Modulation of T4 gene 32 protein DNA binding activity by the recombination mediator protein UvsY

Abstract

Bacteriophage T4 UvsY is a recombination mediator protein that promotes assembly of the UvsX-ssDNA presynaptic filament. UvsY helps UvsX to displace T4 gene 32 protein (gp32) from ssDNA, a reaction necessary for proper formation of the presynaptic filament. Here we use DNA stretching to examine UvsY interactions with single DNA molecules in the presence and absence of gp32 and a gp32 C-terminal truncation (*I), and show that in both cases UvsY is able to destabilize gp32-ssDNA interactions. In these experiments UvsY binds more strongly to dsDNA than ssDNA due to its inability to wrap ssDNA at high forces. To support this hypothesis, we show that ssDNA created by exposure of stretched DNA to glyoxal is strongly wrapped by UvsY, but wrapping occurs only at low forces. Our results demonstrate that UvsY interacts strongly with stretched DNA in the absence of other proteins. In the presence of gp32 and *I, UvsY is capable of strongly destabilizing gp32-DNA complexes in order to facilitate ssDNA wrapping, which in turn prepares the ssDNA for presynaptic filament assembly in the presence of UvsX. Thus, UvsY mediates UvsX binding to ssDNA by converting rigid gp32-DNA filaments into a structure that can be strongly bound by UvsX.

Fig. 1

Proteolytic fragments of gene 32 protein. *I is obtained by trypsin cleavage of full length gp32 at residue 253, while *III results from cleavage at residues 21 and 253. A MOLSCRIPT representation of a *III-oligonucleotide complex is shown at its location within the protein sequence. The protein is pictured in ribbon mode, with the major lobe green, the minor (Zn-containing) lobe blue, and the residue 198–239 flap red. The bound oligonucleotide, in sticks mode, is red, and the coordinated Zn²⁺, in space-filling mode, is yellow. The position of the oligodeoxynucleotide, pTTAT, is approximate; it was modeled by Shamoo et al. to maximally overlap excess electron density in the trough . The Protein Data Bank entry for core domain (without the oligonucleotide) is 1gpc.pdb.

Fig. 2

a) In a dual beam optical tweezers instrument, two laser beams are focused to a small spot, creating an optical trap that attracts polystyrene beads. Single DNA molecules are attached at one end to a bead in the trap, while the other end is attached to another bead held by a glass micropipette. As the DNA molecule is stretched by moving the micropipette, the resulting force on the bead in the trap is measured. b) Typical force extension curves for double stranded DNA are shown as dotted lines. As the stretching force is increased, dsDNA reveals an entropic elastic response, followed by the overstretching region. The data in purple shows typical data for a full cycle of extension and relaxation, including some hysteresis upon reannealing. The data in blue and cyan show the response of the resulting single strands to yet higher forces, as the strands finally separate near 150 pN (thus there are no relaxation curves). The solid lines are DNA models for ssDNA and dsDNA, as described previously .

Fig. 3

Stretching (solid line) and relaxation curves (symbols) for λ-DNA and UvsY in 10mM Hepes, 50 mM Na⁺ (45 mM NaCl and 5 mM NaOH) and pH 7.5. Main panel : Absence of protein (black), 16nM UvsY (pink), 50nM UvsY (red) 100 nM UvsY (blue) and 200nM (green, stretching curve only). Inset: Overstretching force of DNA (F_ov) as a function of UvsY concentration in 50mM Na⁺. While each curve shown on the main panel represents one stretching experiment, the error bars on the insert show the standard deviation of measured forces for at least three stretches, demonstrating that the stretching results are reproducible within an error of approximately ± 1.5 pN.

Fig. 4

a) Stretching (solid line) and relaxation (dash line) curves for λ-DNA in the absence of protein (black) at a pulling rate of 250nm/s, 200nM UvsY (red and blue) at a pulling rate of 100nm/s. b) Stretching (solid line) and relaxation (dash line) curves for λ-DNA in the absence of protein (black) at a pulling rate of 250nm/s, 0.5mM Glyoxal (brown) at a pulling rate of 100nm/s, obtained after exposing DNA held at 0.45 nm/bp to Glyoxal for 30 minutes, 200nM UvsY (red) at a pulling rate of 100nm/s, 200nM UvsY (blue, green) at a pulling rate of 25nm/s. c) Stretching (solid line) and relaxation (dash line) curves for λ-DNA in the absence of protein (black) at a pulling rate of 250nm/s, 0.5mM Glyoxal (brown), obtained after exposing DNA held at 0.45 nm/bp for 30 minutes, 200nM gp32 (red, blue, green) at a pulling rate of 100nm/s. All data in the figure was obtained in 10mM Hepes, 100 mM Na⁺ (95 mM NaCl and 5 mM NaOH) and pH 7.5 and is representative data from a single DNA molecule chosen from a set of similar curves obtained on at least 3 different DNA molecules.

Fig. 5

Stretching and relaxation curves for λ-DNA and various combinations of UvsY, gp32 and *I in 10 mM Hepes,100 mM Na⁺ (95mM NaCl and 5mM NaOH) and pH 7.5. Data are shown in the absence of protein (dark blue), 200 nM gp32 (red), 200 nM gp32 and 100nM UvsY (pink open diamonds), 200nM *I (blue), and 200 nM of *I and 100 nM UvsY (green solid diamonds). The time for stretching and relaxation is approximately two minutes. The reproducibility of these measurements for individual stretching curves is similar to that obtained for the data in Fig. 3.

Fig. 6

Stretching (solid line) and relaxation curves (symbols) for λ-DNA in 10 mM Hepes, pH 7.5, 50mM [Na⁺] (45 mM NaCl and 5 mM NaOH) in the absence of protein (dark blue) and in presence of 200 nM gp32 (red), 200 nM gp32 and 50nM UvsY (light blue), 200nM gp32 and 100nM UvsY( orange) and 200nM gp32 and 200nM UvsY (blue). The reproducibility of these measurements for individual stretching curves is similar to that obtained for the data in Fig. 3.

Fig. 7

Dependence of DNA melting force (F) as a function of time. Data was obtained in the presence of 4nM *I (black), 4 nM *I and 32 nM UvsY (red), 4 nM *I and 50 nM UvsY (blue), and 4 nM *I and 150nM UvsY (green) in 50 mM Na⁺, 10 mM Hepes, pH 7.5.

Fig. 8

UvsY concentration dependence of the apparent cooperative equilibrium binding constant to ssDNA (K_ssω^App) of *I. The error bars are determined from the standard error of at least 3 measurements.

Fig. 9

Schematic diagram showing two mechanisms for the role of UvsY in synaptic filament formation, which are valid under different solution conditions. a) In the concerted mechanism, which is likely valid in high salt when K_ss,UvsY ≈ K_ss,gp32, UvsY initially binds to gp32-coated ssDNA and wraps the ssDNA, forming a cofilament structure to which gp32 remains bound. UvsX and ATP are subsequently required for removal of gp32 from the filament. b) In the step-wise mechanism, which is likely valid in low salt when K_ss,UvsY ≫ K_ss,gp32 , UvsY competes directly with gp32 for ssDNA binding, therefore removing gp32 from ssDNA. UvsY subsequently wraps the ssDNA tightly, forming patches of UvsY lacking gp32 completely. UvsX and ATP are then required to form the final synaptic filament. Pre-synaptic filament formation might occur via a combination of both mechanisms, denoted via dotted arrows (see Discussion for details).