DNA RECOMBINATION. Base triplet stepping by the Rad51/RecA family of recombinases

Abstract

DNA strand exchange plays a central role in genetic recombination across all kingdoms of life, but the physical basis for these reactions remains poorly defined. Using single-molecule imaging, we found that bacterial RecA and eukaryotic Rad51 and Dmc1 all stabilize strand exchange intermediates in precise three-nucleotide steps. Each step coincides with an energetic signature (0.3 kBT) that is conserved from bacteria to humans. Triplet recognition is strictly dependent on correct Watson-Crick pairing. Rad51, RecA, and Dmc1 can all step over mismatches, but only Dmc1 can stabilize mismatched triplets. This finding provides insight into why eukaryotes have evolved a meiosis-specific recombinase. We propose that canonical Watson-Crick base triplets serve as the fundamental unit of pairing interactions during DNA recombination.

Fig. 1. Structure of the presynaptic complex and experimental overview

(A) Structure of the RecA-ssDNA filament highlighting the base triplet organization of the presynaptic RS-ssDNA (9). (B) Cartoon illustration of base triplet stepping for S. cerevisiae Rad51. Quantized reductions in binding energy are proposed to coincide with the third base of each triplet. (C) Experimental outline for measuring the survival probability of fluorescently tagged dsDNA oligonucleotides bound to the presynaptic complex. (D) Example of a kymograph showing the binding of single Atto565-dsDNA molecules to a ScRad51 presynaptic complex. White arrowheads highlight individual dsDNA dissociation events.

Fig. 2. Conservation of base triplet stepping among Rad51/RecA family members

(A) Schematic of the 70-bp dsDNA substrates (set #2; see table S1). All substrates contain an internal 8- to 15-nt tract of microhomology (as indicated) flanked by nonhomologous sequence. (B) Atto565-dsDNA dissociation rates (mean ± SD) for ScRad51 plus ATP. (C) EcRecA plus ATPγS. (D) hRad51 plus ATP. (E) ScDmc1 plus ATP. (F) ScRad51 plus AMP-PNP In (B) to (F), each data point was calculated from an average of ~150 molecules (N = 70 to 250); the color-coded shading highlights each base triplet; magenta shading indicates the minimum 8 nt necessary for binding; purple shading corresponds to additional homologous nucleotides; arrows indicate stepwise reductions in dissociation rates coincident with recognition of the third base of each triplet; dashed lines report the mean rate for each step; and the free energy changes (ΔΔG^‡) associated with each triplet step are indicated.

Fig. 3. Effects of mismatches on base triplet recognition

(A) Schematic highlighting the design of dsDNA substrates (set #2; see table S3) bearing mismatched bases within the fourth nucleotide triplet, corresponding to the second strand exchange step; each mismatch position is highlighted as an underlined magenta X. (B) Mismatch substrate binding for ScRad51, EcRecA, hRad51, and ScDmc1, as indicated. All indicated ΔΔG^‡ values are relative to the step 1 binding data for the substrate bearing 9 nt of microhomology (see Fig. 2); each data point was calculated from an average of ~150 molecules. The green and blue dashed lines correspond to data obtained for non-mismatched substrates with each of the four different recombinases (see Fig. 2, B to F). (C) Substrate design (set #2; see table S4) for testing whether Rad51/RecA is capable of stepping over a mismatched triplet. (D) Triplet binding data illustrating how base mismatches within the fourth triplet affect recognition of the fifth triplet. (E) Schematic highlighting the different models predicted for mismatched substrates in reactions with RecA and Rad51 versus Dmc1.

Fig. 4. RS-DNA governs triplet stepping during strand exchange

(A) Molecular dynamics snapshots of B-DNA annealing intermediates. Paired bases are numbered 1 to 3 for comparison to RS-DNA triplets. (B) Sample annealing trajectory for B-DNA. (C) Lifetimes of B-DNA intermediates calculated from 50 separate simulation runs (5 × 10⁸ simulation steps total). (D) Molecular dynamics snapshots of RS-DNA. (E) Sample annealing trajectory for RS-DNA. (F) Lifetimes of RS-DNA intermediates calculated from 50 separate simulation runs (5 × 10⁸ simulation steps total). In (A) and (D), the number of arrows between each image corresponds to the number of steps necessary to reach the depicted state. The y axes in (B) and (E) denote the position of the most distal paired base relative to the pre-annealed end of the DNA. The data in (C) and (F) do not include the terminal triplets.