Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

Abstract

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.

Figure 1. DNA pairing by RecA, imaged using single-molecule TIRFM, suggests that the three-dimensional conformation of target dsDNA is important in the homology search

a, DNA substrates. b, DNA pairing between λ DNA (green) and RecA filament assembled on 430 nt ssDNA (red): i) ensemble reaction examined by TIRFM; ii-iv) in situ reactions, dsDNA attached prior to pairing: ii) doubly-attached extended DNA, iii) singly-attached DNA, and iv) doubly-attached DNA with ends in proximity. Homologously paired products were observed in iii and iv when DNA was relaxed by stopping flow, and then flow-extended for visualization. White bar = 2.4 μm.

Figure 2. Visualization of RecA-promoted DNA pairing with an individual optically-trapped DNA-dumbbell, imaged by epifluorescence

a, Four channel flowcell with a flow-free reservoir. b, DNA-dumbbell assembly and RecA-pairing reaction: 1) trap two beads (yellow); 2) capture λ DNA molecule (green) on one bead; 3) capture free DNA end with second bead using steerable optical trap; 4) set center-to-center bead distance, and remove YOYO-1; 5) incubate DNA-dumbbell in reservoir with RecA nucleoprotein filaments (red); and 6) extend DNA to visualize products. c, Images of pairing products with 430 and 1,762 nt nucleoprotein filaments.

Figure 3. DNA three-dimensional conformation and nucleoprotein filament length contribute to homology search

a, Effect of DNA end-to-end distance; 430 nt substrate (2 min); error bars: standard error of mean (SEM) from multiple experiments, n =10 to 29). b, Time course; 430 nt substrate: 2 μm (squares) and 6 μm (triangles) separation; respective pairing rates, 0.023 (±0.002) s^-1 (n≥10) and 0.0056 (±0.0006) s^-1 (n≥5). c, Effect of ssDNA length; 162 nt (triangles; n=5, 6, 4, and 2 at times indicated), 430 nt (squares; same data as Figure 3b; n≥10), and 1,762 nt (circles; n≥10); error bars, SEM; 2 μm separation; respective rates: zero, 0.023 (±0.002) s^-1, and 0.086 (±0.026) s^-1.

Figure 4. RecA nucleoprotein filaments exhibit transient non-homologous interactions and loop-release events

a, Kymograph of DNA-dumbbell during bead separation (Figure 2b, step 6). Distance scale (top) and tick marks show positions of beads (green) and nucleoprotein filaments (red); illustration depicts dissociation of heterologously bound filament. Fraction of dsDNA-dumbbells with, b, non-homologously bound intermediates and, c, loop release events; 430 nt (blue) and 1,762 nt (green) filaments; n = 50 and 30, respectively; error bars indicate SEM. d, Model for RecA homology search by intersegmental contact sampling; for simplicity, only two simultaneous points of interaction are depicted.