Figure 2: Magnetic tweezers resolves the kinetics of PFV intasome strand transfer.

(a) Experimental analysis of PFV intasome strand transfer kinetics. DNA bound to an SPM bead and the surface of the flow cell (left) is supercoiled (left centre) by clockwise rotation of ferromagnets under low extension force. PFV intasome interaction with the supercoiled target DNA results in integration where the first strand transfer event creates a strand scission that releases the supercoils (right centre). The second strand transfer event creates a double stand break (DSB) that results in SPM bead dissociation from the target DNA tethered to the surface (right). (b) (top panel) A representative SPM bead image during a concerted integration event. (middle panel) A 3D (x,y,z) trace of the SPM bead position. (bottom panel) The rate of SPM bead position change during a concerted integration event. The first strand transfer (green arrow; at 1.2 s) alters the SPM bead height in the z-direction as a result of DNA relaxation. The second event (orange arrow; at 2.0 s) alters both the x- and y-direction, while the z-direction could not be clearly resolved because the SPM bead leaves the field of view (see Supplementary Fig. 3a for additional examples). The definition of strand transfer time (τ_ST) is shown. (c) The kinetics of DNA relaxation (τ_RE) as a result of one strand transfer event during half-site integration (top panel) and the kinetics of two strand transfer (τ_ST) events as a result of concerted integration. See Methods for the analytical definitions of τ_RE and τ_ST. The integration analysis shown was performed at a fixed applied force (F_D=0.3 pN) on the supercoiled DNA (-10 turns). (d) The distribution of relaxation times (τ_RE) and the times between the two strand transfer events (τ_ST) at various force values (Supplementary Fig. 3b). X-axis error bars indicate s.e. of force measurements. Y-axis error bars indicate s.e. and s.d. of τ_RE and τ_ST, respectively. The frame rate for smMT was 100–200 ms.