Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase

Abstract

Structural and topological data suggest that serine site-specific DNA recombinases exchange duplex DNAs by rigid-body relative rotation of the two halves of the synapse, mediated by a flat protein-protein interaction surface. We present evidence for this rotational motion for a simple serine recombinase, the Bxb1 phage integrase, from a single-DNA-based supercoil-release assay that allows us to follow crossover site cleavage, rotation, religation, and product release in real time. We have also used a two-DNA braiding-relaxation experiment to observe the effect of synapse rotation in reactions on two long molecules. Relaxation and unbraiding are rapid (averaging 54 and 70 turns/s, respectively) and complete, with no discernible pauses. Nevertheless, the molecular friction associated with rotation is larger than that of type-I topoisomerases in a similar assay. Surprisingly we find that the synapse can stay rotationally "open" for many minutes.

Fig. 1.

Experimental design, calibration of extension vs. ΔLk and examples of relaxation events observed for ΔLk = -30. (A) An 11.4 kb DNA carrying an attP⁺ site (filled arrow) is attached at one end to a glass cover slip, and at the other to a magnetic bead (filled circle), allowing it to be supercoiled and extended. (i) Four Int molecules (open circles) plus one attB DNA (open arrow) can form a synapse with the tethered attP⁺ site. (ii) Following synapsis, Int can cleave the DNAs; at this point the complex is held together only by protein-protein interactions. If cycles of strand exchange occur, then supercoils will be relaxed, and the bead will be observed to move away from the glass as the molecule extends. (iii) Following strand exchange, cleaved attP⁺ and attB⁺ sites can recombine to form attL and attR sites, which are not able to form a stable synaptic complex with Bxb1 Int; subsequent release of protein-protein interactions will release the tether. Recombination cannot occur for the mismatch mutant attB-CT. (iv) The complex alternately may religate the DNAs in the parental state, leading to reappearance of torsional stiffness in the tether. Reappearance of torsional stiffness is expected to be the dominant outcome for attB-CT where recombination is forbidden. (B) Calibration data for tether extension as a function of ΔLk, for 0.46 pN force, in buffer with no Int or partner DNA present. At zero supercoiling, the tether extends to about 2.75 µm; for less than about 12 turns, there is little or no change in extension. Beyond 12 turns, the molecule buckles and plectonemic regions are generated, leading to a simple linear reduction of extension with ΔLk. At 0.46 pN force, there is no DNA denaturation and the slopes of the linear regions for positive and negative supercoiling are nearly the same (for ΔLk < -14, slope is 53.8 ± 0.3 nm/ΔLk; for ΔLk > 12, slope is -56.3 ± 0.4 nm/ΔLk). (C) Relaxation event for attB⁺ DNA partner, force = 0.54 pN. Fit slope (2.9 ± 0.4 μm/s for this event) is converted to rotational velocity via calibration data (Fig. 1B). (D) Relaxation event for attB-CT DNA partner, force = 0.42 pN, slope 3.1 ± 0.2 μm/s.

Fig. 2.

Supercoil relaxation and substrate cleavage by Bxb1 Int in ensemble reactions. Synapsis of attP⁺ and attB-CT by Int completely relaxes substrate supercoils in a single encounter. pAttP⁺ supercoiled plasmid DNA (50 nM) was reacted with either attB-CT or attB⁺ oligo substrates (10 nM) and 1 μM Int for the times indicated. As indicated by the large amount of unreacted plasmid supercoils, the 5∶1 plasmid:oligo ratio and the low att site concentration ensure that the majority of pAttP products result from a single encounter between the two parental att sites. After treatment with SDS and proteinase K, samples were analyzed by agarose gel electrophoresis. (A) The gel was stained with ethidium bromide and imaged to show the ethidium-stained DNA (false color—red) and the fluorescent dye used to label the attB oligo substrates (false color—green); the images were superimposed so the recombinant species show as yellow bands. Relaxed pAttP⁺ topoisomers cluster around the nicked substrate circles (and linear recombinants). (B) After imaging, the gel of (A) was subjected to further electrophoresis, which separates the initially relaxed (but covalently closed) species (converted into positively supercoiled molecules by the intercalated ethidium) from the nicked circles. Note the absence of partially relaxed supercoils in both (A) and (B) which would migrate close to the fully supercoiled pAttP⁺ substrate (s.c.). (C) The cleaved state of synaptic complex is readily detected and has a substantial half-life. Linear attP-TA substrate (2,731 bp; about 40 nM) was reacted with an attB-TA oligo substrate (312 bp, 400 nM) and 2 μM Int for the times indicated. Products were analyzed by agarose gel electrophoresis. Cleavage products (1,365 bp) are readily detected even though both synaptic orientations are recombinationally proficient. Quantitative analysis of formation and decay of the cleaved state for the recombinationally proficient reaction indicated its half-life was about 1.8 min (Fig. S2).

Fig. 3.

Relaxation velocities for Int and comparison to other DNA-relaxing enzymes. (A) Rotation rate during supercoil relaxation (angular velocity) for 0.5 pN force. A peaked distribution is obtained with mean relaxation rate of 54 ± 5 turns/s, corresponding to a linear velocity of 2.9 ± 0.3 μm/s. (B) Average rates of rotation during torsional relaxation of DNA by different enzymes, for 0.5 pN. Left-most bar shows the velocity for the Int relaxation of supercoiled DNA; second bar shows velocity for braided DNAs. Int relaxes scDNA at about half the rate of vaccinia topo IB (third bar) and topo V (fourth bar), and about one-third of the rate of nicking endonuclease Nt.AlwI (fifth bar).

Fig. 4.

Unbraiding assay and an example of relaxation events for pNG1179 attP⁺× attB⁺. (A) Two identical nicked 6.2 kb linear DNAs carrying both attP⁺ (filled arrow) and attB⁺ (open arrow) sites (inverted orientation, separated by 441 bp) tethered between a paramagnetic bead and the cover slip form a supercoilable two-DNA tether. When sufficiently twisted, the braid will buckle and undergo plectonemic supercoiling. (B) Extension vs. Ca for a typical double-DNA tether, for 0.47 pN force, in protein-free buffer. A sharp drop from 1.63 μm to 1.51 μm (center) is found at the beginning of rotation in both positive and negative directions. Extension decreases slowly and smoothly as more Ca is introduced. For Ca > +18 and < -17 extension depends linearly on Ca and can be fit with a slope of 63.7 μm/turn and -60.7 μm/turn for negative and positive wings, respectively, indicating plectonemic supercoiling of the braid. (C) Synapsis of attP⁺ and attB⁺ sites in the recombinationally inappropriate orientation by Int (open circles) and cleavage of all four strands permits previously stored catenation number (Ca) to be released. (D) Synapsis of attP⁺ and attB⁺ in the opposite, recombinationally competent, orientation simply partitions Ca into two domains, and does not permit braid relaxation. (E) Int-mediated relaxation process for the tether of (B). Regions I (starting at 0 s and ∼512 s, respectively) correspond to the initially supercoiled plectonemic state, with Ca = -25 and 0.47 pN force. Both regions I end with relaxation of the DNA, to extension ∼1.5 μm corresponding to an X-shaped structure containing a synapse. Then, the synapse opens (start of regions III), leading to a ∼1.65 μm-long braid, indicating a return to the parental state. Region IV begins with extension increase to ∼1.8 μm; at this point torsional stiffness was lost indicating breakage of one of the two tethers.