DNA-binding properties of T4 UvsY recombination mediator protein: polynucleotide wrapping promotes high-affinity binding to single-stranded DNA

Abstract

To carry out homologous recombination events in the cell, recombination proteins must be able to recognize and form presynaptic filaments on single-stranded DNA (ssDNA) in the presence of a vast excess of double-stranded DNA (dsDNA). Therefore recombination machineries stringently discriminate between ssDNA and dsDNA lattices. Recent single-molecule studies of bacteriophage T4 recombination proteins revealed that, surprisingly, the UvsY recombination mediator protein binds stronger to stretched dsDNA molecules than to stretched ssDNA. Here, we show that for relaxed DNA lattices, the opposite is true: UvsY exhibits a 1000-fold intrinsic affinity preference for ssDNA over dsDNA at moderate salt concentrations. This finding suggests that UvsY preferentially loads UvsX recombinase onto ssDNA under physiological conditions. The biochemical basis for high-affinity UvsY-ssDNA binding was investigated by hydrodynamic and cross-linking methods. Results show that UvsY forms ring-like hexamers in solution, and that ssDNA binds to multiple subunits within each hexamer, consistent with ssDNA wrapping. The data support a model in which ssDNA wrapping by UvsY protein is important for the selective nucleation of presynaptic filaments on ssDNA versus dsDNA, and for the coordinated transfer of ssDNA from Gp32 (SSB) to UvsY (RMP) to UvsX (recombinase) during filament assembly.

Figure 1.

Salt-dependent elution of UvsY protein from dsDNA– and ssDNA–cellulose columns. Elution profiles are presented in the format of Log P_ci versus fraction number as described in ‘Materials and Methods’ section. UvsY (4 nmol) was loaded onto and eluted from dsDNA–cellulose columns (A) or ssDNA–cellulose columns (B) in buffer A plus various concentrations of NaCl as denoted in the figure. The fractions were quantified by intrinsic tryptophan fluorescence (excitation 295 nm, emission 340 nm) and calibrated against known concentrations of UvsY. All data points are averaged values from three independent experiments and standard deviations are shown as error bars. At low NaCl concentrations, the error bars are too small to be visualized on the plots.

Figure 2.

Salt-dependent elution of UvsY_K58A single mutant protein from dsDNA– and ssDNA–cellulose columns. UvsY_K58A (4 nmol) was loaded onto and eluted from dsDNA–cellulose columns (A) or ssDNA–cellulose columns (B) in buffer A plus various concentrations of NaCl as denoted in the figure. All other experimental details were identical to those reported in Figure 1.

Figure 3.

Salt-dependent elution of UvsY_K58A,R60A double mutant protein from dsDNA– and ssDNA–cellulose columns. UvsY_K58A,R60A (4 nmol) was loaded onto and eluted from dsDNA–cellulose columns (A) or ssDNA–cellulose columns (B) in buffer A plus various concentrations of NaCl as denoted in the figure. All other experimental details were identical to those reported in Figure 1.

Figure 4.

Ion effects on the intrinsic association constants of UvsY for ssDNA and dsDNA, and of UvsY_K58A for ssDNA, respectively. The log–log plots are made using K_ss and K_ds values and corresponding NaCl concentrations listed in Table 1. The error bars represent the standard deviation from three independently determined K_ss or K_ds values for UvsY–ssDNA (diamonds), UvsY–dsDNA (triangles) and UvsY_K58A–ssDNA (squares) interactions. The data in each series have been fitted to a line yielding slopes representing the dlog K_{(ss or ds)}/dlog[NaCl] values for UvsY–dsDNA, UvsY–ssDNA and UvsY_K58A–ssDNA interactions. These dlog K_{(ss or ds)}/dlog[NaCl] values (slopes) are summarized in Table 2.

Figure 5.

Meniscus depletion of dT₂₄ oligonucleotide by UvsY protein. Meniscus depletion sedimentation equilibrium experiments were carried out as described in ‘Materials and Methods’ section. (A) Depletion experiments conducted under non-DNA-binding conditions (buffer B plus 500 mM NaCl) show no co-sedimentation of oligonucleotide dT₂₄ with UvsY. (B) Similar experiments conducted under permissive conditions for DNA binding (buffer B plus 300 mM NaCl) show a ∼1: 1 stoichiometry of UvsY₆:dT₂₄. In both experiments, oligonucleotide dT₂₄ alone (squares), UvsY alone (circle) or both (triangles) were centrifuged to equilibrium in a Beckman Optima XL-I analytical ultracentrifuge while scanning the absorbance at 260 and 280 nm. Co-sedimentation of dT₂₄ with UvsY under permissive binding conditions lowers absorbance at the meniscus, allowing calculation of complex stoichiometry.

Figure 6.

Photochemical crosslinking of UvsY–dT₂₄ complex. UvsY was crosslinked to 5′-[³²P]-labeled dT₂₄ by 254 nm UV exposure as described in ‘Materials and Methods’ section. The crosslinked species (black arrows) were separated from free 5′-[³²P]-labeled dT₂₄ (white arrow) by SDS–PAGE and visualized by autoradiography. Different exposure times (0, 15 and 30 s) were applied as indicated and pre-stained molecular weight standards (left) were used to estimate the molecular weight of the crosslinked species.

Figure 7.

Hydrodynamic modeling of hexameric assemblies of spherical beads. The frictional ratio f / f ₀ was calculated for modeled assemblies as described in ‘Materials and Methods’ and ‘Result’ sections. Assemblies are assumed to be rigid, and composed of identical rigid, incompressible spheres. R is the equivalent radius of each sphere. The UvsY hexamer (f / f ₀ = 1.2) shows similarity to the open lockwasher (A) and hexagonal (C) models.

Figure 8.

A double hand-off model for the mechanism of mediator protein UvsY in T4 presynaptic filament assembly. Adapted with modifications from ref. (25). UvsY protein facilitates the loading of UvsX recombinase onto ssDNA and the concomitant displacement of Gp32 ssDNA-binding protein from ssDNA. The figure shows UvsX loading and Gp32 displacement from the perspective of a single UvsY hexamer, as if looking down the helical axis of a nascent presynaptic filament. The cooperative binding of Gp32 to ssDNA extends the polynucleotide lattice. The first hand-off occurs as hexameric UvsY recognizes and binds to the extended ssDNA (step 1), then converts it into a wrapped conformation(s) (steps 2 and 3), destabilizing Gp32–ssDNA interactions in the process. The UvsY-wrapped ssDNA complex is postulated to be in equilibrium between ‘closed’ and ‘open’ conformations (step 3), the latter of which is recognized by the ATP-bound form of UvsX protein to nucleate presynaptic filament assembly (step 4) while displacing Gp32. (A) Steps 3 and 4 constitute a step-wise mechanism for Gp32 displacement and UvsX loading by UvsY, which may occur under low-salt conditions. (B) Under high-salt conditions UvsY does not displace Gp32 from ssDNA directly, so filament assembly likely occurs by a concerted mechanism in which synergistic action of UvsY and ATP-bound UvsX is required to displace Gp32. See text for details.