of pages9
JournalJournal of Biomolecular Screening
Volume1
Issue number3
DOIs
StatePublished - 1996

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biotechnology
  • Biochemistry
  • Molecular Medicine
  • Pharmacology
  • Drug Discovery

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