Direct imaging of RecA nucleation and growth on single molecules of SSB-coated ssDNA

Abstract

Escherichia coli RecA is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. To function, filaments of RecA must nucleate and grow on single-stranded DNA (ssDNA) in direct competition with ssDNA-binding protein (SSB), which rapidly binds and continuously sequesters ssDNA, kinetically blocking RecA assembly. This dynamic self-assembly on a DNA lattice, in competition with another protein, is unique for the RecA family compared to other filament-forming proteins such as actin and tubulin. The complexity of this process has hindered our understanding of RecA filament assembly because ensemble measurements cannot reliably distinguish between the nucleation and growth phases, despite extensive and diverse attempts. Previous single-molecule assays have measured the nucleation and growth of RecA--and its eukaryotic homologue RAD51--on naked double-stranded DNA and ssDNA; however, the template for RecA self-assembly in vivo is SSB-coated ssDNA. Using single-molecule microscopy, here we directly visualize RecA filament assembly on single molecules of SSB-coated ssDNA, simultaneously measuring nucleation and growth. We establish that a dimer of RecA is required for nucleation, followed by growth of the filament through monomer addition, consistent with the finding that nucleation, but not growth, is modulated by nucleotide and magnesium ion cofactors. Filament growth is bidirectional, albeit faster in the 5'→3' direction. Both nucleation and growth are repressed at physiological conditions, highlighting the essential role of recombination mediators in potentiating assembly in vivo. We define a two-step kinetic mechanism in which RecA nucleates on transiently exposed ssDNA during SSB sliding and/or partial dissociation (DNA unwrapping) and then the RecA filament grows. We further demonstrate that the recombination mediator protein pair, RecOR (RecO and RecR), accelerates both RecA nucleation and filament growth, and that the introduction of RecF further stimulates RecA nucleation.

Figure 1. Direct visualization of RecA filament assembly on single molecules of SSB-coated ssDNA reveals that RecA nucleates as a dimer

(a) RecA^f filament assembly with ATPγS on a single molecule of SSB-coated ssDNA tethered within a microfluidic flow chamber was visualized using TIRF microscopy; montage is rendered into a topographic fluorescent intensity map. (b) Schematic and (c) fluorescent intensity profile from panel A. (d) The number of RecA clusters increases linearly with time; slope is the nucleation rate (n = 18–93 clusters for each concentration; (±s.d.)). (e) Nucleation rate increases with [RecA] according to, J=k[RecA]ⁿ, where n is 2.2 (±0.6) (ATPγS) and 1.5 (±0.1) (ATP) (error from the linear fits in (d) is smaller than the symbols).

Figure 2. RecA filaments grow via rapid addition of monomers

(a) Lengths for RecA cluster were measured and (b) analyzed (same colors as in a, plus additional data) to determine the rate (slope) and lag time (x-intercept) (100 nM RecA and ATPγS). (c) Distribution of growth rates at increasing [RecA]. (d) Mean growth rates increase linearly with [RecA]; slope is 0.16 (±0.08) nm min⁻¹ nM⁻¹ (ATPγS, black) (or 1.1 (±0.6) × 10⁸ M⁻¹min⁻¹ at 1.5 nm/RecA) and 0.07 (±0.01) nm min⁻¹ nM⁻¹ (ATP, red) (or 0.47 (±0.3) × 10⁸ M⁻¹min⁻¹). (e) Lag time distribution at increasing [RecA]. (f) Lag times plotted with respect to [RecA] and fit to an inverse power law, J⁻¹=1/k[RecA]ⁿ, where n is 1.9 (±0.3). Lines in (c, e) are Gaussian fits. See Supplementary Table 1 statistics (n = 207 filaments, total; ± s.d.).

Figure 3. RecA filament growth on SSB-coated ssDNA is bidirectional

(a) Growth rates of clusters at the tethered 3′-end of the ssDNA (k_e) were plotted against growth rates of internal clusters on the same molecule (k_i) (n = 18; ± s.e.m.). (b) RecA^Cy3 (50 nM, red) clusters were pre-formed with ATPγS on SSB-coated ssDNA followed by growth with RecA^f (250 nM, green). (c) Visualization of RecA^f (green) growth from pre-formed RecA^Cy3 (red) clusters, showing faster growth from the left side of the RecA^Cy3 clusters in the 5′→3′ direction. (d) The growth rates were 44 (±11) nm min⁻¹ in the 5′→3′ direction and 27 (±12) nm min⁻¹ in the 3′→5′ direction; (n = 4; ± s.d.).

Figure 4. Nucleation is modulated by ligand binding, repressed at physiological pH, and enhanced by RecOR and RecFOR; growth is enhanced by RecOR

RecA nucleation and growth rates plotted as function of (a,b) [Mg(OAc)₂], (c,d) nucleotide cofactors, and (e–f) pH (slope = −2.0 (±0.3) monomers min⁻¹ pH⁻¹). (a–f) Error bars are ± s.d. (g) Image montage of RecA^f filament assembly on gapped DNA. (h) Nucleation enhanced by RecOR and RecFOR; (i) growth accelerated by RecOR, with no addition increase by RecFOR. (j) Kinetic model for RecA nucleation where a RecA dimer binds ssDNA transiently released from SSB through either sliding or unwrapping, which is enhanced by RecOR and RecFOR; growth via monomer addition accelerated by RecOR. [RecA] was 250 nM (a, b), 500 nM (c–f), or 1 μM (h–i).