DNA Strand Exchange Promoted by RecA K72R
1996; Elsevier BV; Volume: 271; Issue: 10 Linguagem: Inglês
10.1074/jbc.271.10.5712
ISSN1083-351X
AutoresQun Shan, Michael M. Cox, Ross B. Inman,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoReplacement 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. 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. INTRODUCTIONThe RecA protein of Escherichia coli is a 352-amino acid polypeptide chain with a predicted molecular weight of 37,842. The protein is found in all bacteria and is critical to the processes of recombinational DNA repair, homologous recombination, induction of the SOS response to DNA damage, SOS mutagenesis, and the partitioning of chromosomes at cell division (Clark and Sandler, 1994; Cox, 1994; Kowalczykowski et al., 1994; Stasiak and Egelman, 1994; Livneh et al., 1993; West, 1992; Roca and Cox, 1990).In vitro, RecA protein promotes a set of DNA strand exchange reactions that mimics its presumed role in recombination and recombinational DNA repair. The reactions can involve either three or four DNA strands (Fig. 1). RecA first forms a nucleoprotein filament on the single-stranded or gapped DNA substrate. This DNA is then aligned with a homologous linear duplex DNA. A strand switch then occurs within the filament producing a nascent region of hybrid DNA, which is extended to generate the products shown. In a normal reaction, strand exchange proceeds unidirectionally, 5′ to 3′ relative to the single-stranded DNA (or the single-stranded region of the gapped DNA) to which the RecA first binds. The reaction also proceeds past a variety of structural barriers in the DNA substrates.RecA protein is a DNA-dependent ATPase, with a monomer kcat of 30 min-1 when bound to ssDNA. ( 1The abbreviations used are: ssDNAsingle-stranded DNAdsDNAdouble-stranded DNAwtRecAthe wild type RecA proteinDTTdithiothreitolAMT4′-aminomethyl-4,5′,8-trimethylpsoralenbpbase pair(s)kbpkilobase pair(s)ATPγSadenosine 5′-O-(3-thio)triphosphateSSBthe single-stranded DNA binding protein of E. coliPAGEpolyacrylamide gel electrophoresis.) ATP is hydrolyzed uniformly throughout the nucleoprotein filament (Brenner et al., 1987). When a homologous duplex DNA is added to the reaction, the kcat drops abruptly to about 20 min-1, and remains at that level throughout the ensuing DNA strand exchange reaction as long as ATP is regenerated (Schutte and Cox, 1987; Ullsperger and Cox, 1995). The reaction is apparently quite inefficient, with about 100 ATPs hydrolyzed per base pair of hybrid DNA created. The function of this ATP hydrolysis is incompletely understood. An important clue was provided by the observation that RecA protein can promote DNA strand exchange under some conditions in the presence of ATPγS, an ATP analog that is not readily hydrolyzed by RecA (Menetski et al., 1990; Rosselli and Stasiak, 1990). This observation was reinforced more recently by work on the RecA mutant K72R (Rehrauer and Kowalczykowski, 1993) and again with wild type RecA protein in the presence of ADP-AlF4- (Kowalczykowski and Krupp, 1995). The Lys → Arg substitution in the K72R mutant occurs in a well conserved nucleotide binding fold, and the mutant protein binds but does not hydrolyze ATP. The mutant will also promote a limited DNA strand exchange, functioning best with dATP (Rehrauer and Kowalczykowski, 1993). These results demonstrate that the RecA filament has an inherent capacity to take up at least three DNA strands and promote DNA strand exchange without ATP hydrolysis, and have been used to argue against an essential role for ATP hydrolysis in DNA strand exchange. The RecA filament tends to stabilize the hybrid DNA products of a DNA strand exchange reaction (Adzuma, 1992).Why, then, does RecA protein hydrolyze ATP? One way to elucidate the function of RecA-mediated ATP hydrolysis is to define the limitations of reactions that occur without it. A series of studies determined that the strand exchange occurring withq ATPγS is limited in extent and bidirectional (Jain et al., 1994; Konforti and Davis, 1992). The ATPγS reactions also did not proceed past structural barriers in the DNA (Kim et al., 1992a; Rosselli and Stasiak, 1991) and did not accommodate four DNA strands (Kim et al., 1992b). ATP hydrolysis therefore appears to confer several important properties to the DNA strand exchange reaction, being required in particular for unidirectional branch movement that can bypass barriers and for four-strand exchange reactions. These observations provide some indirect mechanistic clues but have been obtained only under the conditions used with ATPγS.The properties of RecA-mediated DNA strand exchange in the absence of ATP hydrolysis provoke questions which, if answered, might shed additional light on ATP function and strand exchange mechanism. In studies to date with three-strand exchange reactions using homologous substrates, there is a rapid formation of a limited segment of hybrid DNA product (typically 1-3 kbp). However, at this point the reaction halts or slows dramatically. Since the entire filament is capable of promoting an exchange between homologous substrates, it is surprising that the entire reaction does not proceed to completion. The slowing or cessation of strand exchange implies that discontinuities exist in some component of an early strand exchange intermediate. The one possibility presented to date involves filament discontinuities, with ATP hydrolysis needed to recycle RecA monomers and correct the discontinuities (Menetski et al., 1990; Rehrauer and Kowalczykowski, 1993; Kowalczykowski and Krupp, 1995). As shown below, the only reasonable alternative involves discontinuities in a key DNA structure that serves as a strand exchange intermediate.A similar and related unresolved question can be defined even under conditions in which ATP is being hydrolyzed. Upon addition of a homologous duplex DNA to RecA•ssDNA complexes hydrolyzing ATP, the rate of ATP hydrolysis declines abruptly by up to 30%. The observed decline is directly proportional to the length of homologous sequence in the duplex, providing evidence that the entire length of available homology is detected within a minute or two with direct DNA-DNA interactions occurring over distances of 8 kbp or more (Schutte and Cox, 1987). However, productive strand exchange detectable after RecA removal from the DNA proceeds much slower, requiring 20 min or more to encompass the same 8 kbp. There again appears to be a fast phase of strand exchange in which some short length of hybrid DNA is generated, followed by a slow phase in which the nascent hybrid DNA is extended. The fast phase is sometimes manifested as an apparent burst phase in hybrid DNA formation when ATP is hydrolyzed (Kahn and Radding, 1984; Bedale and Cox, 1996). As in the cases where ATP is not hydrolyzed, it is necessary to explain why the fast phase comes to an end before strand exchange is complete, even though the response of the filament indicates the detection of homology along the entire length of the DNA.In this report, we further explore the properties of the fast and slow reaction phases and present a simple model that explains why the fast phase is limited in extent. The model also explains all properties of the two reaction phases and applies to reactions carried out with or without hydrolysis of ATP. The results complement and/or confirm a number of previous observations obtained with ATPγS, using RecA K72R employed under more classical reaction conditions, and further characterize the RecA K72R mutant protein. To date, many aspects of the DNA strand exchange reaction promoted by the RecA K72R mutant protein remain unexplored, but have the potential to test many of the ideas outlined above about the role of the RecA ATPase activity.MATERIALS AND METHODSEnzymes and BiochemicalsE. coli RecA protein was purified and stored as described previously (Cox et al., 1981). The RecA protein concentration was determined by absorbance at 280 nm using an extinction coefficient of ∈280= 0.59 A280 mg-1 ml (Craig and Roberts, 1981). E. coli single-stranded DNA binding protein (SSB) was purified as described (Lohman et al., 1986) with the minor modification that a DEAE-Sepharose column was added to ensure removal of single-stranded DNA exonucleases. The concentration of SSB protein was determined by absorbance at 280 nm using an extinction coefficient of ∈280 = 1.5 A280 mg-1 ml (Lohman and Overman, 1985). Purified LexA repressor was a generous gift from Dr. John Little (University of Arizona). Oligonucleotides were synthesized by the University of Wisconsin Biochemistry Department Synthesis Facility. The Sequenase version 2.0 sequencing kit was from U. S. Biochemical Corp. Restriction endonucleases, β-agarase, and T4 polynucleotide kinase were purchased from New England Biolabs. Terminal transferase and ATPγS were purchased from Boehringer Mannheim. Ultrapure dATP, DEAE-Sepharose resin, and a Mono Q column were from Pharmacia Biotech Inc. Amino-4,5′,8-trimethylpsoralen (AMT) was from Calbiochem. Tris buffer was from Fisher. ATP, proteinase K, lactic dehydrogenase, pyruvate kinase, phosphoenolpyruvate, and nicotinamide adenine dinucleotide (reduced form, NADH+), creatine phosphokinase, phosphocreatine, and low melting agarose were purchased from Sigma. Hydroxylapatite resin was from Bio-Rad.DNADuplex and ssDNA substrates were derived from bacteriophage ϕX174 (5386 bp) and M13mp8 (7229 bp) (Messing, 1983). ϕX174 supercoiled circular duplex DNA and viral circular ssDNA were purchased from New England Biolabs. Bacteriophage M13mp8.52 (7251 bp) is bacteriophage M13mp8 with a short heterologous sequence (52 bp) originally derived from the plasmid pJFS36 (Senecoff et al., 1985) replacing the 30-bp EcoRI-PstI fragment of bacteriophage M13mp8 (Kim et al., 1992a). Bacteriophage M13mp8.1037 (8266 bp) is bacteriophage M13mp8 with 1037 bp (EcoRV fragment from the E. coli galT gene) inserted into the SmaI site (previously called M13mp8.1041) (Lindsley and Cox, 1990b). Supercoiled circular duplex DNA and circular single-stranded DNA from bacteriophage M13mp8 and its derivatives were prepared as described previously (Davis et al., 1980; Messing, 1983; Neuendorf and Cox, 1986). The concentration of dsDNA and ssDNA stock solutions were determined by absorbance at 260 nm, using 50 and 36 mg ml-1A260-1, respectively, as conversion factors. DNA concentrations are expressed in terms of total nucleotides. Linear duplex DNA substrates were generated by complete digestion of supercoiled DNA by appropriate restriction endonucleases. The protein was removed by extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1) followed by ethanol precipitation. Linear duplex DNA fragments were generated by digesting supercoiled DNA with appropriate restriction endonucleases and isolating from preparative low melting agarose gel using β-agarase or as described (Sambrook et al., 1989). The fragments were then extracted twice with Tris-EDTA-saturated butyl alcohol, followed by 1:1 extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1). The fragments were finally concentrated by ethanol precipitation. Gapped duplex DNA substrates were prepared using a large scale (reaction volume = 1.2 ml) RecA reaction. The reaction contained 20 μM circular M13mp8.1037 ssDNA, 20 μM M13mp8 linear double-stranded DNA (digested with SmaI), 6.7 μM wtRecA protein, 2 μM SSB, and 3 mM ATP. Reactions were carried out under standard strand exchange conditions listed below, for 90 min. The reaction was stopped by adding EDTA, SDS, and proteinase K to 12 mM, 1%, and 1 mg/ml, respectively. The mixture was incubated at 37°C for 30 min. The reaction mixture was then extracted 1:1 with phenol/chloroform/isoamyl alcohol (25:24:1) and chloroform/isoamyl alcohol (24:1), and the DNA was concentrated in a Microcon concentrator (Amicon). The concentrated reaction mixture was electrophoresed overnight in a 0.8% low melting agarose gel at about 2 V cm-1. The gapped duplex DNA was purified from low melting agarose as described above. The resulting gapped duplex species is called GD1037 (formerly called GD1041) (Kim et al., 1992b)BuffersP buffer contained 20 mM potassium phosphate (pH 6.8), 1 mM DTT, 0.1 mM EDTA, 10% (w/v) glycerol. R buffer contained 20 mM Tris-HCl, 80% cation (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 10% (w/v) glycerol.Cloning and Overexpressing the recA K72R GeneThe recA K72R gene was cloned by a polymerase chain reaction-based, site-directed mutagenesis procedure. Briefly, a DNA fragment containing the first 236 bp of the recA coding region was polymerase chain reaction synthesized with one primer containing the mutation code for Arg (CGA) at residue 72 and the internal PstI site within the recA gene. This fragment was cloned into pBluescriptSK(-) (Stratagene). The identity of the mutated recA gene fragment was checked by sequencing, and subsequently the recA gene fragment containing the desired mutation was swapped with corresponding fragment from wild-type RecA gene in pUC18, to create pRecA17 (4.0 kbp). The integrity of the cloned recA K72R gene was confirmed by direct sequencing. To express the recA K72R gene, a 16-liter culture of E. coli strain GE643 (lexA51, ΔrecA1398) (Weisemann and Weinstock, 1985) transformed with pRecA17 was grown in LB medium for 16 h at 37°C with aeration. Cells were collected by centrifugation at 4200 rpm at 4°C in a Beckman JA6 rotor. The cell pellet was washed with cell harvest buffer (50 mM Tris-HCl, 80% cation, pH 7.5, 10% sucrose) and then centrifuged again. The pellet was resuspended in cell harvest buffer to give an OD600 reading of about 400. The cell suspension was quickly frozen in liquid N2 and stored at −70°C.Purification of the RecA K72R ProteinThe RecA K72R protein was purified by a modification of the procedure used for the wild type RecA protein. Briefly, 50 g of cell paste were thawed on ice overnight. Cell lysis, polymin P precipitation, and ammonium sulfate extraction were performed as described by Cox et al.(1981). The supernatant from the ammonium sulfate extraction was precipitated three times by adding solid ammonium sulfate to 0.28 g/ml. The pellet was finally resuspended in 10 ml of R buffer + 50 mM KCl and dialyzed overnight against 2 × 1 liter of the same buffer, giving rise to fraction II (18.8 ml, 368 mg of protein). This was loaded onto a DEAE-Sepharose column (2.5 cm × 14 cm, 70-ml bed volume). The column was washed with R + 50 mM KCl buffer, and the majority of the RecA protein appeared in the flow-through. Peak fractions were identified by SDS-PAGE. Protein in the pooled peak fractions was precipitated by adding solid ammonium sulfate to 0.28 g/ml, collected by low speed centrifugation, resuspended in 10 ml of P buffer, and dialyzed against 2 × 1 liter of P buffer overnight to generate fraction III (10 ml, 140 mg protein). Fraction III was loaded onto a hydroxylapatite column (2.5 cm × 7 cm, 35-ml bed volume). The column was washed with 40 ml of P buffer and developed with 360 ml of linear gradient from 20 to 500 mM phosphate buffer (pH 6.8). Protein-containing fractions were located by SDS-PAGE, pooled, and dialyzed against 3 × 2 liters of R + 50 mM KCl to generate fraction IV (47 ml, 74 mg of protein). Fraction IV was applied to a 1-ml Mono Q fast protein liquid chromatography column for several runs using a superloop, and the column was washed with 3 ml of R + 50 mM KCl buffer and developed with 30 ml of 0.05 to 1 M KCl linear gradient. Peak fractions were pooled and dialyzed against 2 × 3 liters of R buffer, producing fraction V (11 ml). This fraction was frozen in liquid nitrogen and stored at −70°C. The final yield of the RecA K72R protein from this 50-g cell paste was 22 mg. The RecA K72R protein was at least 98% pure as judged by a densitometric scan of a Coomassie Blue-stained SDS-PAGE gel. The concentration of the RecA K72R protein was determined using the same extinction coefficient as wild type RecA protein. The RecA K72R protein was free of detectable endo- or exonucleases.Strand Exchange Reaction ConditionsUnless otherwise specified, all reactions were performed at 37°C in a standard strand exchange reaction buffer containing 25 mM Tris acetate (80% cation, pH 7.5), 10 mM magnesium acetate, 3 mM potassium glutamate, 1 mM DTT, 5% (w/v) glycerol, and an ATP (4.7 mM phosphoenolpyruvate, 5 units ml-1 pyruvate kinase) or a dATP (11.8 mM phosphoenolpyruvate, 10 units ml-1 pyruvate kinase) regeneration system. Linear duplex DNA and circular single-stranded or gapped duplex DNA were preincubated with RecA protein or RecA K72R protein for 10 min before the indicated concentrations of dATP (or ATP) and SSB protein were added to initiate the reaction. A regeneration system was omitted in some experiments as noted. The magnesium acetate and dATP concentrations, as well as the order of addition of RecA K72R and SSB, were varied in some experiments as indicated in the text and figure legends.After the gel was stained with ethidium bromide (1 μg/ml) for at least 30 min and destained for at least 2 h, the gel was then photographed over an ultraviolet transilluminator. The intensities of DNA bands were quantified by scanning the photographic negatives using a Molecular Dynamics Personal Densitometer SI and analyzing the image with ImageQuant software (Version 4.2). In order to correct for variability in sample loading onto the agarose gel, the band corresponding to full-length products and/or the broad smear representing intermediates of the strand exchange reaction were quantified as the fraction of the total fluorescing DNA in a given gel lane.In some experiments, the data was plotted with respect to the concentration of Mg2+ in excess of that involved in a complex with dATP. The concentration of “excess” Mg2+ was calculated based on the reported dissociation constant of 1 × 10-1M for the Mg•ATP complex (Alberty, 1969).Agarose Gel AssaysAliquots (10 μl) of the reactions were removed at each indicated time point, and the reactions were stopped by the addition of 0.25 volume of gel loading buffer (60 mM EDTA, 5% SDS, 25% (w/v) glycerol, 0.2% bromphenol blue). Samples were electrophoresed overnight in a 0.8% agarose gel at 2 V cm-1. In some experiments, aliquots of the reactions were cross-linked with AMT before gel electrophoresis as described below.DNA-dependent ATPase and dATPase AssaysThe ssDNA-dependent ATPase or dATPase activity of the RecA K72R and the wild type RecA protein was measured by a coupled enzyme assay (Lindsley and Cox, 1990a; Morrical et al., 1986). In addition to the appropriate phosphoenolpyruvate/pyruvate kinase regeneration system described above, reactions contained 3 mM NADH and 4.5 units ml-1 lactic dehydrogenase. Absorbances were measured at 380 nm, rather than 340 nm (the absorbance maximum for NADH), to remain within the linear range of the spectrophotometer. An NADH extinction coefficient of є380 = 1.21 mM-1 cm-1 was used to calculate the rate of ATP or dATP hydrolysis. Reaction mixtures (400 μl) also contained 8 μM circular M13mp8 ssDNA and RecA protein as noted, in standard strand exchange reaction buffer. Reactions were started by the addition of SSB protein and ATP or dATP to final concentrations of 0.8 μM and 3 mM, respectively.Electron MicroscopySamples for electron microscopy were obtained by spreading the entire strand exchange reaction mixture. Reaction mixtures were cross-linked with AMT prior to examination by electron microscopy to prevent spontaneous branch migration during sample preparation. Samples were cross-linked by addition of AMT to a final concentration of 30 μg ml-1, incubated at room temperature for 3 min and irradiated with long wave UV light (Umlauf et al., 1990) for 4 min at room temperature. The UV light was generated with two 15-watt fluorescent black light tubes. Samples were placed 8 cm below the UV light source. The cross-linked samples were incubated with proteinase K (1 mg ml-1 final) and SDS (1% final) for 30 min at 37°C. The samples were dialyzed into 20 mM NaCl and 5 mM EDTA for 5 h at room temperature on Millipore type VM (0.05 mm) filters (Jain et al., 1994) and then spread as described previously (Inman and Schnös, 1970). Photography and measurements of the DNA molecules were performed as described previously (Littlewood and Inman, 1982).Three types of information were derived from the electron microscopy experiments. First we wished to confirm that the reaction intermediates observed on gels had the anticipated structure. Representative molecules are shown for some experiments and results described in the text. Second, we wished to determine the proportion of the duplex linear substrates that was involved in DNA strand exchange reactions leading to intermediates. This was done by counting the intermediates and unreacted linear duplex DNA molecules found in a representative sample from each experiment. Complex recombinational events involving more than two DNA substrate molecules and events that were interpreted to arise from broken or nicked substrates (the latter produce low levels of complex reaction products in the reactions) were ignored in these estimates. In some experiments, the grids from different reactions were assigned an undescriptive identification code by Q. S., and were subsequently counted in a random sequence by R. B. I. Third, it was important to estimate the length of hybrid DNA generated for a representative sample of intermediates in some experiments. Because of the large numbers of samples, obtaining accurate measurements of significant numbers of intermediates in all of them was impractical; we have therefore estimated the degree of exchange as described earlier (Jain et al., 1994). Briefly, the ratio of unexchanged to exchanged duplex DNA was judged. These ratios were then converted into base pairs of exchanged DNA using the known length of the linear dsDNA substrate. The data were sorted into 8-10 degrees of exchange and plotted as histograms. Degrees of exchange with the 1.3-kbp DNA fragment used as a substrate in some experiments was divided in this manner into eight equal segments of 165 bp; linear M13mp8.1037 dsDNA was divided into 10 segments of 830 bp.These judgments were checked in two ways. First, two grids each from two different samples were counted and judged “blind” as described above. The four sets of data were then compared by the χ2 two-way contingency test at the 95% confidence level. In both cases, the data sets for the two samples were not significantly different. The judgments have also been checked directly by comparing the data obtained to data from more detailed measurements done on the same samples (Jain et al., 1994). Statistical analysis again showed that there is no significant difference in the result obtained with the two methods.LexA Repressor Cleavage AssayReactions (100 μl) were carried out at 37°C and contained 20 mM Tris-HCl (80% cation, pH 7.5), 50 mM NaCl, 4 mM MgCl2, 1 mM ATP, an ATP regeneration system (12 mM phosphocreatine, 10 units ml-1 creatine phosphokinase), 40 μM circular M13mp8 ssDNA, 8.9 μM LexA repressor, and 1.33 μM wild type RecA or RecA K72R protein. The reactions did not contain SSB (Little, 1984). Additional LexA repressor cleavage assays were performed using dATP (with the dATP regeneration system described above) or ATPγS (1 mM) as a nucleotide cofactor. Aliquots (10 μl) were removed at different time points, followed by addition of 5 μl of SDS-PAGE loading buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM 2-mercaptoethanol, 0.1% bromphenol blue). Samples were heated at 95°C for 3 min before they were analyzed by 13% SDS-PAGE. The destained gels were scanned and quantified using the NIH Image Analysis Program (Version 1.44).Copies of the electron micrographs discussed in this publication are available for viewing or downloading using a World-Wide Web client at the URL: http://phage.bocklabs.wisc.edu/.RESULTSExperimental DesignThe goal of these experiments is a more complete characterization of the DNA strand exchange reaction promoted by RecA K72R as a means to address ATPase function. A few experiments were done to measure basic ATPase and strand exchange activities to ensure that our mutant protein exhibited the properties reported by Rehrauer and Kowalczykowski(1993). Proceeding from this base line, we then characterized the magnesium requirements for RecA K72R-mediated DNA strand exchange in detail as a means to distinguish two phases in the reaction. Finally, we tested the capacity of the K72R mutant protein to promote unidirectional strand exchange, bypass of structural barriers in the DNA during strand exchange, and DNA strand exchange with four DNA strands, using strategies similar to those employed in several recent studies (Jain et al., 1994; Kim et al., 1992a, 1992b). We also explored the capacity of the mutant protein to facilitate the cleavage of LexA protein.The RecA K72R Mutant Protein Does Not Hydrolyze ATP or dATPUsing a coupled spectrophotometric assay, we measured the ssDNA-dependent ATPase activity of both the mutant and wild type proteins with ATP and dATP. The mutant did not produce a rate of ATP or dATP hydrolysis detectable above background. Based on an evaluation of assay sensitivity, we estimated the upper limit for ATP and dATP hydrolysis (kcat) to be 0.12 min-1 and 0.24 min-1, respectively. The actual rate is probably much lower, and the results coincide well with the 600-850-fold reduction in NTP hydrolysis observed by Rehrauer and Kowalczykowski(1993). RecA K72R protein was also tested for dATP hydrolytic activities at 2, 4, 6, and 8 mM Mg2+, with no hydrolysis observed in any case. The measured kcat for the wtRecA protein bound to ssDNA in the presence of SSB was 29 min-1 and 36 min-1 for ATP and dATP hydrolysis, respectively, consistent with published findings.The RecA K72R Protein Promotes Limited DNA Strand Exchange in the Presence of dATPDNA strand exchange reactions between homologous linear double-stranded DNA and circular ssDNA substrates derived from ϕX174 phage in the presence of ATP and dATP under standard reaction conditions (including 10 mM magnesium acetate) were monitored by agarose gel electrophoresis (Fig. 2). In the presence of ATP, no DNA strand exchange reaction was observed with RecA K72R (Fig. 2A). Under the same reaction conditions, the wild type RecA protein promoted an efficient reaction (Fig. 2A). When dATP was used as the nucleotide cofactor, the mutant protein converted much of the substrate DNA to strand exchange intermediates (joint molecules). Very little complete strand exchange
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