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.     

The adenovirus DNA binding protein enhances intermolecular DNA renaturation but inhibits intramolecular DNA renaturation

The Adenovirus DNA binding protein (DBP) imposes a regular, rigid and extended conformation on single stranded DNA (ssDNA) and removes secondary structure. Here we show that DBP promotes renaturation of complementary single DNA strands. Enhancement of intermolecular renaturation is sequence independent, can be observed over a broad range of ionic conditions and occurs only when the DNA strands are completely covered with DBP. When one strand of DNA is covered with DBP and its complementary strand with T4 gene 32 protein, renaturation is still enhanced compared to protein-free DNA, indicating that the structures of both protein-DNA complexes are compatible for renaturation. In contrast to promoting intermolecular renaturation, DBP strongly inhibits intramolecular renaturation required for the formation of a panhandle from an ssDNA molecule with an inverted terminal repeat. We explain this by the rigidity of an ssDNA-DBP complex. These results will be discussed in view of the crystal structure of DBP that has recently been determined.     

Protein interactions in genetic recombination in Escherichia coli. Interactions involving RecO and RecR overcome the inhibition of RecA by single-stranded DNA-binding protein

RecA promotes homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA). This reaction occurs inefficiently if the ssDNA substrate is preincubated with Escherichia coli ssDNA-binding protein (SSB). However, RecO and RecR can act together as accessory factors for RecA to overcome this inhibition by SSB (Umezu, K., Chi, N.-W., and Kolodner, R. D. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3875-3879). To elucidate the mechanism that underlies this process, we examined protein-protein interactions between RecA, RecF, RecO, RecR, and SSB, and characterized the structure and activity of the ssDNA complexes formed with different combinations of these proteins. We obtained the following results. (i) RecO physically interacts with both RecR and SSB. The interaction between RecO and SSB is stronger than the RecO-RecR interaction. (ii) RecO and RecR do not remove SSB from SSB.ssDNA complexes, but instead bind to these complexes. The resulting RecO.RecR.SSB.ssDNA complexes were more active in RecA-mediated joint molecule formation than were SSB.ssDNA complexes. (iii) RecA can nucleate on the RecO.RecR.SSB.ssDNA complexes more efficiently than on SSB.ssDNA complexes. (iv) When RecA presynaptic filaments were formed in the presence of SSB, RecO, and RecR, the protein-DNA complexes obtained contained 70% of the amount of RecA required to saturate ssDNA. These complexes, however, can mediate joint molecule formation and strand exchange as efficiently as presynaptic filaments which are fully saturated with RecA. Based on these results, we propose dual roles for RecO and RecR in joint molecule formation. First, RecO and RecR bind to SSB.ssDNA complexes and modify their structure to allow RecA to nucleate on them efficiently. Second, RecO and RecR are retained in RecA presynaptic filaments and play a role in the subsequent homologous pairing process promoted by RecA.     

Gene 4 helicase of bacteriophage T7 mediates strand transfer through pyrimidine dimers, mismatches, and nonhomologous regions

In bacteriophage T7 the gene 2.5 single-stranded DNA-binding protein and the gene 4 helicase together promote the annealing of homologous regions of two DNA partners to form a joint molecule and subsequent strand transfer. In this reaction T7 gene 2.5 protein is essential for joint molecule formation, but is not required for T7 gene 4 protein-mediated strand transfer. T7 gene 4 helicase alone is able to mediate strand transfer, provided that a joint molecule is available. The present paper shows that, in addition, strand transfer proceeds at a normal rate even when both DNA partners contain ultraviolet-induced pyrimidine dimers (0.6 dimer per 100 nt). An insert of a relatively long (842-nt) segment of nonhomologous DNA in the single-stranded DNA partner has no effect on strand transfer, whereas its presence in the double-stranded partner prevents strand transfer. A short insert (37 nt) can be tolerated in either partner. Thus, DNA helicase is able to participate in recombinational DNA repair through its role in strand exchange, providing a pathway distinct from nucleotide excision repair.     

A hexameric helicase encircles one DNA strand and excludes the other during DNA unwinding

The bacteriophage T7 DNA helicase/primase (gene 4 protein) is a ring-like hexamer that encircles ssDNA and requires forked DNA to catalyze DNA unwinding. We report that optimal rates of unwinding of forked DNA require ssDNA tails of 55 nucleotides on the 5'-to-3' strand and 15 nucleotides on the 3'-to-5' strand. Surprisingly, streptavidin bound to a biotinylated 3'-end fully substitutes for the 3'-to-5' ssDNA tail. This suggests that excluding the 3'-to-5' DNA strand from the center of the helicase is an essential aspect of the mechanism of hexameric helicase-catalyzed DNA unwinding. We also report that streptavidin bound to a biotinylated dT within the 5'-to-3' strand of the duplexed region abolishes DNA unwinding; whereas, streptavidin bound to a biotinylated dT within the duplexed region of the other strand has no effect. These results unambiguously demonstrate that the T7 gene 4 protein is a 5'-to-3' helicase and imply that during DNA unwinding the 5'-to-3' strand transverses the center of the ring while the 3'-to-5' strand is excluded from the center of the ring. Implications for collisions between a helicase and other protein-DNA complexes are discussed.     

Three mechanistic steps detected by FRET after presynaptic filament formation in homologous recombination. ATP hydrolysis required for release of oligonucleotide heteroduplex product from RecA

The Escherichia coli RecA protein promotes DNA strand exchange in homologous recombination and recombinational DNA repair. Stopped-flow kinetics and fluorescence resonance energy transfer (FRET) were used to study RecA-mediated strand exchange between a 30-bp duplex DNA and a homologous single-stranded 50mer. In our standard assay, one end of the dsDNA helix was labeled at apposing 5' and 3' ends with hexachlorofluorescein and fluorescein, respectively. Strand exchange was monitored by the increase in fluorescence emission resulting upon displacement of the fluorescein-labeled strand from the initial duplex. The potential advantages of FRET in study of strand exchange are that it noninvasively measures real-time kinetics in the previously inaccessible millisecond time regime and offers great sensitivity. The oligonucleotide substrates model short-range mechanistic effects that might occur within a localized region of the ternary complex formed between RecA and long DNA molecules during strand exchange. Reactions in the presence of ATP with 0.1 microM duplex and 0.1-1.0 microM ss50mer showed triphasic kinetics in 600 s time courses, implying the existence of three mechanistic steps subsequent to presynaptic filament formation. The observed rate constants for the intermediate phase were independent of the concentration of ss50mer and most likely characterize a unimolecular isomerization of the ternary complex. The observed rate constants for the first and third phases decreased with increasing ss50mer concentration. Kinetic experiments performed with the nonhydrolyzable analogue ATPgammaS showed overall changes in fluorescence emission identical to those observed in the presence of ATP. In addition, the observed rate constants for the two fastest reaction phases were identical in ATP or ATPgammaS. The observed rate constant for the slowest phase showed a 4-fold reduction in the presence of ATPgammaS. Results in ATPgammaS using an alternate fluorophore labeling pattern suggest a third ternary intermediate may form prior to ssDNA product release. The existence of two or three ternary intermediates in strand exchange with a 30 bp duplex suggests the possibility that the step size for base pair switching may be 10-15 bp. Products of reactions in the presence of ATP and ATPgammaS, with and without proteinase K treatment, were analyzed on native polyacrylamide gels. In reactions in which only short-range RecA-DNA interactions were important, ATP hydrolysis was not required for recycling of RecA from both oligonucleotide products. Hydrolysis or deproteinization was required for RecA to release the heteroduplex product, but not the outgoing single strand.     

Binding of double-stranded DNA by Escherichia coli RecA protein monitored by a fluorescent dye displacement assay

We have developed a new assay to characterize the double-stranded DNA (dsDNA) binding properties of RecA protein. This assay is based on measurement of changes in the fluorescence of a 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complex upon RecA protein binding. The binding of RecA protein to a complex of DAPI and dsDNA results in displacement of the bound DAPI, producing a decrease in the observed fluorescence. DAPI displacement is dependent on both RecA protein and ATP; dATP and, to a lesser extent, UTP and dCTP also support the DAPI displacement reaction, but dGTP, GTP, dITP and TTP do not. Binding stoichiometry for the RecA protein-dsDNA complex measured by DAPI displacement is 3 bp per RecA protein monomer in the presence of ATP. These results, taken together with data for mutant RecA proteins, suggest that this DAPI displacement assay monitors formation of the high affinity DNA binding state of RecA protein. Since this state of RecA protein defines the form of the nucleoprotein filament that is active in DNA strand exchange, these findings raise the possibility that the RecA protein-dsDNA filament may possess a homologous pairing capacity.     

The DNA binding properties of Saccharomyces cerevisiae Rad51 protein

Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins. To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homologue the Escherichia coli RecA protein, we analyzed the DNA binding properties of the Rad51 protein. Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively. Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein. Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore, Rad51 protein displays two distinct modes of DNA binding. These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it.     

Formation of a DNA loop at the replication fork generated by bacteriophage T7 replication proteins

Intermediates in the replication of circular and linear M13 double-stranded DNA by bacteriophage T7 proteins have been examined by electron microscopy. Synthesis generated double-stranded DNA molecules containing a single replication fork with a linear duplex tail. A complex presumably consisting of T7 DNA polymerase and gene 4 helicase/primase molecules was present at the fork together with a variable amount of single-stranded DNA sequestered by gene 2.5 single-stranded DNA binding protein. Analysis of the length distribution of Okazaki fragments formed at different helicase/primase concentrations was consistent with coupling of leading and lagging strand replication. Fifteen to forty percent of the templates engaged in replication have a DNA loop at the replication fork. The loops are fully double-stranded with an average length of approximately 1 kilobase. Labeling with biotinylated dCTP showed that the loops consist of newly synthesized DNA, and synchronization experiments using a linear template with a G-less cassette demonstrated that the loops are formed by active displacement of the lagging strand. A long standing feature of models for coupled leading/lagging strand replication has been the presence of a DNA loop at the replication fork. This study provides the first direct demonstration of such loops.     

Visualisation of human rad52 protein and its complexes with hRad51 and DNA

The human Rad52 protein stimulates joint molecule formation by hRad51, a homologue of Escherichia coli RecA protein. Electron microscopic analysis of hRad52 shows that it self-associates to form ring structures with a diameter of approximately 10 nm. Each ring contains a hole at its centre. hRad52 binds to single and double-stranded DNA. In the ssDNA-hRad52 complexes, hRad52 was distributed along the length of the DNA, which exhibited a characteristic "beads on a string" appearance. At higher concentrations of hRad52, "super-rings" (approximately 30 nm) were observed and the ssDNA was collapsed upon itself. In contrast, in dsDNA-hRad52 complexes, some regions of the DNA remained protein-free while others, containing hRad52, interacted to form large protein-DNA networks. Saturating concentrations of hRad51 displaced hRad52 from ssDNA, whereas dsDNA-Rad52 complexes (networks) were more resistant to hRad51 invasion and nucleoprotein filament formation. When Rad52-Rad51-DNA complexes were probed with gold-conjugated hRad52 antibodies, the presence of globular hRad52 structures within the Rad51 nucleoprotein filament was observed. These data provide the first direct visualisation of protein-DNA complexes formed by the human Rad51 and Rad52 recombination/repair proteins.     

Modulation of RecA nucleoprotein function by the mutagenic UmuD'C protein complex

The RecA, UmuC, and UmuD' proteins are essential for error-prone, replicative bypass of DNA lesions. Normally, RecA protein mediates homologous pairing of DNA. We show that purified Umu(D')2C blocks this recombination function. Biosensor measurements establish that the mutagenic complex binds to the RecA nucleoprotein filament with a stoichiometry of one Umu(D')2C complex for every two RecA monomers. Furthermore, Umu(D')2C competitively inhibits LexA repressor cleavage but not ATPase activity, implying that Umu(D')2C binds in or proximal to the helical groove of the RecA nucleoprotein filament. This binding reduces joint molecule formation and even more severely impedes DNA heteroduplex formation by RecA protein, ultimately blocking all DNA pairing activity and thereby abridging participation in recombination function. Thus, Umu(D')2C restricts the activities of the RecA nucleoprotein filament and presumably, in this manner, recruits it for mutagenic repair function. This modulation by Umu(D')2C is envisioned as a key event in the transition from a normal mode of genomic maintenance by "error-free" recombinational repair, to one of "error-prone" DNA replication.     

Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.     

Rad52 protein stimulates DNA strand exchange by Rad51 and replication protein A

The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. We find that Rad52 protein stimulates DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required.     

Kinetic analysis of pairing and strand exchange catalyzed by RecA. Detection by fluorescence energy transfer

RecA is a 38-kDa protein from Escherichia coli that polymerizes on single-stranded DNA, forming a nucleoprotein filament that pairs with homologous duplex DNA and carries out strand exchange in vitro. In this study, we measured RecA-catalyzed pairing and strand exchange in solution by energy transfer between fluorescent dyes on the ends of deoxyribo-oligonucleotides. By varying the position of the dyes in separate assays, we were able to detect the pairing of single-stranded RecA filament with duplex DNA as an increase in energy transfer, and strand displacement as a decrease in energy transfer. With these assays, the kinetics of pairing and strand displacement were studied by stopped-flow spectrofluorometry. The data revealed a rapid, second order, reversible pairing step that was followed by a slower, reversible, first order strand exchange step. These data indicate that an initial unstable intermediate exists which can readily return to reactants, and that a further, rate-limiting step (or steps) is required to effect or complete strand exchange.     

Sequence specific binding of chlamydial histone H1-like protein

Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1. Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation. We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA. Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame. The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively. Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome. Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios. Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand. The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images. Binding was always followed by coiling, shrinking and aggregation of the affected DNA. Very low protein-DNA ratio was required if incubations were carried out in solution. However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1.     

Stimulation by Rad52 of yeast Rad51-mediated recombination

In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homologue of the recA gene and the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologues of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Here we show that Rad52 protein stimulates Rad51 reactions and that binding to Rad51 is necessary for this stimulatory effect. We conclude that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments.     

The activity of topoisomerase I is modulated by large T antigen during unwinding of the SV40 origin

When simian virus 40 (SV40) large T antigen binds to the virus origin of replication, it forms a double hexamer that functions as a helicase to unwind the DNA bidirectionally. We demonstrate in this report that T antigen can unwind and release an origin DNA single strand of less than full length in the presence of purified human topoisomerase I. The sites nicked by topoisomerase I in the strands released by T antigen during DNA unwinding were localized primarily to the "late" side of the origin, and the template for lagging strand synthesis was preferred significantly over the one for leading strand synthesis. Importantly, these sites were, for the most part, different from the sites nicked by topoisomerase I in the absence of T antigen. These data indicate that T antigen activates topoisomerase I nicking at discrete sites and releases these nicked strands during unwinding. We hypothesize that a single molecule of topoisomerase I can form a functional complex with a double hexamer of T antigen to simultaneously relax and unwind double-stranded origin-containing DNA.     

Identification of an androgen-induced C3-P4 DNA binding protein in the cytosol of rat ventral prostate

A DNA binding protein C3-P4 was detected in the rat ventral prostate cytosol by gel retardation assay using a 32P-labeled 31 base pair synthetic oligomers (sequence deduced from the rat prostatic steroid binding protein C3-1 gene promoter -149 to -119) as a probe. The DNA binding activity of C3-P4 DNA binding protein is sequence specific, with preference for single strand and coding strand exclusive. This protein can be detected in many androgen target tissues and controlled well by androgen in the rat ventral prostate, we speculate that this DNA binding protein may function as an accessory factor to androgen receptor (AR) for the regulation of the C3-1 gene expression.     

Purification and characterization of XRad51.1 protein, Xenopus RAD51 homologue: recombinant XRad51.1 promotes strand exchange reaction

BACKGROUND: The RAD51 gene of Saccharomyces cerevisiae is homologous to the Escherichia coli recA gene and plays a key role in genetic recombination and DNA double-strand break repair. To construct an improved experimental system of homologous recombination in higher eukaryotes, we have chosen the South African clawed frog, Xenopus laevis, whose egg extracts might be useful for the in vitro studies. We identified and characterized a Xenopus homologue of RAD51 gene, the XRAD51.1. RESULTS: Recombinant XRad51.1 was expressed in E. coli. The purified XRad51.1 protein showed ssDNA-dependent ATPase activity and promoted the DNA strand exchange reaction between two 55-mer oligonucleotides. The binding stoichiometry of XRad51.1 to ssDNA was determined by fluorescence of poly(d epsilonA), a chemically modified poly(dA), and was found to be about six bases/XRad51.1 monomer in a nucleoprotein filament, a similar value to E. coli RecA protein. The kinetics of the fluorescence change of poly(d epsilonA) after XRad51.1 binding in the presence of ATP was significantly different from that observed with RecA protein. The affinity of XRad51.1 to ssDNA in the presence of ATP was higher than that of RecA protein, and the dissociation of the XRad51.1-ssDNA complex was slower than the RecA-ssDNA complex. CONCLUSIONS: Purified recombinant XRad51.1 protein promoted the strand exchange between short DNA molecules. While the binding stoichiometry of XRad51.1 protein to ssDNA was identical to that of the RecA protein, XRad51.1 has a significantly higher affinity and binding stability to ssDNA than did the RecA protein in the presence of ATP.