uvsY Protein Research Articles

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

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.

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

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.

Crystallization and preliminary X-ray analysis of bacteriophage T4 UvsY recombination mediator protein

Bacteriophage T4 UvsY protein is considered to be the prototype of recombination mediator proteins, a class of proteins which assist in the loading of recombinases onto DNA. Wild-type and Se-substituted UvsY protein have been expressed and purified and crystallized by hanging-drop vapor diffusion. The crystals diffract to 2.4 A using in-house facilities and to 2.2 A at NSLS, Brookhaven National Laboratory. The crystals belong to space group P422, P4(2)22, P42(1)2 or P4(2)2(1)2, the ambiguity arising from pseudo-centering, with unit-cell parameters a = b = 76.93, c = 269.8 A. Previous biophysical characterization of UvsY indicates that it exists primarily as a hexamer in solution. Along with the absence of a crystallographic threefold, this suggests that the asymmetric unit of these crystals is likely to contain either three monomers, giving a solvent content of 71%, or six monomers, giving a solvent content of 41%.

Dynamics of Bacteriophage T4 Presynaptic Filament Assembly from Extrinsic Fluorescence Measurements of Gp32-Single-stranded DNA Interactions

In the bacteriophage T4 homologous recombination system, presynaptic filament assembly on single-stranded (ssDNA) DNA requires UvsX recombinase, UvsY mediator, and Gp32 ssDNA-binding proteins. Gp32 exerts both positive and negative effects on filament assembly: positive by denaturing ssDNA secondary structure, and negative by competing with UvsX for ssDNA binding sites. UvsY is believed to help UvsX displace Gp32 from the ssDNA. To test this model we developed a real-time fluorescence assay for Gp32-ssDNA interactions during presynapsis, based on changes in the fluorescence of a 6-iodoacetamidofluorescein-Gp32 conjugate. Results demonstrate that the formation of UvsX presynaptic filaments progressively disrupts Gp32-ssDNA interactions. Under stringent salt conditions the disruption of Gp32-ssDNA by UvsX is both ATP- and UvsY-dependent. The displacement of Gp32 from ssDNA during presynapsis requires ATP binding, but not ATP hydrolysis, by UvsX protein. Likewise, UvsY-mediated presynapsis strongly requires UvsY-ssDNA interactions, and is optimal at a 1:1 stoichiometry of UvsY to UvsX and/or ssDNA binding sites. Presynaptic filaments formed in the presence of UvsY undergo assembly/collapse that is tightly coupled to the ATP hydrolytic cycle and to stringent competition for ssDNA binding sites between Gp32 and various nucleotide-liganded forms of UvsX. The data directly support the Gp32 displacement model of UvsY-mediated presynaptic filament assembly, and demonstrate that the transient induction of high affinity UvsX-ssDNA interactions by ATP are essential, although not sufficient, for Gp32 displacement. The underlying dynamics of protein-ssDNA interactions within presynaptic filaments suggests that rearrangements of UvsX, UvsY, and Gp32 proteins on ssDNA may be coupled to central processes in T4 recombination metabolism.

Mutations in a Conserved Motif Inhibit Single-stranded DNA Binding and Recombination Mediator Activities of Bacteriophage T4 UvsY Protein

The UvsY recombination mediator protein is critical for homologous recombination in bacteriophage T4. UvsY uses both protein-protein and protein-DNA interactions to mediate the assembly of the T4 UvsX recombinase onto single-stranded (ss) DNA, forming presynaptic filaments that initiate DNA strand exchange. UvsY helps UvsX compete with Gp32, the T4 ssDNA-binding protein, for binding sites on ssDNA, in part by destabilizing Gp32-ssDNA interactions, and in part by stabilizing UvsX-ssDNA interactions. The relative contributions of UvsY-ssDNA, UvsY-Gp32, UvsY-UvsX, and UvsY-UvsY interactions to these processes are only partially understood. The goal of this study was to isolate mutant forms of UvsY protein that are specifically defective in UvsY-ssDNA interactions, so that the contribution of this activity to recombination processes could be assessed independent of other factors. A conserved motif of UvsY found in other DNA-binding proteins was targeted for mutagenesis. Two missense mutants of UvsY were isolated in which ssDNA binding activity is compromised. These mutants retain self-association activity, and form stable associations with UvsX and Gp32 proteins in patterns similar to wild-type UvsY. Both mutants are partially, but not totally, defective in stimulating UvsX-catalyzed recombination functions including ssDNA-dependent ATP hydrolysis and DNA strand exchange. The data are consistent with a model in which UvsY plays bipartite roles in presynaptic filament assembly. Its protein-ssDNA interactions are suggested to moderate the destabilization of Gp32-ssDNA, whereas its protein-protein contacts induce a conformational change of the UvsX protein, giving UvsX a higher affinity for the ssDNA and allowing it to compete more effectively with Gp32 for binding sites.

Biochemical interactions within a ternary complex of the bacteriophage T4 recombination proteins uvsY and gp32 bound to single-stranded DNA.

The presynaptic phase of homologous recombination requires the formation of a filament of single-stranded DNA (ssDNA) coated with a recombinase enzyme. In bacteriophage T4, at least three proteins are required for the assembly of this presynaptic filament. In addition to the T4 recombinase, uvsX protein, the T4 ssDNA binding protein (gp32), and the uvsY recombination accessory protein are also required. Here we report on a detailed analysis of a tripartite filament containing ssDNA bound by stoichiometric quantities of both uvsY and gp32, which appears to be an important intermediate in the assembly of the T4 presynaptic filament. We demonstrate that uvsY and gp32 simultaneously co-occupy the ssDNA in a noncompetitive fashion. In addition, we show that protein-protein interactions between uvsY and gp32 are not required for the assembly of this ternary complex and do not affect the affinity of uvsY for the ssDNA lattice. Finally, we demonstrate that the interaction of gp32 with the ssDNA is destabilized within this complex, in a manner which is independent of gp32-uvsY interactions. The data suggest that the uvsY protein acts to remodel the gp32-ssDNA complex via uvsY-ssDNA interactions. The implications of these findings for the mechanism of presynapsis in the T4 recombination system are discussed.

The uvsY recombination protein of bacteriophage T4 forms hexamers in the presence and absence of single-stranded DNA.

A prerequisite to genetic recombination in the T4 bacteriophage is the formation of the presynaptic filament-a helical nucleoprotein filament containing stoichiometric amounts of the uvsX recombinase in complex with single-stranded DNA (ssDNA). Once formed, the filament is competent to catalyze homologous pairing and DNA strand exchange reactions. An important component in the formation of the presynaptic filament is the uvsY protein, which is required for optimal uvsX-ssDNA assembly in vitro, and essential for phage recombination in vivo. uvsY enhances uvsX activities by promoting filament formation and stabilizing filaments under conditions of low uvsX, high salt, and/or high gp32 (ssDNA-binding protein) concentrations. The molecular properties of uvsY include noncooperative binding to ssDNA and specific protein-protein interactions with both uvsX and gp32. Evidence suggests that all of these hetero-associations of the uvsY protein are important for presynaptic filament formation. However, there is currently no structural information available on the uvsY protein itself. In this study, we present the first characterization of the self-association of uvsY. Using hydrodynamic methods, we demonstrate that uvsY associates into a stable hexamer (s020,w = 6.0, M = 95 kDa) in solution and that this structure is competent to bind ssDNA. We further demonstrate that uvsY hexamers are capable of reversible association into higher aggregates in a manner dependent on both salt and protein concentration. The implications for presynaptic filament formation are discussed.

Characterization of an amino-terminal fragment of the bacteriophage T4 uvsY recombination protein

The uvsY protein plays essential roles in homologous genetic recombination processes in the bacteriophage T4. In vitro, uvsY promotes the formation of presynaptic filaments containing stoichiometric amounts of the T4 uvsX recombinase bound to single-stranded DNA. uvsY protein has intrinsic binding activities towards ssDNA, uvsX, and gp32, the T4-encoded SSB, however, it has not been directly determined which of these activities are essential for uvsY's role in presynapsis. We have therefore sought to generate altered forms of uvsY deficient in uvsX- and/or gp32-binding, in order to assess whether these specific protein-protein interactions are essential for uvsY recombination functions. Limited chymotrypsinolysis of the 16 kDa uvsY protein generates two major fragments: an 11.4 kDa fragment containing the N-terminus of uvsY, and a 4.5 kDa C-terminal fragment. We have expressed and purified the large fragment as a fusion protein containing the N-terminal 101 amino acids of uvsY. We show that this truncated uvsY species, which we call uvsY nt, retains ssDNA-binding activity, but is devoid of both uvsX- and gp32-binding activities. Like native uvsY, uvsY nt stimulates the ssDNA-dependent ATPase activity of the uvsX protein, however, the synergistic effects observed between uvsY, uvsX, and gp32 are not observed with uvsY nt. In addition, uvsY nt weakly stimulates uvsX-catalyzed DNA strand exchange reactions. The latter result is surprising since it suggests that specific interactions with uvsX and/or gp32 are not absolutely essential for uvsY recombination functions. Taken together, the data are consistent with a model in which uvsY-ssDNA interactions alone are capable of promoting the assembly of functional uvsX-ssDNA complexes, while uvsY-protein interactions stabilize uvsX-ssDNA complexes.

Single-stranded DNA binding properties of the uvsy recombination protein of bacteriophage T4

The uvsY protein is an essential component of the bacteriophage T4 general recombination machinery. The properties of this 16 kDa protein include selective binding to ssDNA, as well as specific protein-protein interactions with other T4 recombination proteins including uvsX (general recombinase) and gp32 (ssDNA-binding protein). uvsY promotes the assembly of uvsX-ssDNA filaments, the active species in uvsX-catalyzed DNA rearrangements, apparently by helping uvsX displace gp32 from the ssDNA. To better understand the role of uvsY in the T4 recombination system, here we characterize the thermodynamic and molecular properties of the interaction of the uvsY protein with a model single-stranded polynucleotide, εDNA, which is a fluorescent, etheno-modified form of random-sequence ssDNA. We have found that the binding of uvsY protein enhances the fluorescence of the εDNA lattice and that the maximal amount of fluorescence enhancement observed is dependent on salt concentration. In addition, we have used the εDNA fluorescence enhancement assay to establish thermodynamic parameters of binding and to define some of the molecular details of uvsY-εDNA interactions. We show that uvsY binds to εDNA in a non-cooperative manner, with a binding site size of four nucleotide residues per monomer of uvsY, and that this binding is salt-sensitive and involves the displacement of anions from the uvsY protein. We further show that uvsY protein binds preferentially to εDNA over unmodified ssDNA. The significance of these results is discussed in light of current models of uvsY action in the T4 recombination system.

Inhibition of holliday structure resolving endonuclease VII of bacteriophage T4 by recombination enzymes UvsX and UvsY

Proteins UvsX, UvsY and Endonuclease VII (Endo VII) of bacteriophage T4 are required for DNA recombination, replication and repair. Endo VII is the product of gene 49 (gp49) and essential for resolution of branches from newly made DNA, prior to packaging into preformed heads. The ability of Endo VII to resolve Holliday structures in vitro suggested an in vivo function for the resolution of recombination intermediates, generated by UvsX and UvsY during the early infection cycle. Here we report results which contrast with this hypothesis. It is shown that the potent endonucleolytic activity of Endo VII with branched DNAs is inhibited in strand transfer reactions by the strand transferase UvsX, and more strongly by the accessory protein UvsY in vitro. The inhibitory effect of UvsX or UvsY is also seen in reactions with Endo VII using two synthetic cruciform DNAs and a C/C-mismatch containing substrate. Low concentrations of UvsY protein (12 ng or 0,76 pmol) were sufficient to reduce the cleavage efficiency of 30 units of Endo VII (about 16 fmol) to 50%. The inhibition is due to a direct protein-protein interaction between Endo VII, UvsX and UvsY as suggested by electrophoretic mobility shift assays (EMSAs). These results were confirmed through affinity chromatography, where UvsX and UvsY bound to Endo VII, immobilized on a NHS-activated Sepharose matrix. This is the first identification of phage-encoded proteins which modulate the potent endonucleolytic activity of gp49 in vitro.

Recombination-dependent DNA replication stimulated by double-strand breaks in bacteriophage T4.

We analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.

The purification and characterization of gene 59 protein from bacteriophage T4.

Gene 59 of bacteriophage T4 is involved in genetic recombination and recombination-dependent DNA replication. We purified the protein encoded by gene 59 after cloning the gene in an expression vector. The molecular weight and the sequence of seven N-terminal amino acids of the protein were consistent with the nucleotide sequence of the gene (Hahn, S., Kruse, U., and Ruger, W. (1986) Nucleic Acids Res. 14, 9311-9327). The purified 59 protein appeared to be a monomer under nondenaturing conditions, and it bound to single-stranded DNA in preference to double-stranded DNA. The protein could bind to a 24-nucleotide-long single-stranded DNA molecule that had previously bound either 32 protein (single-stranded DNA-binding protein) or uvsY protein (enhancer of uvsX protein), but it would not bind to this DNA if it had previously bound uvsX protein (synaptase). The binding occurred rapidly with 32 protein-bound DNA and slowly with uvsY protein-bound DNA. Accordingly, 32 protein bound specifically to an agarose matrix containing immobilized 59 protein. The 41 protein (DNA helicase) also bound specifically to this agarose matrix.

The role of protein-protein interactions in the assembly of the presynaptic filament for T4 homologous recombination.

The presynaptic filament is an obligatory intermediate in general genetic recombination. It is composed of a strand transferase protein polymerized along single-stranded DNA. In bacteriophage T4-infected cells, the presynaptic filament is composed of at least three proteins. In addition to the strand transferase (the uvsX protein), the uvsY (an accessory factor), and gene 32 (a helix-destabilizing factor), proteins also bind to the single-stranded DNA. In this report, we probe the assembly of the T4 presynaptic filament and the organization of the proteins in the complex. We find that interactions between the uvsY protein and the C terminus of the gene 32 protein are required to load UvsY onto gene 32 protein-covered DNA. Efficient binding of the uvsY protein to DNA is shown to be necessary for subsequent loading of the uvsX protein.

Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.

The characterization of a complex of three bacteriophage T4 recombination proteins, uvsX protein, uvsY protein, and gene 32 protein, on single-stranded DNA.

Three recombination proteins of bacteriophage T4, uvsX, uvsY, and gene 32 proteins, were examined for the formation of a complex with short single-stranded DNA (ssDNA) molecules containing either 24 or 69 nucleotides. Gel-shift assays revealed that either the uvsX or uvsY protein, when present alone, formed a stable complex only with the 69-mer, while the gene 32 protein bound stably to both ssDNAs. However, a characteristic stable complex formed on the 24-mer when both the uvsX and uvsY proteins were present, and the uvsY protein bound to this DNA in the presence of the gene 32 protein. Isolation of the complexes by centrifugation through a glycerol gradient revealed their protein constituents and showed that the uvsX protein-uvsY protein-24-mer ssDNA complex formed even in the presence of excess gene 32 protein. The possible biological significance of these protein-DNA complexes is discussed.

Functional interactions between phage T4 and E. coli DNA-binding proteins during the presynapsis phase of homologous recombination

The “protein machine” for phage T4 homologous recombination has begun to be assembled in vitro. A particularly heavily studied reaction has been the uvsX protein (a RecA-like strand transferase)-mediated homologous pairing reaction between single and double-stranded DNAs, a key step in the recombination cycle in vivo. A necessary prerequisite for uvsX protein-mediated pairing is the polymerization of this factor along the invading single strand, a process known as presynapsis. Recent work has indicated that at least two other T4 recombination factors are involved in this process as well, the uvsY and gene 32 products. These proteins are also ssDNA-binding factors and exhibit an affinity for UvsX and each other. In order to begin to sort out the potential functional roles played by these protein-protein interactions in presynapsis, I have examined the ability of the uvsX protein to form stable filaments along ssDNA in the presence of these proteins. It is shown that the uvsY protein relieves the inhibition to filament formation due to the presence of the gene 32 protein, but experiments with the E. coli SSB protein (the bacterial analogue of gp32) suggest that this effect does not involve a direct interaction between UvsY and gp32.

The UvsY protein of bacteriophage T4 modulates recombination-dependent DNA synthesis in vitro.

An in vitro system containing the T4 gene 43, 45, 44/62, 32, dda, and uvsX proteins catalyzes DNA synthesis that is dependent on the synapsis step of homologous genetic recombination. The rate of DNA synthesis in this system is highly dependent on the concentration of the uvsX recombinase (a recA-like protein). Here we report the effect of the T4 uvsY protein, a recombination accessory protein, on this reaction. Low concentrations of uvsY protein greatly stimulate DNA synthesis at low concentrations of uvsX protein, but these same concentrations inhibit DNA synthesis at high concentrations of uvsX protein. As a result, the addition of small amounts of uvsY protein lowers the minimum concentration of uvsX protein needed for the reaction 8-fold, and it lowers the uvsX protein concentration for maximum activity 4-fold. The uvsY protein can affect either the initiation or elongation phase of DNA synthesis, depending on the concentration of uvsX protein present. The implications of these results for the function of the uvsY protein in T4 DNA replication in vivo are discussed.

The Phage T4 uvsY Recombination Protein Stabilizes Presynaptic Filaments

The bacteriophage T4 uvsY protein is required for efficient recombination in T4-infected Escherichia coli cells. Previous in vitro work has shown that the purified uvsY protein is an accessory protein; it stimulates homologous pairing catalyzed by the phage uvsX protein (a RecA-like recombinase) under certain conditions. We show here that this effect can be traced, at least in part, to a UvsY-dependent stabilization of uvsX protein-single-stranded DNA complexes. These presynaptic filaments are one of the early obligatory intermediates in the strand exchange reaction between homologous single- and double-stranded DNAs. The mechanism of filament stabilization seems to involve a slower loss of UvsX subunits. A model that accounts for the data is presented in which both recombination proteins are incorporated into the presynaptic filament.

Synergistic action of three recombination gene products of bacteriophage T4, uvsX, uvsY, and gene 32 proteins

Previously we proved that uvsX protein catalyzes reactions similar to those thought to occur during T4 recombination (Yonesaki, T., Ryo, Y., Minagawa, T., and Takahashi, H. (1985) Eur. J. Biochem. 148, 127-134; Yonesaki, T., and Minagawa, T. (1985) EMBO J. 4, 3321-3327). Now we have found that uvsY protein stabilizes binding of uvsX protein and single-stranded DNA and weakly stimulates those reactions catalyzed by uvsX protein, although it has little activity, if any, by itself. Gene 32 protein also stimulates them weakly at low concentrations, but is strongly inhibitory at high concentrations (Yonesaki, T., and Minagawa, T. (1985) EMBO J. 4, 3321-3327; Formosa, T., and Alberts, B. M. (1986) J. Biol. Chem. 261, 6107-6118). This inhibition is counteracted by uvsY protein. The highest efficiency of uvsX protein is achieved by the co-existence of a small amount of uvsY protein and a large amount of gene 32 protein. We discuss the mechanism and the role of the three proteins in recombination.

UvsY protein of bacteriophage T4 is an accessory protein for in vitro catalysis of strand exchange

The uvsX and uvsY genes are essential to genetic recombination, recombination-dependent DNA synthesis and to the repair of DNA damage in bacteriophage T4. Purified UvsX protein has been shown to catalyze strand exchange and D-loop formation in vitro, but the role of UvsY protein has been unclear. We report that UvsY protein enhances strand exchange by UvsX protein by interacting specifically with UvsX protein: gene 32 protein (gp32) is not necessary for this effect and UvsY protein has no similar effect on the RecA protein of E. coli. UvsY protein, like UvsX protein, protects single-stranded DNA from digestion by nucleases, but, unlike UvsX protein, shows no ability to protect double-stranded DNA. UvsY protein enhances the rate of single-stranded-DNA-dependent ATP hydrolysis by UvsX protein, particularly in the presence of gp32 or high concentrations of salt, factors that otherwise reduce the ATPase activity of UvsX protein. The enhancement of ATP hydrolysis by UvsY protein is shown to result from the ability of UvsY protein to increase the affinity of UvsX protein for single-stranded DNA.