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 journalArticle

3 Citations (Scopus)

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 - Jan 1 1996

Fingerprint

Bacterial DNA
Type I DNA Topoisomerase
Scintillation
Assays
Throughput
DNA
Immobilized Enzymes
Fusion reactions
Topoisomerase Inhibitors
Topoisomerase I Inhibitors
Superhelical DNA
Affinity chromatography
Escherichia
Streptavidin
Avidin
Enzymes
Affinity Chromatography
Escherichia coli
Proteins

ASJC Scopus subject areas

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

Cite this

High throughput screen for inhibitors of bacterial DNA topoisomerase I using the scintillation proximity assay. / Lerner, Claude G.; Chiang Saiki, Anne Y.; Mackinnon, A. Craig; Xuei, Xiaoling.

In: Journal of Biomolecular Screening, Vol. 1, No. 3, 01.01.1996, p. 135-143.

Research output: Contribution to journalArticle

Lerner, Claude G. ; Chiang Saiki, Anne Y. ; Mackinnon, A. Craig ; Xuei, Xiaoling. / High throughput screen for inhibitors of bacterial DNA topoisomerase I using the scintillation proximity assay. In: Journal of Biomolecular Screening. 1996 ; Vol. 1, No. 3. pp. 135-143.
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