Fig. 3.

Single-molecule measurement of DNA translocation for WT and mutant HsdR subunits by magnetic tweezers. For all experiments, HsdR was at 160 nM, MTase was at 20 nM and F = 1.5 pN. (a) Sample single-molecule profile showing DNA length as a function of time. The inset shows a cartoon illustrating the principle of the assay, with DNA shown in red, the EcoR124I recognition site shown as a white box, the MTase shown as a light orange ovoid, the HsdR subunits shown as green circles and the magnetic bead shown in gray. See the main text for further details. The WT profile shows a period of inactivity, initiation of translocation of one HsdR subunit, initiation of translocation of a second HsdR subunit so that both motors are running simultaneously, dissociation of one subunit to leave a single translocating HsdR and, finally, dissociation of the remaining HsdR. (b) Histograms showing the translocation rates obtained from individual events binned in 25 s windows. Events are scored as either a single translocating HsdR (R₁) or the sum of two translocating HsdR subunits (R₂) (see the main text and Materials and Methods). Red lines are the fits of the R₁ data to a single Gaussian (WT, E165D) or double Gaussian (D151A, E165A, E165H, K167A). Mean translocation rates for R₁ population (from the single Gaussian fits) or both R₁ and R_1,slow populations (from the double Gaussian fits) are given in Table 2. (c) Histogram showing the average time for a translocation event (T_event) when either one HsdR subunit (R₁) or two HsdR subunits (R₂) are translocating. Error bars represent the standard deviation of the mean. For HsdR subunits with bimodal R₁ populations, T_event represents the average of both subpopulations.