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A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria

[ Vol. 16 , Issue. 3 ]

Author(s):

Rasmus N. Klitgaard and Anders Løbner-Olesen*   Pages 272 - 277 ( 6 )

Abstract:


Background: One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.

Materials and Methods: Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.

Results: The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.

Conclusion: We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

Keywords:

DNA replication initiation, inhibitors, DnaA, high throughput screen, fluorescence, microbial extracts.

Affiliation:

Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen

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