Binding of Rad51p to DNA

Like RecA, Saccharomyces cerevisiaeRad51p promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). We have investigated several parameters characteristic of the interaction of Rad51p with ssDNA and dsDNA, particularly the effects of the nucleotide cofactors ATP and ADP and the analogs adenosine 5′-O-(thiotriphosphate) (ATPγS) and adenylyl-imidodiphosphate (AMP-PNP). Rad51p binding to both 1-N 6-ethenoadenosine and 3-N 4-ethenocytidine ssDNA (εDNA) and dsDNA requires the presence of Mg2+ and ATP; no binding occurs in the presence of ADP, AMP-PNP, or ATPγS. Binding of Rad51p to dsDNA also requires ATP; ADP is ineffective, whereas ATPγS and AMP-PNP are considerably less able to promote binding and only at elevated concentrations of Rad51p. ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA. The Kd values for ATP for promoting binding of Rad51p to ssDNA and dsDNA are 1 and 3 μm, respectively. Rad51p binding occurs with a stoichiometry of one monomer of Rad51p per ∼6.3 nucleotides of εDNA and ∼3.3 base pairs of dsDNA. Once formed, Rad51p·ssDNA complexes are stable so long as sufficient ATP levels are maintained. ATP hydrolysis causes dissociation of Rad51p from DNA. Moreover, the preformed complex is stable in the presence of a 10-fold excess of ADP or AMP-PNP over ATP. ATPγS, however, in the same -fold excess over ATP causes dissociation of the Rad51p·ssDNA complex. Like RecA, Saccharomyces cerevisiaeRad51p promotes strand exchange between circular single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA). We have investigated several parameters characteristic of the interaction of Rad51p with ssDNA and dsDNA, particularly the effects of the nucleotide cofactors ATP and ADP and the analogs adenosine 5′-O-(thiotriphosphate) (ATPγS) and adenylyl-imidodiphosphate (AMP-PNP). Rad51p binding to both 1-N 6-ethenoadenosine and 3-N 4-ethenocytidine ssDNA (εDNA) and dsDNA requires the presence of Mg2+ and ATP; no binding occurs in the presence of ADP, AMP-PNP, or ATPγS. Binding of Rad51p to dsDNA also requires ATP; ADP is ineffective, whereas ATPγS and AMP-PNP are considerably less able to promote binding and only at elevated concentrations of Rad51p. ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA. The Kd values for ATP for promoting binding of Rad51p to ssDNA and dsDNA are 1 and 3 μm, respectively. Rad51p binding occurs with a stoichiometry of one monomer of Rad51p per ∼6.3 nucleotides of εDNA and ∼3.3 base pairs of dsDNA. Once formed, Rad51p·ssDNA complexes are stable so long as sufficient ATP levels are maintained. ATP hydrolysis causes dissociation of Rad51p from DNA. Moreover, the preformed complex is stable in the presence of a 10-fold excess of ADP or AMP-PNP over ATP. ATPγS, however, in the same -fold excess over ATP causes dissociation of the Rad51p·ssDNA complex. The Saccharomyces cerevisiae RAD51 gene is a member of the RAD52 epistasis group, that is required for efficient DNA double strand break repair and genetic recombination (1Petes T.D. Malone R.E. Symington L.S. Broach J. Pringle J. Jones E. The Molecular and Cellular Biology of the Yeast Saccharomyces: Genome Dynamics, Protein Synthesis, and Energetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1991: 407-521Google Scholar). The amino acid sequence of RAD51 strongly resembles that of the Escherichia coli RecA protein (2Aboussekhra A. Chanet R. Adjiri A. Fabre F. Mol. Cell. Biol. 1992; 12: 3224-3234Crossref PubMed Scopus (320) Google Scholar, 3Basile G. Aker M. Mortimer R.K. Mol. Cell. Biol. 1992; 12: 3235-3246Crossref PubMed Scopus (239) Google Scholar, 4Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1052) Google Scholar), and like RecA, Rad51p catalyzes joint molecule formation and strand exchange between circular ssDNA 1The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; εDNA, 1-N 6-ethenoadenosine and 3-N 4-ethenocytidine ssDNA; ATPγS, adenosine 5′-O-(thiotriphosphate); AMP-PNP, adenylyl-imidodiphosphate; Mes, 2-(N-morpholino)ethanesulfonic acid. and linear dsDNA (5Cox M.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 3433-3437Crossref PubMed Scopus (222) Google Scholar, 6Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar, 7Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). The reaction promoted by Rad51p also requires Mg2+, ATP, and a single-stranded DNA-binding protein, yeast replication protein A, being the most efficient (6Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar, 7Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). However, Rad51p-promoted strand exchange has certain striking differences compared with RecA. For example, RecA has the unique ability to pair homologous ssDNA and dsDNA to form three-stranded joint molecules, which are then resolved into heteroduplex DNA; this pairing can occur even between DNAs without ends (9Cox M.M. Soltis D.A. Livneh Z. Lehman I.R. J. Biol. Chem. 1983; 258: 2577-2585Abstract Full Text PDF PubMed Google Scholar, 10Cunningham R.P. Wu A.M. Shibata T. DasGupta C. Radding C.M. Cell. 1981; 24: 213-223Abstract Full Text PDF PubMed Scopus (78) Google Scholar, 11DasGupta C. Shibata T. Cunningham R.P. Radding C.M. Cell. 1980; 22: 437-446Abstract Full Text PDF PubMed Scopus (96) Google Scholar). In the reaction promoted by Rad51p, joint molecules are formed only between ssDNA and linear dsDNA having either 3′- or 5′-overhanging complementary ends; joint molecules are not formed with linear DNA having blunt or recessed complementary ends (8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). As a consequence of being able to use either a 3′- or 5′-overhanging complementary strand to initiate joint molecule formation, the direction of strand exchange depends on which end initiates the reaction (8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). The interactions of RecA with ssDNA and dsDNA are important determinants for strand exchange (12Kowalczykowski S.C. Burk D.L. Krupp R.A. J. Mol. Biol. 1989; 207: 719-733Crossref PubMed Scopus (30) Google Scholar, 13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google Scholar, 14Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar). To understand the molecular mechanism of Rad51p-promoted strand exchange, we have examined the interactions between Rad51p and single- and double-stranded DNA, particularly the role of nucleotides and salt in these interactions. Rad51p binding to ssDNA was followed by measuring the increased fluorescence of chemically modified 1-N 6-ethenoadenosine and 3-N 4-ethenocytidine ssDNA (etheno-DNA, εDNA) that accompanies protein binding (13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google Scholar, 15Kowalczykowski S.C. Lonberg N. Newport J.W. von Hippel P.H. J. Mol. Biol. 1981; 145: 75-104Crossref PubMed Scopus (259) Google Scholar); binding to dsDNA was followed by protection of the DNA against DNase I degradation (14Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar) and, in some cases, by nitrocellulose filter binding (16McEntee K. Weinstock G.M. Lehman I.R. J. Biol. Chem. 1981; 256: 8835-8844Abstract Full Text PDF PubMed Google Scholar). Our data indicate that ATP and Mg2+ are essential for Rad51p binding to both kinds of DNA and that ADP, adenosine 5′-O-thiotriphosphate (ATPγS), and adenylyl imidodiphosphate (AMP-PNP) do not satisfy this requirement. The ATP analog ATPγS, which is unable to promote Rad51p binding to DNA, nevertheless causes dissociation of the Rad51p·εDNA complex. The results of dissociation of the Rad51p·εDNA complex at different temperatures indicate that ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA. Yeast Rad51p was expressed in and purified from insect cells as described previously (8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). The concentration of Rad51p was determined using an extinction coefficient (determined from amino acid composition) of 1.26 × 104 at 280 nm. Unless otherwise noted, the standard reaction buffer used in all experiments contained 40 mmK-Mes (pH 6.5), 4 mm MgCl2, 1 mmdithiothreitol, and 5% glycerol. In experiments testing pH dependence, K-Mes was replaced by 25 mm Tris-HCl (pH 7.5 or 8.5). ATP and ADP were obtained from Pharmacia Biotech Inc., and ATPγS and AMP-PNP were from Boehringer Mannheim. All reactions were carried out at 30 and 37 °C. The concentrations of the DNA substrates are expressed as nucleotide equivalents. Etheno-M13 DNA (εDNA) was prepared as described (17Zlotnick A. Mitchell R.S. Steed R.K. Brenner S.L. J. Biol. Chem. 1993; 268: 22525-22530Abstract Full Text PDF PubMed Google Scholar), and the concentration was determined using an extinction coefficient of 7000 m−1cm−1 (18Menetski J.P. Kowalczykowski S.C. J. Biol. Chem. 1987; 262: 2085-2092Abstract Full Text PDF PubMed Google Scholar). Uniform 3H-labeled pBluescript SK+ DNA was prepared with SssI methylase and S-[methyl-3H]adenosyl-l-methionine. Linear DNA was prepared by digestion of 3H-labeled pBluescript SK(+) dsDNA with PstI restriction endonuclease. The binding of Rad51p to ssDNA was monitored by measuring the change in fluorescence of εDNA at 410 nm following excitation at 305 nm using an AMINCO-Bowman luminescence spectrometer. Sample volumes were normally 0.6 ml, and additions did not exceed 40 μl. The titrant was either Rad51p in “forward” titrations or concentrated sodium chloride solution in the “salt-back” titration. When ATP, ADP, ATPγS, and AMP-PNP were included in the titrations, their concentrations were 100 μm each. Following the addition of each titrant, the fluorescence was measured until the value reached a stable plateau. In experiments measuring the stability of the Rad51p·εDNA complex, the complex was formed in the presence of 100 μm ATP, and the change in fluorescence was monitored after the addition of 1000 μm ADP, ATPγS, or AMP-PNP. Measurements were performed at a constant temperature, maintained by a circulating water bath. Corrections for fluorescence of the protein were made from the values obtained after adding Rad51p in the absence of εDNA. The fluorescence values observed in these control titrations were subtracted from the measured values made in the presence of εDNA. 3H-Labeled linear dsDNA (10 μm) and variable amounts of Rad51p were incubated in the standard reaction buffer containing 0.5 mmATP, ADP, ATPγS, or AMP-PNP at 30 °C for 10 min. Aliquots (20 μl) of the reaction mixtures were incubated with 1 μl (12 units) of pancreatic DNase I (Boehringer Mannheim) under standard conditions for 1 min. The reaction was stopped by adding 10 μl of a solution containing 2 mg/ml salmon sperm DNA (heat-denatured) and 0.375m EDTA followed by 0.9 ml of ice-cold 10% trichloroacetic acid. The samples remained on ice for 30 min and then were filtered through Whatman GF/C filter discs. The filters were washed twice with 1 ml of 10% trichloroacetic acid followed by 2 ml of 95% ethanol. The filters were dried and assayed for radioactivity in a liquid scintillation counter. For each experiment, complete protection was equivalent to the value obtained without DNase I digestion. Complete digestion was equivalent to the value obtained after DNase I digestion when Rad51p was omitted from the binding reaction. The degree of protection was obtained by dividing the amount of acid-precipitable3H for each sample by the value obtained without DNase I digestion after the background (the value without addition of Rad51p) was subtracted from both. This background was ∼10% of the value for 100% Rad51p binding to DNA was also measured by the of DNA on nitrocellulose filters μm, K. Weinstock G.M. Lehman I.R. Proc. Natl. Acad. Sci. U. S. A. 1980; PubMed Scopus Google Scholar). Aliquots (20 μl) of the reaction mixtures were in 1 ml of the same reaction buffer and filtered through filter discs. The filters were washed with 1 ml of same buffer and and the radioactivity was measured in a liquid scintillation counter. The ATP of Rad51p binding to ssDNA was determined by measuring the increased fluorescence of εDNA in the standard reaction buffer containing μm εDNA, 1 μm Rad51p, and levels of ATP with an The of ATP concentration on the binding of Rad51p to dsDNA was with the protection using the same reaction conditions that the contained 10 linear dsDNA and 2 μm Rad51p. Measurements of the of Mg2+ on Rad51p binding to εDNA were performed by the concentration of the presence of or μm ATP. The of Mg2+ on Rad51p binding to dsDNA was determined by the concentration in the presence of μm ATP, and binding was measured by the nitrocellulose filter binding All reactions were carried out at 30 °C. was measured as described 1983; PubMed Scopus Google Scholar). 1 μm Rad51p was to μm εDNA in a reaction containing μm ATP, 10 10 mm and 100 μm in the standard reaction of was measured as a in at nm. Rad51p binding to ssDNA was followed by measuring the in fluorescence with binding of the protein to chemically modified ssDNA (εDNA) G. PubMed Scopus Google Scholar). The in fluorescence is to a consequence of the DNA and the in the Rad51p·ssDNA complex (13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google S.C. Lonberg N. Newport J.W. von Hippel P.H. J. Mol. Biol. 1981; 145: 75-104Crossref PubMed Scopus (259) Google Scholar). Rad51p to εDNA only when both ATP and Mg2+ were ATP or with ADP or AMP-PNP the ATPγS did not Rad51p binding to εDNA at 37 did at Rad51p concentrations at 30 °C. was no in the or of the binding or in the for ATP or Mg2+ over the pH not on the stoichiometry of Rad51p and εDNA the fluorescence is we a binding of and at 37 and 30 a we do not the of we nucleotides as the of the Rad51p binding of the in the titrations carried out in the presence of salt we did not the and parameters for Rad51p interactions. we the value of the of using the binding of nucleotides for the and 1 of Kowalczykowski S.C. L.S. Lonberg N. Newport J.W. von Hippel P.H. PubMed Scopus Google Scholar) for binding the value of for binding at 30 and 37 °C is × Rad51p a with dsDNA that is to the by when RecA with dsDNA T. A. Science. 1993; PubMed Scopus Google Scholar). To several parameters the interaction of Rad51p with dsDNA, different were protection of 3H-labeled dsDNA against degradation by DNase I and nitrocellulose filter RecA (14Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar), Rad51p binding to dsDNA the DNA to degradation by DNase protection was complete even after 1 min of and the complex was stable for at 1 2 As in the of ssDNA, binding of Rad51p to dsDNA was most in the presence of ATP. 1 μm Rad51p, ADP, ATPγS, and AMP-PNP did not promote binding, at concentrations of Rad51p, these nucleotides promoted some binding 2 ATP was also required for Rad51p binding to dsDNA in the nitrocellulose filter binding 2 protection of the dsDNA against DNase I in the presence of ATP reached a value of ∼3.3 base RecA, which to dsDNA most at pH (14Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar, K. Weinstock G.M. Lehman I.R. J. Biol. Chem. 1981; 256: 8835-8844Abstract Full Text PDF PubMed Google Scholar, G.M. K. Lehman I.R. J. Biol. Chem. 1981; 256: Full Text PDF PubMed Google Scholar, G.M. K. Lehman I.R. J. Biol. Chem. 1981; 256: Full Text PDF PubMed Google Scholar), Rad51p dsDNA with the same at pH in the presence of ATP not as in the of ssDNA, we an constant for Rad51p binding to dsDNA of × which is to the value for Rad51p binding to 3 the of Rad51p binding to DNA on ATP The Kd value for ATP for Rad51p binding to εDNA is μm, and that for binding to dsDNA is However, binding to ssDNA and dsDNA was measured in the presence of 1 and 2 μm Rad51p, these values the stoichiometry of binding of Rad51p and ATP a which considerably The concentration of Mg2+ required for promoting Rad51p binding to εDNA with the amount of ATP in the reaction or Rad51p binding with μm ATP, however, was or no of Mg2+ until concentration reached 1 This that ATP is in Rad51p binding to ssDNA and that an complex is the for promoting In the presence of 3 mm ATP, Mg2+ on Rad51p binding to εDNA until most or all of the ATP was This the that ATP is and even with by the complex. The binding of Rad51p to dsDNA the same on the amounts of ATP and not The data indicate that a excess of Mg2+ over ATP is to form the of the complex, we the that Mg2+ is also to the binding in with Rad51p or the DNA Once formed in the presence of 100 μm ATP and maintained in this the Rad51p·εDNA complex was stable for an of However, the complex was formed with μm ATP, was min at 30 °C. This is most to of ATP by hydrolysis and not to the of ADP even a 10-fold excess of ADP over ATP did not the binding AMP-PNP, which was also unable to complex formation also to the Rad51p·εDNA complex formed with ATP the Rad51p·εDNA complex was in the presence of ATPγS even ATPγS was unable to Rad51p binding to εDNA 1 We the dissociation of the complex to the ability of ATPγS to with ATP for binding to Rad51p and the of the complex to εDNA. The of ADP, ATPγS, or AMP-PNP to promote Rad51p binding to DNA that ATP hydrolysis for DNA To this we measured both Rad51p binding to εDNA and the of ATP hydrolysis at and 10 °C. 30 dissociation of the formed Rad51p·εDNA complex with ATP hydrolysis and was complete after 1000 Rad51p to εDNA with the same dissociation of the complex at the the of ATP 10 Rad51p with εDNA to an even and as as at 30 in the absence of ATP hydrolysis, the complex was results that Rad51p requires with ATP, not hydrolysis, for DNA binding and that hydrolysis of the ATP causes dissociation of Rad51p from DNA. The formation of the Rad51p·εDNA complex was by sodium chloride concentrations 100 mm that both the and of Rad51p·εDNA complex formation are by were required for the formation of 95% of the complex without whereas were required to form the same amount of complex in the presence of mm of Once formed, however, the complex was stable at salt concentrations from their effects on complex formation that the are different in and that in mm sodium or no complex was formed by 10 of the complex remained 10 min after the addition of mm salt to the preformed complex and after 10 min in mm is to the Rad51p·εDNA complex at salt concentrations is to the The sequence between S. cerevisiaeRad51p and RecA on that or in recombination (2Aboussekhra A. Chanet R. Adjiri A. Fabre F. Mol. Cell. Biol. 1992; 12: 3224-3234Crossref PubMed Scopus (320) Google Scholar, 3Basile G. Aker M. Mortimer R.K. Mol. Cell. Biol. 1992; 12: 3235-3246Crossref PubMed Scopus (239) Google Scholar, 4Shinohara A. Ogawa H. Ogawa T. Cell. 1992; 69: 457-470Abstract Full Text PDF PubMed Scopus (1052) Google Scholar). both promote strand exchange between a linear DNA and a homologous circular single-stranded DNA to form a double-stranded and a linear the of their (6Sung P. Science. 1994; 265: 1241-1243Crossref PubMed Scopus (756) Google Scholar, 7Sung P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). We are in the Rad51p-promoted reaction in particularly the binding of Rad51p to the kinds of DNA To this we examined the binding of Rad51p to a modified single-stranded DNA, which has used previously to RecA binding to ssDNA (13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google Scholar). Protein binding to εDNA is monitored by an in the fluorescence of the DNA. Our results that Rad51p binding to εDNA is strongly on the presence of ATP and 37 ADP, ATPγS, and AMP-PNP for ATP in promoting Rad51p binding to however, at 30 in the presence of concentrations of Rad51p, ATPγS a of RecA to ssDNA without ATP, ATP a to a binding form (13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google Scholar). The analogs ATPγS and AMP-PNP also the of RecA for DNA, the complex is stable in the presence of these nucleotides (13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google Scholar). the stoichiometry of Rad51p with εDNA a binding of and of × on the value at This binding is different from the previously value of nucleotides determined from of the strand exchange reaction P. Robberson D.L. Cell. 1995; 82: 453-461Abstract Full Text PDF PubMed Scopus (425) Google Scholar, 8Namsaraev E.A. Berg P. Mol. Cell. Biol. 1997; 17: 5359-5368Crossref PubMed Scopus (58) Google Scholar). The in the binding stoichiometry can the Rad51p monomer has binding each of which can in binding stoichiometry when measuring the binding stoichiometry of RecA and εDNA and the binding at which the is these were and (17Zlotnick A. Mitchell R.S. Steed R.K. Brenner S.L. J. Biol. Chem. 1993; 268: 22525-22530Abstract Full Text PDF PubMed Google Scholar). The between RecA and Rad51p has in their ability to with dsDNA. RecA to dsDNA at pH and the is increased at pH or with ATPγS (14Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar, K. Weinstock G.M. Lehman I.R. J. Biol. Chem. 1981; 256: 8835-8844Abstract Full Text PDF PubMed Google Scholar, G.M. K. Lehman I.R. J. Biol. Chem. 1981; 256: Full Text PDF PubMed Google Scholar, G.M. K. Lehman I.R. J. Biol. Chem. 1981; 256: Full Text PDF PubMed Google Scholar). Rad51p, however, to dsDNA in the presence of ATP and Mg2+ over the pH of ATPγS and AMP-PNP are able to binding only at concentrations of Rad51p, whereas ADP is only The binding from the is ∼3.3 base with a binding of × these values are the same as for RecA binding to dsDNA (14Pugh B.F. Cox M.M. J. Biol. Chem. 1987; 262: 1326-1336Abstract Full Text PDF PubMed Google Scholar). Binding of Rad51p to εDNA is at μm ATP and at 1 for binding to dsDNA, the values are respectively. However, binding to ssDNA and dsDNA was measured in the presence of 1 and 2 μm Rad51p, the Kd value for ATP binding considerably The amount of Mg2+ is by the amount of ATP; at μm ATP, 2 mm Mg2+ is with excess ATP binding requires mm This that the for promoting Rad51p binding to ssDNA and dsDNA is a complex, which requires for formation a excess of Mg2+ over ATP. however, Mg2+ has an role in the binding by with the DNA or by on the protein concentrations the binding of Rad51p to εDNA is The degree of with salt concentration until complete occurs at We have noted, not investigated that and are the chloride Once formed at levels of the Rad51p·εDNA complex is as the salt concentration is at salt the complex is considerably stable from the of that salt concentration on complex Once formed, the Rad51p·εDNA complex is stable so long as ATP and Mg2+ are maintained. of either causes the complex to of ADP or AMP-PNP at levels 10 the ATP not the of the complex. However, the addition of ATPγS to a preformed Rad51p·εDNA complex at a concentration 10 that of the ATP concentration causes a dissociation of the complex. We that ATPγS with ATP for binding to Rad51p, the complex is unable to to εDNA, dissociation of the Rad51p·εDNA complex The results of dissociation of the Rad51p·εDNA complex formed in the presence of that ATP binding, not ATP hydrolysis, is required for Rad51p binding to DNA. ATP hydrolysis to Rad51p the DNA. The in the ability of Rad51p to DNA in the presence of ATP and ATPγS that only the interaction with ATP the change to to DNA. has also to RecA for DNA binding (13Menetski J.P. Kowalczykowski S.C. J. Mol. Biol. 1985; 181: 281-295Crossref PubMed Scopus (252) Google S.C. Krupp R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). as was the for the strand exchange, is only a between Rad51p and RecA Rad51p a with DNA to the one formed with RecA, is of to ATP and Mg2+ are required to Rad51p for binding or to promote the formation of of Rad51p that then to DNA or ATP and the binding of to of Rad51p. that to investigated is like RecA, the and dissociation of Rad51p with DNA occurs with a unique We M. for the εDNA, R. Lehman for of the and E. for