Using a Single-Molecule Method to Visualize RecBCD Helicase Translocation along Single-Stranded DNA

Abstract

The E. coli RecBCD helicase initiates the repair of double strand DNA break in the homologous recombination pathway. RecBCD is a heterotrimeric enzyme composed of two helicase motors with different polarities: RecB is a 3’-to- 5’ helicase and RecD is a 5’-to-3’ helicase. How RecBCD unwinds and translocates along duplex DNA is not clearly defined. Here we used a single-molecule tethered particle motion (TPM) experiment to visualize the RecBCD helicase translocation over long distance single-stranded (ss) DNA. We first prepared DNA substrates containing a > 200 nt long, unstructured ssDNA gap flanked by double-stranded DNA for RecBCD loading. In the TPM experiments, the bead-labeled, biotinylated RecBCD helicases are found to recognize and bind to the blunt, double-stranded DNA end, and successfully translocate along the duplex/single-stranded/duplex DNA substrate, resulting in a gradual decrease in the bead Brownian motion amplitude. Successful observation of RecBCD translocation over long ssDNA gap in either 3’-to- 5’ or 5’-to-3’ direction indicates that wild-type RecBCD functions an ssDNA translocase.