RecA polymerization on double-stranded DNA by using single-molecule manipulation: the role of ATP hydrolysis

Abstract

The polymerization of RecA on individual double-stranded DNA molecules is studied. A linear DNA (lambda DNA, 48.5 Kb), anchored at one end to a cover glass and at the other end to an optically trapped 3-micrometers diameter polystyrene bead, serves as a template. The elongation caused by RecA assembly is measured in the presence of ATP and ATP[gammaS]. By using force extension and hydrodynamic recoil, a value of the persistence length of the RecA-DNA complex is obtained. In the presence of ATP, the polymer length is unstable, first growing to saturation and then decreasing. This suggests a transient dynamics of association and dissociation for RecA on a double-stranded DNA, the process being controlled by ATP hydrolysis. Part of this dynamics is suppressed in the presence of ATP[gammaS], leading to a stabilized RecA-DNA complex. A one-dimensional nucleation and growth model is presented that may account for the protein assembly.

Figure 1

Schematic of the experimental setup.

Figure 2

The force exerted by DNA as a function of the extension at the various stages of RecA assembly. (a) ATP, (b) ATP[γS]. Curves for naked DNA (circles) and DNA fully covered with RecA (diamonds) are shown for both polymerization conditions. In a, the force-extension behavior for partially covered DNA at t₂ = 15 min (squares) and the one obtained after depolymerization at t₄ = 300 min (triangles) are also shown.

Figure 3

The dynamics of recoil of DNA-bead initially stretched by a 6-pN force. Both extension and time are normalized by the contour length (L) of the DNA. Even though the naked DNA curves (bullets, triangles and squares) correspond to slightly different lengths, in the normalized variables they coincide within experimental error. The recoil is much slower when DNA is fully covered with RecA-ATP[γS] (diamonds).

Figure 4

The relative change in the length of DNA during the assembly and disassembly of RecA on DNA in the presence of ATP (bullets).

Figure 5

Comparison of the relative change in the length of DNA during the assembly of RecA on DNA in the presence of (a) ATP (bullets) and (b) ATP[γS] (bullets). In a, the line corresponds to a linear fit to the growth phase of the ATP assembly. In b, the curve represents the best fit of Eq. 7 to the data. A schematic illustration of the corresponding mechanisms is shown in the insets of both a and b.

Figure 6

(a) The disassembly of RecA from DNA in the presence of ATP (bullets). The curve represents the best fit of Eq. 10. (b) [ATP]/[ADP] ratio as obtained from the ³²P radioactive ATPase assay (bullets). The line represents the best exponential fit.