Studying RecBCD Helicase Translocation along Chi-DNA Using Tethered Particle Motion with a Stretching Force

Abstract

E. coli RecBCD helicase unwinds blunt-end duplex DNA to repair damaged DNA molecules in the homologous recombination pathway. Previous single-molecule experiments show that RecBCD recognizes an 8 nt DNA sequence, chi, and lowers its unwinding rate afterwards under saturating ATP condition. We have developed a single-molecule Force Tethered Particle Motion (FTPM) method, which is modified from the conventional TPM method, and applied it to study RecBCD motion in details. In the FTPM experiment, a stretching force is applied to the DNA-bead complex, and suppressed bead's Brownian motion, resulting in an improved spatial resolution at long DNA substrates. Based on the equipartition theorem, the mean square displacement (MSD) of the bead Brownian motion measured by FTPM correlates linearly to DNA extension length with a predicted slope, circumventing the difficulties, such as non-linearality and low resolution of long DNA substrates in conventional TPM experiments. The FTPM method offers the best resolution (56 bp at 433 bp long DNA) in the presence of only a small stretching force (0.20 pN). We have used the FTPM method to investigate the RecBCD helicase motion along 4.1 kb long chi-containing duplex DNA molecules, and observed that translocation rate of RecBCD changes after chi sequence under limited ATP concentrations. This suggests that chi recognition by RecBCD does not require saturating ATP conditions, contrary to what have been previously reported.