A complementary pair of rapid molecular screening assays for RecA activities.
The bacterial RecA protein has been implicated in the evolution of antibiotic resistance in pathogens, which is an escalating problem worldwide. The discovery of small molecules that can selectively modulate RecA's activities can be exploited to tease apart its roles in the de novo development and transmission of antibiotic resistance genes. Toward the goal of discovering small-molecule ligands that can prevent either the assembly of an active RecA-DNA filament or its subsequent ATP-dependent motor activities, we report the design and initial validation of a pair of rapid and robust screening assays suitable for the identification of inhibitors of RecA activities. One assay is based on established methods for monitoring ATPase enzyme activity and the second is a novel assay for RecA-DNA filament assembly using fluorescence polarization. Taken together, the assay results reveal complementary sets of agents that can either suppress selectively only the ATP-driven motor activities of the RecA-DNA filament or prevent assembly of active RecA-DNA filaments altogether. The screening assays can be readily configured for use in future automated high-throughput screening projects to discover potent inhibitors that may be developed into novel adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index.
Lee
Andrew M
AM
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7360, USA.
Wigle
Tim J
TJ
Singleton
Scott F
SF
D000251:Adenosine Triphosphatases / Q000032:analysis; D003124:Colorimetry; D004268:DNA-Binding Proteins / Q000032:analysis*; D004791:Enzyme Inhibitors / Q000032:analysis*; D029968:Escherichia coli Proteins / Q000032:analysis*; D005456:Fluorescent Dyes; D009243:NAD / Q000378:metabolism; D011938:Rec A Recombinases / Q000032:analysis* / Q000037:antagonists & inhibitors
D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D009243:NAD; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases