Investigating direct interaction between Escherichia coli topoisomerase I and RecA

• E. coli topoisomerase I (EcTOP1) physically interacts with RecA.
• EcTOP1 can interact with RecA directly in the absence of DNA.
• RecA interacts with the N-terminal domain of EcTOP1 that forms the active site region.
• RecA interaction with EcTOP1 is stimulated by ATP.

Protein–protein interactions are of special importance in cellular processes, including replication, transcription, recombination, and repair. Escherichia coli topoisomerase I (EcTOP1) is primarily involved in the relaxation of negative DNA supercoiling. E. coli RecA, the key protein for homologous recombination and SOS DNA-damage response, has been shown to stimulate the relaxation activity of EcTOP1. The evidence for their direct protein–protein interaction has not been previously established. We report here the direct physical interaction between E. coli RecA and topoisomerase I. We demonstrated the RecA-topoisomerase I interaction via pull-down assays, and surface plasmon resonance measurements. Molecular docking supports the observation that the interaction involves the topoisomerase I N-terminal domains that form the active site. Our results from pull-down assays showed that ATP, although not required, enhances the RecA-EcTOP1 interaction. We propose that E. coli RecA physically interacts with topoisomerase I to modulate the chromosomal DNA supercoiling.