Integrating multi-scale data on homologous recombination into a new recognition mechanism based on simulations of the RecA-ssDNA/dsDNA structure

Abstract

RecA protein is the prototypical recombinase. Members of the recombinase family can accurately repair double strand breaks in DNA. They also provide crucial links between pairs of sister chromatids in eukaryotic meiosis. A very broad outline of how these proteins align homologous sequences and promote DNA strand exchange has long been known, as are the crystal structures of the RecA-DNA pre- and postsynaptic complexes; however, little is known about the homology searching conformations and the details of how DNA in bacterial genomes is rapidly searched until homologous alignment is achieved. By integrating a physical model of recognition to new modeling work based on docking exploration and molecular dynamics simulation, we present a detailed structure/function model of homology recognition that reconciles extremely quick searching with the efficient and stringent formation of stable strand exchange products and which is consistent with a vast body of previously unexplained experimental results.

Figure 1.

Schematic of the homology searching process. The initiating, complementary, and outgoing strands are shown in orange, purple and cyan, respectively. RecA protein monomers are excluded for simplicity.

Figure 2.

Structures known from crystallography. The initiating and complementary strands are shown in orange and purple, respectively. The residues in the C-terminal domain are shown in pink. (A) Schematic showing the initiating ssDNA. (B) I. Active filament seen from the bottom with the protein with alternating monomers shown in white and blue. (B) II. The residues in the C-terminal domain are shown in transparent pink except for lysine residues K280, K282, K286 and K302, which are shown in green. K232 is shown in silver. Site II residues R226, R227, R243, and K245 are shown in red, and the L2 loop (198–206) is shown in yellow. (B) III. Same as I., but seen from the side with the 5′ end of the initiating strand at the top. (B) IV. Same as II., but from the same angle as (B-III). (B) V. Close-up of the initiating strand and I199. (C and D (I. to III)). Analogous to (A) and (B), respectively. (D) IV. shows I199 in yellow and M164 in royal blue highlighting their intercalation in the rises in the initiating and complementary strands, respectively. (D) V. is analogous to V. in (B), showing M164 (royal blue) intercalating in the rise in the complementary strand.

Figure 3.

A detailed view of the active filament bound to dsDNA stabilized by interactions with RecA C-terminal domain and site II residues. The initiating, complementary, and outgoing strands are shown in orange, purple, and cyan, respectively. The lysine residues K280, K282, K286 and K302 are shown in green. K232 is shown in silver. The secondary DNA-binding site residues R226, R227, R243 and K245 are shown in red, and the L2 loop residues M202 and F203 are shown in yellow. The black arrows point to the site II residues at the beginning and end of the extended and untwisted region. F203 intercalates the bound dsDNA, and some simulations showed M202 can intercalate as well (see also Supplementary Figure S5).

Figure 4.

Illustration of base-pair homology testing using a duplet that is nearly in site I, while the third base of the triplet at the 3′ end of the triplet remains positioned in site II. (A) Structure of full filament with stretched and unwounded dsDNA bound to site II. The transparent and solid structures represent the conformations before and after 14 ns of simulation. (B) Structures before and after 14 ns simulation (10 ns conventional MD and 4 ns accelerated MD). (B) I. The complementary backbone in the postsynaptic filament is shown in silver. After flipping, the backbone near the flipped duplet shown in purple is nearly in postsynaptic position. (B) II. and III. Top and schematic views of the complementary and initiating bases at the 5′ end of a triplet before and after flipping. The transparent structures indicate the initial positions. The purple arrow indicates the relocation of the complementary strand backbone. (C) Postsynaptic dsDNA in site I; side view and top view are shown in I. and II., respectively.

Figure 5.

Trajectories for structures with 2 base pair triplets initially bound to site II. (A) I. Side view of the dsDNA. II. Rotated 90^o view of I. III. Structure at a later simulation time when the duplet at the 5′ end has begun to flip. IV. Flipped duplet paired with the initiating strand. The flipping of the base at the 3′ end is sterically hindered by the L2 loop. (B) Base pairing parameters D1 and θ₁ are the C1′ and C1′ distance between complementary and initiating strands and C1′–C1′–N9 projection angle onto the base-flipping plane, respectively. The graphs show the histogram of D₁ and θ₁ for a paired dsDNA in the stable postsynaptic filament (Supplementary Figure S9). The bottom three histograms show results for a triplet bound in the site II. The left and middle one corresponds to the base-flipping process shown in (A). (C) Illustration of the separation of the outgoing strand from the site II residues that occurs as the bases begin to flip. Top panel illustrates the initial time and bottom panel is 11 ns later when the bases are flipped, the outgoing strand is locally separated from the site II, and in the region near the flipped duplet the complementary strand is nearly in the postsynaptic conformation. Accelerated MD was carried out after 10 ns of conventional MD simulation. (D) Top diagram illustrates the backbone of the complementary progress toward the position of the postsynaptic filament. Bottom diagram shows the measured distance of the outgoing strand locally separating from site II.

Figure 6.

Proposed transition to the more stable configuration. The outgoing, complementary, and initiating strands are shown in cyan, purple, and orange respectively. (A) I. Schematic of the base pairing in a structure with three successive strand exchanged duplets. The red region indicates the position of the site II residues. (A) II. A structure showing one flipped duplet in nearly the postsynaptic conformation, followed by an unflipped duplet, followed by a duplet beginning to flip. The resulting distortion of the complementary strand backbone is visible. (B) I. Schematic of a conformation with 2 complete triplets in the very stable postsynaptic conformation. (B) II. Same as II. in (A), but with all of the bases in the postsynaptic conformation.

Figure 7.

Overview of the proposed searching process. (A) Illustration of the known homology recognition process. The initiating, complementary, and outgoing strands are shown in orange, purple and cyan, respectively. (B) Overall structural transition during homology testing including the known structures (I and IV) and structures obtained using modeling and simulations (II and III). We propose that the structures correspond to the illustration shown above them in (A). The upper and central panels of (B) show end views of the filament, while the bottom panels show side views. The residues in the C-terminal domain are shown in pink except for lysine residues K280, K282, K286, and K302 which are shown in green. K232 is shown in silver. Site II residues R226, R227, R243, and K245 are shown in red, and the L2 loop (198–206) is shown in yellow.