Changes in the tension in dsDNA alter the conformation of RecA bound to dsDNA-RecA filaments

Abstract

The RecA protein is an ATPase that mediates recombination via strand exchange. In strand exchange a single-stranded DNA (ssDNA) bound to RecA binding site I in a RecA/ssDNA filament pairs with one strand of a double-stranded DNA (dsDNA) and forms heteroduplex dsDNA in site I if homology is encountered. Long sequences are exchanged in a dynamic process in which initially unbound dsDNA binds to the leading end of a RecA/ssDNA filament, while heteroduplex dsDNA unbinds from the lagging end via ATP hydrolysis. ATP hydrolysis is required to convert the active RecA conformation, which cannot unbind, to the inactive conformation, which can unbind. If dsDNA extension due to RecA binding increases the dsDNA tension, then RecA unbinding must decrease tension. We show that in the presence of ATP hydrolysis decreases in tension induce decreases in length whereas in the absence of hydrolysis, changes in tension have no systematic effect. These results suggest that decreases in force enhance dissociation by promoting transitions from the active to the inactive RecA conformation. In contrast, increases in tension reduce dissociation. Thus, the changes in tension inherent to strand exchange may couple with ATP hydrolysis to increase the directionality and stringency of strand exchange.

Figure 1.

Effects of dynamics on dsDNA molecules completely and partially covered by RecA. (A) Schematic representation of dsDNA (black lines) bound to RecA (blue) where the circles represent the ATP-RecA conformation and the ovals the ADP-form. (B) Extension versus force curves for a control in the absence of RecA (gray), a full filament in ATPγS (magenta) and 1 µM RecA, and cycles in buffer containing 4 mM ATP (purple) and 10 mM ATP (green) and 1 µM RecA, where the dark colors show the first half of the cycles. The arrows indicate the direction of the force change.

Figure 2.

Dynamics effects take place during sequences of constant force steps. (A) Extension (black) and force (orange) versus time data in 10 mM ATP and 10 µM ATPγS after exchanging the buffer containing 1 µM RecA and 10 mM ATP using flow, along with a gray curve showing the corresponding extension for a control flow experiment in 1 mM ATPγS. Expanded views are shown in the circles. Linear fits to the curves shown in red and blue are −0.4 ± 0.05 nm/s and −6.4 ± 0.14 nm/s, respectively. The slope during the intervening 120 s is 4.0 nm ± 0.02 nm/s. (B) Plot analogous to that shown in (A) for dsDNA partially covered by RecA in 1 mM ATP and 1 µM RecA. Expanded views are shown in the circles with the y-axis offset between the two views; the black lines show the fits to the slopes during the last 90 s. The measured slopes and the standard deviations in the slopes are 12.1 ± 0.25 nm/s for the first 30 s after the force is increased to 30 pN, and 4.6 ± 0.09 nm/s for the following 90 s. Similarly, the values for the first 30 s and last 90 s after the force is decreased from 55 to 30 pN are −2.6 ± 0.25 and 4.4 ± 0.07 nm/s, respectively. The slopes for the corresponding time interval of the 55 pN second force are 7 ± 0.3 and 4.3 ± 0.06 nm/s.

Figure 3.

Extension versus time for dsDNA pulled by a sequence of constant forces in conditions where hydrolysis is negligible. (A) Extension (black) and force (gray) as a function of time data in RecA buffer, 1 µM RecA and 1 mM ATPγS. (B) A more detailed sequence during ΔF = +25 pN and ΔF = –25 pN. (C) Extension (black) and force (gray) as a function of time in 1 µM RecA, 10 mM CaCl₂ and 1 mM ATP. (D) More detailed sequence during ΔF = +20 pN and ΔF = –20 pN.

Figure 4.

Effect of dynamics on many incomplete filaments. (A) Results for 0–30 s. (B) Results for 30–120 s. The x and y coordinates are dL/dt after a ΔF > 0 and after a ΔF < 0, respectively. In ATP, the F_c ranges are orange ≤20 pN, green 20–30 pN and blue >30 pN. All forces for controls, samples in ATPγS and ATP/CaCl₂ are shown as black squares, magenta triangles and gray triangles, respectively. The standard deviations for the individual slopes vary, but the average standard deviation is 0.17 nm/s.

Figure 5.

Quasi-continuous measurements. (A) Selected cycles of the complete sequence shown in Supplementary Figure S7 (1 µM RecA and 1 mM ATP), where the extension as a function of force in the absence of RecA has been subtracted. The up cycles are shown with solid lines and the down cycles with dashed lines. (B) Same as (A) but in 1 µM RecA and 1 mM ATPγS. (C) Change in extension as a function of force in 1 mM ATP for the curves shown in (A), where the solid lines correspond to the up cycles and the dashed lines correspond to the down cycles. (D) Same as (C) but in 1 mM ATPγS. (E) Each point corresponds to two successive measurements of the slope at a given force F_c, where the x and y values corresponds to the slopes when the force is increasing and decreasing, respectively, in ATP. (F) Same as (E) in ATPγS. The colors correspond to the cycle number of the complete series of cycles shown in Supplementary Figure S7: black (first), orange (second) and blue (fourth). The variations in slope are ∼±3 nm/s.

Figure 6.

Results for quasi continuous force changes. The gray diamonds show data for measurements in 1 µM RecA and 1 mM ATPγS. Black circles and squares correspond to forces <30 pN and ≥30 pN in 1 µM RecA and 1 mM ATP, respectively. The variations in slope are ∼±3 nm/s.

Figure 7.

Extension versus force curves for different pulling techniques in 1 mM ATP showing significant dynamics effects and large changes in slope at the force minimum. (A) Pulling from 3′3′-ends. (B) 5′5′-ends. (C) 3′5′-ends. (D) Both ends. For these cycles force was initially increased in (A) and (B) and initially decreased in (C) and (D).