Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently

The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination. Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA. We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state. As with RecA protein, these two states have a large difference in pitch. Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments. We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament. Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA. This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways.     

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.     

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

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.     

Crystal structures of Escherichia coli RecA in a compressed helical filament

The X-ray crystal structure of uncomplexed Escherichia coli RecA protein has been determined in three new crystal forms at resolutions of 1.9 A, 2.0 A, and 2.6 A. The RecA protein used for this study contains the extra residues Gly-Ser-His-Met at the N terminus, but retains normal ssDNA-dependent ATPase and coprotease activities. In all three crystals, RecA is packed in a right-handed helical filament with a pitch of approximately 74 A. These RecA filaments are compressed relative to the original crystal structure of RecA, which has a helical pitch of 82.7 A. In the structures of the compressed RecA filament, the monomer-monomer interface and the core domain are essentially the same as in the RecA structure with the 83 A pitch. The change in helical pitch is accommodated by a small movement of the N-terminal domain, which is reoriented to preserve the contacts it makes at the monomer-monomer interface. The new crystal structures show significant variation in the orientation and conformation of the C-terminal domain, as well as in the inter-filament packing interactions. In crystal form 2, a calcium ion is bound closely to a beta-hairpin of the C-terminal domain and to Asp38 of a neighboring filament, and residues 329-331 of the C-terminal tail become ordered to contact a neighboring filament. In crystal forms 3 and 4, a sulfate ion or a phosphate anion is bound to the same site on RecA as the beta-phosphate group of ADP, causing an opening of the P-loop. Altogether, the structures show the conformational variability of RecA protein in the crystalline state, providing insight into many aspects of RecA function.     

ATP-mediated conformational changes in the RecA filament

The crystal structure of the E. coli RecA protein was solved more than 10 years ago, but it has provided limited insight into the mechanism of homologous genetic recombination. Using electron microscopy, we have reconstructed five different states of RecA-DNA filaments. The C-terminal lobe of the RecA protein is modulated by the state of the distantly bound nucleotide, and this allosteric coupling can explain how mutations and truncations of this C-terminal lobe enhance RecA's activity. A model generated from these reconstructions shows that the nucleotide binding core is substantially rotated from its position in the RecA crystal filament, resulting in ATP binding between subunits. This simple rotation can explain the large cooperativity in ATP hydrolysis observed for RecA-DNA filaments.     

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.     

Structural data suggest that the active and inactive forms of the RecA filament are not simply interconvertible.

We have used electron microscopy to examine the two major conformational states of the helical filament formed by the RecA protein of Escherichia coli. The compressed filament, formed in the absence of a nucleotide cofactor either as a self-polymer or on a single-stranded DNA molecule, is characterized in solution by about 6.1 subunits per turn of a 76 A pitch helix, and appears to be inactive with respect to all RecA activity. The active state of the filament, formed with ATP or an ATP analog on either a single or double-stranded DNA substrate, has about 6.2 subunits per turn of a 94 A pitch helix. Measurements of the contour length of RecA-covered single-stranded DNA circles in ice, formed in the absence of nucleotide cofactor, indicate that each RecA subunit binds five bases, in contrast to the three bases or base-pairs per subunit in the active state. The different stoichiometries of DNA binding suggests that the two polymeric forms are not interconvertible, as has been suggested on biochemical grounds. A three-dimensional reconstruction of the inactive state shows the same general features as the 83 A pitch filament present in the RecA crystal. This structural similarity and the fact that the crystal does not contain ATP or DNA suggests that the crystal structure is more similar to the compressed filament than the active, extended filament.     

Allosteric movements in eubacterial RecA

The action of RecA, an important eubacterial protein involved in recombination and repair, involves the transition from an inactive filament in the absence of DNA to an active filament formed in association with DNA and ATP. The structure of the inactive filament was first established in Escherichia coli RecA (EcRecA). The interaction of RecA with non-hydrolysable ATP analogues and ADP has been thoroughly characterized and the DNA binding loops visualized based on the crystal structures of the RecA proteins from Mycobacterium tuberculosis (MtRecA) and Mycobacterium smegmatis (MsRecA). A switch residue, which triggers the transformation of the information on ATP binding to the DNA binding regions, has been identified. The 20-residue C-terminal stretch of RecA, which is disordered in all other relevant crystal structures, has been defined in an MsRecA-dATP complex. The ordering of the stretch is accompanied by the generation of a new nucleotide binding site which can communicate with the original nucleotide binding site of an adjacent molecule in the filament. The plasticity of MsRecA and its mutants involving the switch residue has been explored by studying crystals grown under different conditions at two different temperatures and, in one instance, at low humidity. The structures of these crystals and those of EcRecA and Deinococcus radiodurans RecA (DrRecA) provide information on correlated movements involving different regions of the molecule. These correlated movements appear to be important in the allosteric transitions of RecA during its action.     

Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase

Homologous recombination is important for the error-free repair of DNA double-strand breaks and for replication fork restart. Recombinases of the RecA/Rad51 family perform the central catalytic role in this process. UvsX recombinase is the RecA/Rad51 ortholog of bacteriophage T4. UvsX and other recombinases form presynaptic filaments on ssDNA that are activated to search for homology in dsDNA and to perform DNA strand exchange. To effectively initiate recombination, UvsX must find and bind to ssDNA within an excess of dsDNA. Here we examine the binding of UvsX to ssDNA and dsDNA in the presence and absence of nucleotide cofactor, ATP. We also examine how the binding of one DNA substrate is affected by simultaneous binding of the other to determine how UvsX might selectively assemble on ssDNA. We show that the two DNA binding sites of UvsX are regulated by the nucleotide cofactor ATP and are coordinated with each other such that in the presence of ssDNA, dsDNA binding is significantly reduced and correlated with its homology to the ssDNA bound to the enzyme. UvsX has high affinity for dsDNA in the absence of ssDNA, which may allow for sequestration of the enzyme in an inactive form prior to ssDNA generation.     

Phe217 regulates the transfer of allosteric information across the subunit interface of the RecA protein filament

BACKGROUND: ATP-mediated cooperative assembly of a RecA nucleoprotein filament activates the protein for catalysis of DNA strand exchange. RecA is a classic allosterically regulated enzyme in that ATP binding results in a dramatic increase in ssDNA binding affinity. This increase in ssDNA binding affinity results almost exclusively from an ATP-mediated increase in cooperative filament assembly rather than an increase in the inherent affinity of monomeric RecA for DNA. Therefore, certain residues at the subunit interface must play an important role in transmitting allosteric information across the filament structure of RecA. RESULTS: Using electron microscopic analysis of RecA polymer formation in the absence of DNA, we show that while wild-type RecA undergoes a slight decrease in filament length in the presence of ATP, a Phe217Tyr substitution results in a dramatic ATP-induced increase in cooperative filament assembly. Biosensor DNA binding measurements reveal that the Phe217Tyr mutation increases ATP-mediated cooperative interaction between RecA subunits by more than 250-fold. CONCLUSIONS: These studies represent the first identification of a subunit interface residue in RecA (Phe217) that plays a critical role in regulating the flow of ATP-mediated information throughout the protein filament structure. We propose a model by which conformational changes that occur upon ATP binding are propagated through the structure of a RecA monomer, resulting in the insertion of the Phe217 side chain into a pocket in the neighboring subunit. This event serves as a key step in intersubunit communication leading to ATP-mediated cooperative filament assembly and high affinity binding to ssDNA.     

Crystal structure of RecA from Deinococcus radiodurans: insights into the structural basis of extreme radioresistance

The resistance of Deinococcus radiodurans (Dr) to extreme doses of ionizing radiation depends on its highly efficient capacity to repair dsDNA breaks. Dr RecA, the key protein in the repair of dsDNA breaks by homologous recombination, promotes DNA strand-exchange by an unprecedented inverse pathway, in which the presynaptic filament is formed on dsDNA instead of ssDNA. In order to gain insight into the remarkable repair capacity of Dr and the novel mechanistic features of its RecA protein, we have determined its X-ray crystal structure in complex with ATPgammaS at 2.5A resolution. Like RecA from Escherichia coli, Dr RecA crystallizes as a helical filament that is closely related to its biologically relevant form, but with a more compressed pitch of 67 A. Although the overall fold of Dr RecA is similar to E.coli RecA, there is a large reorientation of the C-terminal domain, which in E.coli RecA has a site for binding dsDNA. Compared to E.coli RecA, the inner surface along the central axis of the Dr RecA filament has an increased positive electrostatic potential. Unique amino acid residues in Dr RecA cluster around a flexible beta-hairpin that has also been implicated in DNA binding.     

Structure of RadB recombinase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1: an implication for the formation of a near-7-fold helical assembly

The X-ray crystal structure of RadB from Thermococcus kodakaraensis KOD1, an archaeal homologue of the RecA/Rad51 family proteins, have been determined in two crystal forms. The structure represents the core ATPase domain of the RecA/Rad51 proteins. Two independent molecules in the type 1 crystal were roughly related by 7-fold screw symmetry whereas non-crystallographic 2-fold symmetry was observed in the type 2 crystal. The dimer structure in the type 1 crystal is extended to construct a helical assembly, which resembles the filamentous structures reported for other RecA/Rad51 proteins. The molecular interface in the type 1 dimer is formed by facing a basic surface patch of one monomer to an acidic one of the other. The empty ATP binding pocket is located at the interface and barely concealed from the outside similarly to that in the active form of the RecA filament. The model assembly has a positively charged belt on one surface bordering the helical groove suitable for facile binding of DNA. Electron microscopy has revealed that, in the absence of ATP and DNA, RadB forms a filament with a similar diameter to that of the hypothetical assembly, although its helical properties were not confirmed.     

Mechanism of presynaptic filament stabilization by the bacteriophage T4 UvsY recombination mediator protein

UvsY is the recombination mediator protein (RMP) of bacteriophage T4, which promotes homologous recombination by facilitating presynaptic filament assembly. The results of previous studies suggest that UvsY promotes the assembly of presynaptic filaments in part by stabilizing interactions between T4 UvsX recombinase and single-stranded DNA (ssDNA). To test this hypothesis, we studied the interactions of UvsX and UvsY with a fluorescein-derivatized oligonucleotide. This assay distinguishes between bipartite UvsX- or UvsY-ssDNA and tripartite UvsX-UvsY-ssDNA complex formation via differential fluorescence quenching effects. Salt stabilities of the three complexes were measured at equilibrium in the presence and absence of various nucleotide ligands of the UvsX protein and also under steady-state conditions for UvsX-catalyzed ssDNA-dependent ATP hydrolysis. The results demonstrate that UvsY globally stabilizes UvsX-ssDNA complexes, consistent with an increase in the apparent equilibrium binding affinity, K(ss)omega, of the UvsX-ssDNA interactions. The UvsY-mediated affinity increase is observed at equilibrium in the presence of ADP, ATPgammaS, or in the absence of the nucleotide and also at steady-state in the presence of ATP. Intriguingly, the stabilizing effects of UvsY and ATPgammaS on UvsX-ssDNA interactions are synergistic, indicating nonredundant mechanisms for UvsX-ssDNA complex stabilization by RMP versus nucleoside triphosphate effectors. Experiments with UvsY missense mutants defective in ssDNA binding demonstrate that UvsY-ssDNA interactions are of major importance in stabilizing UvsX-ssDNA complexes, whereas UvsY-UvsX protein-protein interactions provide residual stabilization energy. Together, the data is consistent with a mechanism in which UvsY stabilizes presynaptic filaments by organizing the ssDNA lattice into a structure that is favorable for UvsX-ssDNA interactions.     

Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.     

Conformational flexibility of RecA protein filament: transitions between compressed and stretched states

RecA protein is a central enzyme in homologous DNA recombination, repair and other forms of DNA metabolism in bacteria. It functions as a flexible helix-shaped filament bound on stretched single-stranded or double-stranded DNA in the presence of ATP. In this work, we present an atomic level model for conformational transitions of the RecA filament. The model describes small movements of the RecA N-terminal domain due to coordinated rotation of main chain dihedral angles of two amino acid residues (Psi/Lys23 and Phi/Gly24), while maintaining unchanged the RecA intersubunit interface. The model is able to reproduce a wide range of observed helix pitches in transitions between compressed and stretched conformations of the RecA filament. Predictions of the model are in agreement with Small Angle Neutron Scattering (SANS) measurements of the filament helix pitch in RecA::ADP-AlF(4) complex at various salt concentrations.     

Structure of the hDmc1-ssDNA Filament Reveals the Principles of Its Architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down''s, Klinefelter''s and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca²⁺, Mg²⁺, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic 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.     

Structural studies on MtRecA-nucleotide complexes: insights into DNA and nucleotide binding and the structural signature of NTP recognition

RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg(+2)-ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single-stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA-ADP complex, complexes with ATPgammaS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP-AlF4 confirms an expansion of the P-loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPgammaS rather than ADP-AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA-binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition.     

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.     

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.