Homologous pairing activities of Arabidopsis thaliana RAD51 and DMC1.

In eukaryotes, homologous recombination plays a pivotal role in both genome maintenance and generation of genetic diversity. Eukaryotic RecA homologues, RAD51 and DMC1, are key proteins in homologous recombination that promote pairing between homologous DNA sequences. Arabidopsis thaliana is a prominent model plant for studying eukaryotic homologous recombination. However, A. thaliana RAD51 and DMC1 have not been biochemically characterized. In the present study, we purified A. thaliana RAD51 (AtRAD51) and DMC1 (AtDMC1). Biochemical analyses revealed that both AtRAD51 and AtDMC1 possess ATP hydrolyzing activity, filament formation activity and homologous pairing activity in vitro. We then compared the homologous pairing activities of AtRAD51 and AtDMC1 with those of the Oryza sativa and Homo sapiens RAD51 and DMC1 proteins.

Co-expression and purification of the RadA recombinase with the RadB paralog from Haloferax volcanii yields heteromeric ring-like structures.

The study of archaeal proteins and the processes to which they contribute poses particular challenges due to the often extreme environments in which they function. DNA recombination, replication and repair proteins of the halophilic euryarchaeon, Haloferax volcanii (Hvo) are of particular interest as they tend to resemble eukaryotic counterparts in both structure and activity, and genetic tools are available to facilitate their analysis. In the present study, we show using bioinformatics approaches that the Hvo RecA-like protein RadA is structurally similar to other recombinases although is distinguished by a unique acidic insertion loop. To facilitate expression of Hvo RadA a co-expression approach was used, providing its lone paralog, RadB, as a binding partner. At present, structural and biochemical characterization of Hvo RadA is lacking. Here, we describe for the first time co-expression of Hvo RadA with RadB and purification of these proteins as a complex under in vitro conditions. Purification procedures were performed under high salt concentration (>1 M sodium chloride) to maintain the solubility of the proteins. Quantitative densitometry analysis of the co-expressed and co-purified RadAB complex estimated the ratio of RadA to RadB to be 4 : 1, which suggests that the proteins interact with a specific stoichiometry. Based on a combination of analyses, including size exclusion chromatography, Western blot and electron microscopy observations, we suggest that RadA multimerizes into a ring-like structure in the absence of DNA and nucleoside co-factor.

The cohesin-like RecN protein stimulates RecA-mediated recombinational repair of DNA double-strand breaks.

RecN is a cohesin-like protein involved in DNA double-strand break repair in bacteria. The RecA recombinase functions to mediate repair via homologous DNA strand invasion to form D-loops. Here we provide evidence that the RecN protein stimulates the DNA strand invasion step of RecA-mediated recombinational DNA repair. The intermolecular DNA tethering activity of RecN protein described previously cannot fully explain this novel activity since stimulation of RecA function is species-specific and requires RecN ATP hydrolysis. Further, DNA-bound RecA protein increases the rate of ATP hydrolysis catalysed by RecN during the DNA pairing reaction. DNA-dependent RecN ATPase kinetics are affected by RecA protein in a manner suggesting a specific order of protein-DNA assembly, with RecN acting after RecA binds DNA. We present a model for RecN function that includes presynaptic stimulation of the bacterial repair pathway perhaps by contributing to the RecA homology search before ternary complex formation.

Discovery and characterization of RecA protein of thermophilic bacterium Thermus thermophilus MAT72 phage Tt72 that increases specificity of a PCR-based DNA amplification.

The recA gene of newly discovered Thermus thermophilus MAT72 phage Tt72 (Myoviridae) was cloned and overexpressed in Escherichia coli. The 1020-bp gene codes for a 339-amino-acid polypeptide with an Mr of 38,155 which shows 38.7% positional identity to the E. coli RecA protein. When expressed in E. coli, the Tt72 recA gene did not confer the ability to complement the ultraviolet light (254nm) sensitivity of an E. coli recA mutant. Tt72 RecA protein has been purified with good yield to catalytic and electrophoretic homogeneity using a three-step chromatography procedure. Biochemical characterization indicated that the protein can pair and promote ATP-dependent strand exchange reaction resulting in formation of a heteroduplex DNA at 60°C under conditions otherwise optimal for E. coli RecA. When the Tt72 RecA protein was included in a standard PCR-based DNA amplification reaction, the specificity of the PCR assays was significantly improved by eliminating non-specific products.

A recombinase paralog from the hyperthermophilic crenarchaeon Sulfolobus solfataricus enhances SsoRadA ssDNA binding and strand displacement.

Homologous recombination (HR) is a major pathway for the repair of double-strand DNA breaks, a highly deleterious form of DNA damage. The main catalytic protein in HR is the essential RecA-family recombinase, which is conserved across all three domains of life. Eukaryotes and archaea encode varying numbers of proteins paralogous to their main recombinase. Although there is increasing evidence for the functions of some of these paralog proteins, overall their mechanism of action remains largely unclear. Here we present the first biochemical characterization of one of the paralog proteins, SsoRal3, from the crenarchaeaon Sulfolobus solfataricus. The SsoRal3 protein is a ssDNA-dependent ATPase that can catalyze strand invasion at both saturating and subsaturating concentrations. It can bind both ssDNA and dsDNA, but its binding preference is altered by the presence or absence of ATP. Addition of SsoRal3 to SsoRadA nucleoprotein filaments reduces total ATPase activity. Subsaturating concentrations of SsoRal3 increase the ssDNA binding activity of SsoRadA approximately 9-fold and also increase the persistence of SsoRadA catalyzed strand invasion products. Overall, these results suggest that SsoRal3 functions to stabilize the SsoRadA presynaptic filament.

RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

BACKGROUND: Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown. RESULTS: In this study, recA genes from Deinococcus geothermalis and Deinococcus murrayi, bacteria that are slightly thermophilic and extremely γ-radiation resistant, were isolated, cloned and expressed in E. coli. After production and purification, the biochemical properties of DgeRecA and DmuRecA proteins were determined. Both proteins continued to exist in the solutions as heterogenous populations of oligomeric forms. The DNA binding by DgeRecA and DmuRecA proteins is stimulated by Mg2+ ions. Furthermore, both proteins bind more readily to ssDNA when ssDNA and dsDNA are in the same reaction mixture. Both proteins are slightly thermostable and were completely inactivated in 10 s at 80°C. Both proteins hydrolyze ATP and dATP in the presence of ssDNA or complementary ssDNA and dsDNA, but not in the absence of DNA or in the presence of dsDNA only, and dATP was hydrolyzed more rapidly than ATP. They were also able to promote DNA strand exchange reactions by a pathway common for other RecA proteins. However, we did not obtain DNA strand exchange products when reactions were performed on an inverse pathway, characteristic for RecA of D. radiodurans. CONCLUSIONS: The characterization of DgeRecA and DmuRecA proteins made in this study indicates that the unique properties of D. radiodurans RecA are probably not common among RecA proteins from Deinococcus sp.

Purification and characterization of the RecA protein from Neisseria gonorrhoeae.

The strict human pathogen Neisseria gonorrhoeae is the only causative agent of the sexually transmitted infection gonorrhea. The recA gene from N. gonorrhoeae is essential for DNA repair, natural DNA transformation, and pilin antigenic variation, all processes that are important for the pathogenesis and persistence of N. gonorrhoeae in the human population. To understand the biochemical features of N. gonorrhoeae RecA (RecA(Ng)), we overexpressed and purified the RecA(Ng) and SSB(Ng) proteins and compared their activities to those of the well-characterized E. coli RecA and SSB proteins in vitro. We observed that RecA(Ng) promoted more strand exchange at early time points than RecA(Ec) through DNA homologous substrates, and exhibited the highest ATPase activity of any RecA protein characterized to date. Further analysis of this robust ATPase activity revealed that RecA(Ng) is more efficient at displacing SSB from ssDNA and that RecA(Ng) shows higher ATPase activity during strand exchange than RecA(Ec). Using substrates created to mimic the cellular processes of DNA transformation and pilin antigenic variation we observed that RecA(Ec) catalyzed more strand exchange through a 100 bp heterologous insert, but that RecA(Ng) catalyzed more strand exchange through regions of microheterology. Together, these data suggest that the processes of ATP hydrolysis and DNA strand exchange may be coupled differently in RecA(Ng) than in RecA(Ec). This difference may explain the unusually high ATPase activity observed for RecA(Ng) with the strand exchange activity between RecA(Ng) and RecA(Ec) being more similar.

The Mycoplasma pneumoniae MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange.

BACKGROUND: Mycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms. RESULTS: The MPN229 ORF has the capacity to encode a 166-amino acid protein with a calculated molecular mass of 18.4 kDa. The amino acid sequence of this protein (Mpn SSB) is most closely related to that of the protein predicted to be encoded by the MG091 gene from Mycoplasma genitalium (61% identity). The MPN229 ORF was cloned, and different versions of Mpn SSB were expressed in E. coli and purified to > 95% homogeneity. The purified protein was found to exist primarily as a homo-tetramer in solution, and to strongly and selectively bind single-stranded DNA (ssDNA) in a divalent cation- and DNA substrate sequence-independent manner. Mpn SSB was found to bind with a higher affinity to ssDNA substrates larger than 20 nucleotides than to smaller substrates. In addition, the protein strongly stimulated E. coli Recombinase A (RecA)-promoted DNA strand exchange, which indicated that Mpn SSB may play an important role in DNA recombination processes in M. pneumoniae. CONCLUSION: The M. pneumoniae MPN229 gene encodes a protein, Mpn SSB, which selectively and efficiently binds ssDNA, and stimulates E. coli RecA-promoted homologous DNA recombination. Consequently, the Mpn SSB protein may play a crucial role in DNA recombinatorial pathways in M. pneumoniae. The results from this study will pave the way for unraveling these pathways and assess their role in antigenic variation of M. pneumoniae.

Purification, crystallization and preliminary crystallographic analysis of RecA superfamily ATPase PH0284 from Pyrococcus horikoshii OT3.

Circadian (daily) protein clocks are found in cyanobacteria, where a complex of the KaiA, KaiB and KaiC proteins generates circadian rhythms. The 28.09 kDa KaiC homologue PH0284 protein from Pyrococcus horikoshii OT3 was cloned and expressed and the purified protein was crystallized by the oil-microbatch method at 295 K. X-ray diffraction data from the crystal were collected to 2.0 angstroms resolution using synchrotron radiation at 100 K. The crystal belongs to the trigonal space group P3(2)21, with unit-cell parameters a = b = 96.06, c = 298.90 angstroms. Assuming the presence of one hexamer in the asymmetric unit gives a V(M) value of 2.36 angstroms3 Da(-1) and a solvent content of 47.9%. A cocrystal with ATP was prepared and a diffraction data set was collected at 2.3 angstroms resolution.

Intein-mediated affinity-fusion purification of the Escherichia coli RecA protein.

The RecA protein of Escherichia coli plays important roles in homologous recombination, recombinational DNA repair, and SOS induction. Because its functions are conserved among the phylogenetic kingdoms, RecA investigations have provided a paradigm for understanding these biological processes. The RecA protein has been overproduced in E. coli and purified using a variety of purification schemes requiring multiple, time-intensive steps. The purification schemes share a dependence on appropriate RecA structure and/or function at one or more steps. In this report, we used a modified protein splicing element (intein) and a chitin-binding domain, fused to the C-terminus of RecA, to facilitate a one-step affinity purification of RecA protein without modification of the native protein sequence. Following the single chromatographic step, RecA protein that is greater than 95% physical purity at a concentration of greater than microM was obtained. The protein displays in vitro activities that are identical to those of protein isolated using classical procedures. The purification strategy described here promises to yield mutant RecA proteins in sufficient quantity for rigorous biophysical characterization without dependence on intrinsic RecA function.

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization.

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

Characterization of RecA424 and RecA670 proteins from Deinococcus radiodurans.

RecA protein is considered to be the most important participant in the radiation resistance of Deinococcus radiodurans. However, it is still unclear how RecA contributes to the resistance. In this study, we identified a new recA mutation (recA424) in the DNA-repair deficient mutant strain KI696, the phenotype of which is remarkably different from mutant strain rec30 carrying recA670. The properties of the gene products from the recA mutants were compared. recA424 could not complement the deficiency in Escherichia coli RecA, as found for recA670. In vitro, neither RecA424 nor RecA670 could promote DNA strand exchange under conditions in which wild-type RecA promoted the reaction, indicating that both RecA424 and Rec670 are defective in recombination activity. RecA424 promoted the autocleavage reaction of LexA in vitro, whereas RecA670 did not. The intracellular LexA level in KI696 was decreased following gamma-irradiation. However, the LexA level in strain rec30 was constant irrespective of irradiation. These results indicate that RecA424 retains co-protease activity, whereas RecA670 does not. While strain rec30 is extremely radiation sensitive, strain KI696 is only slightly sensitive. Together, these observations suggest that the co-protease activity rather than the recombination activity of RecA contributes to radiation resistance in D. radiodurans.

Purification and characterization of the RecA protein from Streptococcus pneumoniae.

Streptococcus pneumoniae is a naturally transformable bacterium that is able to take up single-stranded DNA from its environment and incorporate the exogenous DNA into its genome. This process, known as transformational recombination, is dependent upon the presence of the recA gene, which encodes an ATP-dependent DNA recombinase whose sequence is 60% identical to that of the RecA protein from Escherichia coli. We have developed an overexpression system for the S. pneumoniae RecA protein and have purified the protein to greater than 99% homogeneity. The S. pneumoniae RecA protein has ssDNA-dependent NTP hydrolysis and NTP-dependent DNA strand exchange activities that are generally similar to those of the E. coli RecA protein. In addition to its role as a DNA recombinase, the E. coli RecA protein also acts as a coprotease, which facilitates the cleavage and inactivation of the E. coli LexA repressor during the SOS response to DNA damage. Interestingly, the S. pneumoniae RecA protein is also able to promote the cleavage of the E. coli LexA protein, even though a protein analogous to the LexA protein does not appear to be present in S. pneumoniae.

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.

An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing.

RecA protein and its homologs catalyze homologous pairing of dsDNA and ssDNA, a critical reaction in homologous genetic recombination in various organisms from a virus, microbes to higher eukaryotes. In this reaction, RecA protein forms a nucleoprotein filament on ssDNA, which in turn binds to naked dsDNA for homology search. We suggested that the C-terminal domain of RecA protein plays a role in capturing the dsDNA. Here, we isolated the C-terminal domain as a soluble form and determined the solution structure by NMR spectroscopy. The overall folding of the NMR structure agrees with that of the corresponding part of the reported crystal structure, but a remarkable difference was found in a solvent-exposed region due to intermolecular contacts in the crystal. Then, we studied the interaction between the C-terminal domain and DNA, and found that significant chemical shift changes were induced in a specific region by titration with dsDNA. SsDNA induced a much smaller chemical shift perturbation. The difference of DNA concentrations to give the half-saturation of the chemical shift change showed a higher affinity of the C-terminal region toward dsDNA. Combined with our previous results, these provide direct evidence that the defined region in the C-terminal domain furnishes a binding surface for DNA.

Cloning, characterization and expression of the recA gene of Aeromonas salmonicida.

The Aeromonas salmonicida A449 recA gene has been cloned, sequenced and expressed in vitro. The predicted amino acid sequence of A. salmonicida RecA was determined and, when compared to other RecA, was found to possess a number of domains identical to those characterized in Escherichia coli RecA. The A. salmonicida recA was mobilized into an E. coli recA mutant strain and was shown to allow increased survival in the presence of the chemical mutagen MMS and after ultraviolet (UV) irradiation. The A. salmonicida recA also possesses a potential regulatory SOS box in the DNA 5' of the gene. The rate of A. salmonicida-mediated recombination in E. coli was increased by exposure to UV light, which suggests that SOS induction in A. salmonicida parallels that of E. coli.

Isolation, characterization, and magnesium-induced self-association kinetics of discrete aggregates of RecA protein from Escherichia coli.

Dynamic and static intensity light scattering techniques were employed to identify conditions allowing preparation of homogeneous solutions of distinct oligomeric states of RecA protein. These hydrodynamically distinguishable oligomer populations of RecA protein were obtained in homogeneous pure quantities sufficient for physical studies. Results indicate two fairly narrow distributions of RecA oligomers comprised on average of 42 +/- 3 and 18 +/- 1 RecA monomers. These structures, denoted RecA42 and RecA18, respectively, could be obtained reproducibly in milligram quantities and were stable for at least one week. This enabled reliable characterizations of their hydrodynamic properties by dynamic and total intensity light scattering. These measurements revealed RecA42 had an average translational diffusion coefficient, D20(L) = 8 +/- 2 x 10(-8) cm2/s, molecular weight, M(r) = 1.6 +/- 0.1 x 10(6), and radius of gyration, RG = 465 +/- 29 A. The smaller aggregate, RecA18, had D20(S) = 20.5 +/- 2.5 x 10(-8) cm2/s, M(r) = 7.0 +/- 0.4 x 10(5), and RG = 300 +/- 20 A. Heating RecA18 at 37 degrees C overnight resulted in conversion to a species with hydrodynamic properties indistinguishable from RecA42, called RecA18/42. Conversion of RecA42 to RecA18 occurred almost instantaneously by 50% dilution at 38 degrees C or very slowly with incubation at 4 degrees C for at least 39 days. Self-association reactions of the three starting oligomeric states (RecA18, RecA42, and RecA18/42) induced by MgCl2 were monitored at several temperatures by dynamic light scattering. Results of these experiments provided evaluations of kinetic activation parameters of the self-association reactions. The activation parameters found for each starting oligomeric state of the protein were significantly different, revealing the variable influence of MgCl2 on the activation barriers to RecA self-association. Highly aggregated equilibrium solutions that ultimately form in solutions of each starting oligomeric species, incubated in MgCl2 at 38 degrees C for four days, were characterized by total intensity light scattering. Interpretations of these data in terms of characteristic behavior of random polymers suggests the surface morphologies of these highly associated equilibrium states formed from RecA42 and RecA18/42 are similar but contrast with that of RecA18. Calculated values of the translational diffusion coefficient D0 were obtained for oligomeric structures modeled as helical arrays of connected monomer spheres. Best agreement with experimentally determined diffusion coefficients required that constituent monomer spheres of RecA42 have radii 33-40% larger than the monomer spheres of RecA18. Results suggest the hydrodynamically distinct oligomeric forms of RecA may reside in conformational states with different surface exposure of hydrophobic residues, which results in substantial differences in local solvation/hydration.

DNA strand exchange promoted by RecA K72R. Two reaction phases with different Mg2+ requirements.

Replacement of lysine 72 in RecA protein with arginine produces a mutant protein that binds but does not hydrolyze ATP. The protein nevertheless promotes DNA strand exchange (Rehrauer, W. M., and Kowalczykowski, S. C. (1993) J. Biol. Chem. 268, 1292-1297). With RecA K72R protein, the formation of the hybrid DNA product of strand exchange is greatly affected by the concentration of Mg2+ in ways that reflect the concentration of a Mg.dATP complex. When Mg2+ is present at concentrations just sufficient to form the Mg.dATP complex, substantial generation of completed product hybrid DNAs over 7 kilobase pairs in length is observed (albeit slowly). Higher levels of Mg2+ are required for optimal uptake of substrate duplex DNA into the nucleoprotein filament, indicating that the formation of joint molecules is facilitated by Mg2+ levels that inhibit the subsequent migration of a DNA branch. We also show that the strand exchange reaction promoted by RecA K72R, regardless of the Mg2+ concentration, is bidirectional and incapable of bypassing structural barriers in the DNA or accommodating four DNA strands. The reaction exhibits the same limitations as that promoted by wild type RecA protein in the presence of adenosine 5'-O-(3-thio)triphosphate. The Mg2+ effects, the limitations of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis, and unusual DNA structures observed by electron microscopy in some experiments, are interpreted in the context of a model in which a fast phase of DNA strand exchange produces a discontinuous three-stranded DNA pairing intermediate, followed by a slow phase in which the discontinuities are resolved. The mutant protein also facilitates the autocatalytic cleavage of the LexA repressor, but at a reduced rate.

Evidence for the coupling of ATP hydrolysis to the final (extension) phase of RecA protein-mediated DNA strand exchange.

RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis. When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 +/- 20 bp min-1 at 37 degrees C. A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis. The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45 degrees C that is, within experimental error, identical. This provides new evidence that the two processes are coupled. Arrhenius activation energies derived from the work are 13.3 +/- 1.1 kcal mole-1 for DNA strand exchange, and 14.4 +/- 1.4 kcal mole-1 for ATP hydrolysis during strand exchange. The rate of branch movement in the extension phase (base pair min-1) is related to the kcat for ATP hydrolysis during strand exchange (min-1) by a factor equivalent to 18 bp throughout the temperature range examined. The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates. RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor.