Forward and reverse motion of single RecBCD molecules on DNA

Abstract

RecBCD is a processive, DNA-based motor enzyme with both helicase and nuclease activities. We used high-resolution optical trapping to study individual RecBCD molecules moving against applied forces up to 8 pN. Fine-scale motion was smooth down to a detection limit of 2 nm, implying a unitary step size below six basepairs (bp). Episodes of constant-velocity motion over hundreds to thousands of basepairs were punctuated by abrupt switches to a different speed or by spontaneous pauses of mean length 3 s. RecBCD occasionally reversed direction, sliding backward along DNA. Backsliding could be halted by reducing the force, after which forward motion sometimes resumed, often after a delay. Elasticity measurements showed that the DNA substrate was partially denatured during backsliding events, but reannealed concomitant with the resumption of forward movement. Our observations show that RecBCD-DNA complexes can exist in multiple, functionally distinct states that persist for many catalytic turnovers: such states may help tune enzyme activity for various biological functions.

FIGURE 1

(a) Cartoon illustrating the experimental geometry (not to scale). A RecBCD-bio molecule (B, C, and D) linked to a glass coverslip unwinds duplex DNA (red). The distal end of the DNA is attached to a bead (blue) held at initial height h by the focused beam of an optical trap (pink) centered a lateral distance x_st from the enzyme. As the enzyme moves, the stage is automatically repositioned (gray) to maintain the displacement of the bead from the trap center (x_bd), and thereby the force. (b) Operation of the force clamp. k_opt (purple) is the trap stiffness; x_bd (black) is the bead displacement from the trap center; F = k_opt × x_bd (red) is the applied force; L (blue) is the remaining contour length of the DNA molecule, equivalent to the instantaneous position of RecBCD on the DNA. At t = 33 s, the force set point was raised to 5.5 pN by increasing the laser intensity, transiently deflecting the bead toward the center; the clamp responded by moving the stage until x_bd returned to its preset value of 70 nm. (c) Force (red trace) and position (blue trace) records of a RecBCD molecule moving on DNA at 15 μM ATP. Black lines show average velocity over the indicated intervals. Discontinuities in L at t = 91s and 93 s and the increased noise levels in this interval are a consequence of the extremely low (0.2 pN) force setting. Elasticity-centering measurements performed during indicated periods (gray shading) yielded (p, L, and Δ) of (48 nm, 1459 nm, and 1.8 nm), (23 nm, 1596 nm, and 0.3 nm), and (46 nm, 1607 nm, and 4.5 nm), respectively, where Δ represents stage drift since the previous measurement. Data in b are from the first 50 s of this record.

FIGURE 2

Peak backsliding velocities are roughly proportional to the applied force. Peak velocity v_max was computed over a minimal distance of 35 nm for each of the 11 backsliding events detected at 15 μM ATP. Line is the unweighted linear fit to the data.

FIGURE 3

RecBCD-bio degrades one DNA strand into small fragments during unwinding. RecBCD degradation of uniformly ³²P-labeled DNA lacking χ-sites was detected by denaturing gel electrophoresis (lower inset). At increasing reaction times, concentration of full-length DNA strands (○) decreases, whereas intermediate (□) and short (▵) DNA fragment concentrations increase. Concentrations are calculated by integrating the bracketed areas of the gel lanes (inset) and normalizing to the full-length DNA band at zero time. Analysis of identical samples on a nondenaturing gel (upper inset) reveals concomitant loss of full-length dsDNA (•). The concentration of full-length DNA strands is predicted (——) to decrease at half the rate of full-length dsDNA loss if degradation of one of the two strands is simultaneous with unwinding. This prediction agrees well with the measured full-length DNA concentrations corrected (see Materials and Methods) for postunwinding degradation of full-length ssDNA (×).

FIGURE 4

Contour length records at various loads and ATP concentrations reveal diverse enzyme behaviors. (a) A trace displaying relatively uniform motion (blue) irrespective of the external load (red) up to 8 pN. Line fits over indicated intervals (black lines) show average velocities. The minor discontinuity in L at 47 s as F was doubled to 6.3 pN corresponds to a 1% error in determining L. (b) Record showing spontaneous variations in enzyme speed. (c–e) Examples of records with abrupt velocity changes. Some changes are spontaneous; others are concomitant with changes in applied force. A backslide occurred during the interruption in e.

FIGURE 5

Fine-scale motion. (a) Control trace of a bead-DNA complex attached directly to the coverglass without RecBCD illustrates the low drift and noise of the system. Filter bandwidth, 13 Hz; force, 7.5 pN. (b) RecBCD motion at 4 μM ATP (black). There is no evidence for uniform steps > 2 nm, and even isolated abrupt displacements > 2 nm are infrequent. Filter bandwidth, 13 Hz; force, 7.4 pN. The microscope stage position (gray) was adjusted every 1 s by the feedback system to maintain constant force. The stage motions are not detected as either pauses or abrupt changes in contour length, demonstrating the validity of the derived contour length measurement.

FIGURE 6

Scheme of state switching inferred from single-molecule experiments, together with hypothesized structures of the RecBCD-DNA complex in the different states (see text). The enzyme moves leftward relative to the DNA during unwinding and rightward during backsliding.