Functional complementation of UvsX and UvsY mutations in the mediation of T4 homologous recombination

Abstract

Bacteriophage T4 homologous recombination events are promoted by presynaptic filaments of UvsX recombinase bound to single-stranded DNA (ssDNA). UvsY, the phage recombination mediator protein, promotes filament assembly in a concentration-dependent manner, stimulating UvsX at stoichiometric concentrations but inhibiting at higher concentrations. Recent work demonstrated that UvsX-H195Q/A mutants exhibit decreased ssDNA-binding affinity and altered enzymatic properties. Here, we show that unlike wild-type UvsX, the ssDNA-dependent ATPase activities of UvsX-H195Q/A are strongly inhibited by both low and high concentrations of UvsY protein. This inhibition is partially relieved by UvsY mutants with decreased ssDNA-binding affinity. The UvsX-H195Q mutant retains weak DNA strand exchange activity that is inhibited by wild-type UvsY, but stimulated by ssDNA-binding compromised UvsY mutants. These and other results support a mechanism in which the formation of competent presynaptic filaments requires a hand-off of ssDNA from UvsY to UvsX, with the efficiency of the hand-off controlled by the relative ssDNA-binding affinities of the two proteins. Other results suggest that UvsY acts as a nucleotide exchange factor for UvsX, enhancing filament stability by increasing the lifetime of the high-affinity, ATP-bound form of the enzyme. Our findings reveal new details of the UvsX/UvsY relationship in T4 recombination, which may have parallels in other recombinase/mediator systems.

Figure 1.

UvsY effects on ssDNA-dependent ATPase activities of UvsX, UvsX-H195A and UvsX-H195Q. Reaction velocities were measured by coupled spectrophotometric assay as described in Materials and Methods section. Reactions contained 0.4 µM UvsX (closed circles), UvsX-H195A (closed squares) or UvsX-H195Q (closed diamonds). ssDNA concentration was 4.5 µM and ATP concentration was 2 mM in all reactions, and UvsY concentration varied as indicated. All other conditions were as described in Materials and Methods section.

Figure 2.

Effects of wild-type and mutant UvsY proteins on ADP and AMP production by UvsX ssDNA-dependent ATPase activity. Velocities of ADP and AMP production were measured by TLC assay as described in Materials and Methods section. All reactions contained 0.45 µM UvsX, 4.5 µM ssDNA and 4 mM α-[³²P]-ATP. All other conditions were as described in Materials and Methods section. (A) Velocity of ADP production by wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration. (B) Velocity of AMP production by wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration. Note that (B) is plotted on an expanded scale compared to (A). (C) ADP/AMP product ratio for wild-type UvsX protein as a function of UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds) concentration.

Figure 3.

Effects of wild-type and mutant UvsY proteins on ADP production by (A) UvsX-H195A mutant and (B) UvsX-H195Q mutant ssDNA-dependent ATPase activities. Velocities of ADP production were measured by TLC assay as described in Materials and Methods section. All reactions contained 0.45 µM recombinase, 4.5 µM ssDNA, 4 mM α-[³²P]-ATP and variable concentrations of either UvsY (closed circles), UvsY-SM (closed squares) or UvsY-DM (closed diamonds). All other conditions were as described in Materials and Methods section.

Figure 4.

DNA strand exchange reactions promoted by different combinations of wild-type and mutant UvsX and UvsY proteins in the absence of Gp32. Reactions were performed at low UvsX concentration where strand exchange is codependent on UvsX and UvsY. (A) Agarose gel electrophoresis assays for DNA strand exchange were carried out as described in Materials and Methods section. All reactions contained 0.5 µM recombinase (wild-type, H195A or H195Q as indicated above the lanes), 10 µM M13mp18 ssDNA, 10 µM 5′-[³²P]-labeled M13mp18 RFIII DNA and 3 mM ATP. Lanes 1–4—control reactions lacking UvsY. Lanes 5–16—reactions containing 0.5 µM UvsY. Lanes 17–28—reactions containing 0.5 µM UvsY-SM. Lanes 29–40—reactions containing 0.5 µM UvsY-DM. All other conditions were as described in Materials and Methods section. Gel mobility positions of aggregates (agg), joint molecules (jm) and linear dsDNA (RFIII) substrate are shown to the left of the gel. (B) Quantification of results for reactions containing UvsY-wt and either UvsX-wt (filled circles), UvsX-H195A (filled squares) or UvsX-H195Q (filled diamonds), as determined by phosphorimaging of gel in (A), lanes 5–16. On the y-axis, % products denote percentage of total DNA migrating as joint molecules and aggregates. (C) Quantification of results for reactions containing UvsY-SM and either UvsX-wt (filled circles), UvsX-H195A (filled squares) or UvsX-H195Q (filled diamonds), as determined by phosphorimaging of gel in (A), lanes 17–28. (D) Quantification of results for reactions containing UvsY-DM and either UvsX-wt (filled circles), UvsX-H195A (filled squares) or UvsX-H195Q (filled diamonds), as determined by phosphorimaging of gel in Panel A, lanes 29–40.

Figure 5.

DNA strand exchange reactions promoted by different combinations of wild-type and mutant UvsX and UvsY proteins in the presence of Gp32. (A) Agarose gel electrophoresis assays for DNA strand exchange were carried out as described in Materials and Methods section. All reactions contained 0.5 µM recombinase (wild-type, H195A, or H195Q as indicated above the lanes), 1 µM Gp32, 10 µM M13mp18 ssDNA, 10 µM 5′-[³²P]-labeled M13mp18 RFIII DNA and 3 mM ATP. Lanes 1–7—control reactions lacking UvsY. Lanes 8–19—reactions containing 0.5 µM UvsY. Lanes 20–31—reactions containing 0.5 µM UvsY-SM. Lanes 32–43—reactions containing 0.5 µM UvsY-DM. All other conditions were as described in Materials and Methods section. Gel mobility positions of aggregates (agg), joint molecules (jm) and linear dsDNA (RFIII) substrate are shown to the left of the gel. (B) Quantification of results for reactions containing UvsY-wt and either UvsX-wt (filled circles), UvsX-H195A (filled squares) or UvsX-H195Q (filled diamonds), as determined by phosphorimaging of gel in (A), lanes 8–19. On the y-axis,% products denote percentage of total DNA migrating as joint molecules and aggregates. (C) Quantification of results for reactions containing UvsY-SM and either UvsX-wt (filled circles), UvsX-H195A (filled squares) or UvsX-H195Q (filled diamonds), as determined by phosphorimaging of gel in (A), lanes 20–31. (D) Quantification of results for reactions containing UvsY-DM and either UvsX-wt (filled circles), UvsX-H195A (filled squares) or UvsX-H195Q (filled diamonds), as determined by phosphorimaging of gel in (A), lanes 32–43.

Figure 6.

Models for UvsY effects on UvsX–ssDNA dynamics. (A) Formation of competent presynaptic filaments requires a hand-off of ssDNA from UvsY to UvsX, with the efficiency of the hand-off controlled by the relative ssDNA-binding affinities of the two proteins. A step-wise mechanism (upper path) is postulated in which UvsY hexamers interact with ssDNA to form a tightly wrapped ‘closed’ complex in equilibrium with a loosely wrapped ‘open’ complex. High UvsY concentrations shift the equilibrium toward the closed form that is inaccessible to UvsX protein. In contrast, UvsX captures ssDNA from the open complex, effectively shifting the closed/open equilibrium to the right. Mutations that weaken UvsX–ssDNA interactions, such as H195Q/A, cannot capture ssDNA from the open complex. On the other hand, mutations that weaken UvsY–ssDNA interactions, such as K58A/R60A, shift the closed/open equilibrium to the right, complementing UvsX mutations by allowing more efficient ssDNA hand-off and neutralizing the inhibitory effects of high UvsY concentrations. Alternatively, ssDNA hand-off could occur by a concerted mechanism as shown in the lower path. See text for additional details. (B) A hypothetical model for UvsY suppression of UvsX AMP production. Kinetics data indicate that ssDNA-bound UvsX hydrolyzes ATP to AMP via a processive, step-wise mechanism (11). UvsY may short circuit this process, causing ADP to release from the active site prior to hydrolysis to AMP. UvsY may therefore act as a nucleotide exchange factor for UvsX–ssDNA presynaptic filaments, and stimulate recombination by increasing the lifetime of UvsX in a high ssDNA-binding affinity state. See text for additional details.