Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
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`The FEBS Journal
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`Volume 259, Issue 3
`February 1999
`Pages 592–601
`Characterization of thermostable RecA protein and analysis of its interaction with single-stranded
`DNA
`Ryuichi Kato, Seiki Kuramitsu
`
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`First published:
`February 1999 Full publication history
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`DOI:
`10.1046/j.1432-1327.1999.00044.x View/save citation
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`S. Kuramitsu, Department of Biology, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560 0043, Japan. Tel.:
`+ 81 6 850 5433. Fax: + 81 6 850 5442.
`E-mail: kuramitu@bio.sci.osaka-u.ac.jp
`
`Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 °C but not at 37 °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 °C. The kinetic parameters, K and k
`, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 m M and
`m
`cat
`0.60 s at 65 °C, and 0.34 m M and 0.28 s at 37 °C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill
`–1
`–1
`coefficient was about 2. At 65 °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 °C. At 37 °C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 °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 µM at 25 °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.
`
`ATP-γ-S
`adenosine 5′- O -(3-thiotriphosphate)
`CD
`circular dichroism
`ecRecA
`E. coli RecA protein
`IPTG
`isopropyl-β- D(–)-thiogalactopyranoside
`ttRecA
`T. thermophilus RecA protein
`
`UV
`ultraviolet.
`
`RecA protein is essential for homologous recombination and recombinational repair in Escherichia coli [1–3]. Many RecA homologues have been
`isolated not only from prokaryotes [4] but also from eukaryotes [5]. This suggests that the mechanism of DNA recombination and repair is
`conserved in most living organisms, and that RecA protein may play a central role in the system. The properties of E. coli RecA protein (ecRecA)
`have been extensively studied, and it has been shown that this relatively low-molecular-mass protein (38 kDa) has four markedly different activities
`in vitro [1–3]. First, RecA monomers aggregate by themselves and polymerize on DNA to form a nucleoprotein helical filament. Second, RecA
`protein promotes a DNA strand exchange reaction. Third, RecA protein has single-stranded DNA (ssDNA)-dependent ATPase activity. Finally, RecA
`protein has coprotease activity to induce an SOS response. The fact that these different activities are all shown by this single molecule is of
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`Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
`
`http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00044.x/full
`
`considerable interest, and it is important to elucidate the molecular mechanisms involved.
`In order to elucidate the mechanism of recombination by RecA protein, X-ray crystallographic studies of the complexes between RecA protein and
`DNA and/or adenosine triphosphate (ATP) are necessary. Although the structure of ecRecA without DNA has been clarified [6, 7], cocrystallization
`of RecA protein with DNA and/or ATP has not yet been performed, and the binding domain and binding mode of RecA protein with DNA remain to
`be elucidated. Therefore, cocrystallization of RecA protein with DNA and/or ATP is required in order to study the protein–DNA interaction. Thermus
`thermophilus is an extremely thermophilic Gram-negative eubacterium with an optimum growth temperature of about 70 °C [8]. The proteins from
`T. thermophilus are well suited for research on protein biochemistry and physicochemistry because they are physically stable and easily
`crystallized. The recA gene of T. thermophilus has already been cloned and sequenced [9, 10]. The deduced amino acid sequence showed 61%
`identity with that of E. coli , and the T. thermophilus recA gene complemented the ultraviolet (UV) light sensitivity of an E. coli recA mutant. These
`features suggest that T. thermophilus RecA protein (ttRecA) and ecRecA have a common function in vivo . In order to obtain information on the
`enzymatic functions of ttRecA at the molecular level, we have overproduced ttRecA in E. coli , purified it to homogeneity and performed in vitro
`analysis. Crystallization of the protein with DNA is now underway.
`The enzymatic activities of RecA protein, which are DNA strand exchange and coprotease for repressor cleavage, require both ssDNA and ATP. The
`observation of ssDNA-dependent ATP hydrolysis by RecA protein indicates that the DNA binding ability and ATPase activity are interdependent and
`cannot be split. RecA protein activated by binding with ssDNA promotes strand exchange of homologous DNA molecules using free energy obtained
`from ATP hydrolysis. On the other hand, it has been reported that DNA strand exchange can occur without hydrolysis of ATP [11, 12]. It has been
`reported that only a 20-amino-acid peptide derived from ecRecA can promote homologous DNA pairing without ATP [13]. Yeast Rad51 protein,
`which is a eukaryotic homologue of RecA protein, can also catalyze DNA pairing and strand exchange in the presence of nonhydrolyzable ATP
`analogues [14]. It is necessary to know how the energy obtained by ATP hydrolysis is used during DNA recombination and repair. Although it has
`been reported that some thermostable RecA proteins, including that of T. thermophilus , have ATPase activity [10], no kinetic studies of these
`ATPase activities have yet been performed. Detailed kinetic studies of ATPase activity may be useful to elucidate this problem. In this report, we
`show that ttRecA has ATPase activity with substrate cooperativity and determined its kinetic parameters.
`It has been shown that ATPase activity of ecRecA is activated to differing extents by different species of ssDNA [15]. In this report, we show that
`ATPase activity of ttRecA is stimulated by ssDNA, and that the order of the stimulation is: poly(dC) > poly(dT) > M13 ssDNA > poly(dA).
`Interestingly, poly(dA) and M13 ssDNA activated the ATPase at 65 °C but not at 37 °C. It has been reported that poly(dA) shows a characteristic
`positive band at around 192 nm by circular dichroism (CD) spectrometry [16]. This CD band in the region of vacuum UV indicates that poly(dA) does
`not have a random, but rather a specific, structure. In order to know the relationship between the structure and the ATPase stimulating activity, we
`measured the CD spectra of ssDNA and showed that poly(dA) and M13 ssDNA have a specific structure whereas poly(dC) and poly(dT) do not. We
`also showed that the positive CD band decreases as the temperature rises. From these observations, we surmise that the specific structure of
`ssDNA prevents activation of ttRecA to express the ATPase activity.
`Unlike the case of ttRecA, ecRecA expresses ATPase activity to varying degrees in the presence of various kinds of ssDNAs [15, 17]. This
`suggests that the characteristic of ecRecA for ssDNA differs from that of ttRecA. The temperature dependency of the ATPase activity of ttRecA is
`similar to that of the structural alteration of poly(dA) and M13 ssDNA. In order to elucidate whether RecA protein can induce structural change in the
`ssDNA, we carried out CD measurement of poly(dA) in the vacuum UV region in the presence of various concentrations of ttRecA or ecRecA at
`lower temperatures, and showed that ecRecA was able to change the specific structure of poly(dA) whereas ttRecA could not. On the basis of these
`observations, we discuss the difference between ecRecA and ttRecA with regard to the capability of modifying the specific structure of ssDNA
`necessary for the binding.
`
`Enzymes and chemicals
`
`The sources of enzymes and reagents were as follows: DNA modification enzymes including restriction enzymes were from Takara Shuzo, Nippon
`Gene, Toyobo, Japan and New England Biolabs (USA); isopropyl-β- D(–)-thiogalactopyranoside (IPTG) was from Wako Pure Chemical (Japan);
`phosphocellulose (type P11) and DEAE-cellulose (type DE52) were from Whatman Biochemicals (Germany); plastic-backed polyethyleneimine
`cellulose sheets (MN-Polygram CEL300PEI/UV) were from Machery and Nagel (England); [α- P]ATP was from ICN (USA); rabbit muscle pyruvate
`32
`kinase (type II) was from Sigma (USA); pig heart lactate dehydrogenase (grade II) was from Toyobo; adenosine 5′- O -(3-thiotriphosphate) (ATP-γ-S)
`was from Boehringer Mannheim (Germany); poly(dT), poly(dC), poly(dA) and poly(dA)·poly(dT) were from Pharmacia (USA). ecRecA, M13 ssDNA
`and M13 double-stranded DNA (dsDNA) were provided by T. Mikawa (Osaka University).
`
`Media, bacterial strains, plasmids and DNA manipulation
`
`The E. coli strains used were DH5α[18] for plasmid DNA preparation and BL21(DE3) harboring the pLysE plasmid [19] for overproduction of
`ttRecA. They were grown on Terrific broth or LB medium at 37 °C [18]. The plasmids used were pTA3, which contains the entire T. thermophilus
`recA gene [9] in the pET3a expression vector [19]. DNA manipulation was carried out by standard procedures [18].
`
`Determination of nucleotide and polydeoxyribonucleic acid concentrations
`
` = 15 000·M ·cm ,
` = 15 400 M ·cm or ε
`The concentration of ATP or ATP-γ-S was determined using the molar absorption coefficient of ε
`–1
`–1
`–1
`–1
`259
`259
`respectively [20]. The residue molar concentrations of polydeoxyribonucleic acids were determined using the following molar absorption coefficients:
` = 8520 M ·cm for poly(dT) [21]; ε
` = 7400 M ·cm for poly(dC) [21]; ε
` = 8600 M ·cm for poly(dA) [22]; ε
`ε
` = 6000 M ·cm for
`−1
`–1
`–1
`–1
`–1
`–1
`–1
`–1
`264
`268
`257
`264
`poly(dA)·poly(dT) [22].
`
`Overexpression of the T. thermophilus recA gene in E. coli
`
`To create a Nde I restriction enzyme site in the first ATG codon of the T. thermophilus recA gene, a pair of DNA primers was synthesized
`(MilliGen/Biosearch , USA, Cyclone Plus DNA synthesizer). These were 5′-CCTGAGAGGTG CATATGGACGAGAG-3′ and 5′-GCCCCCTTGCC
`GAACTCCTTCTCA-3′, the underlining indicating the Nde I site and the Xmn I site, respectively. A DNA fragment of about 80 bp was amplified by
`polymerase chain reaction under the same conditions as those described previously [9] using pTA3 as a template. The amplified fragment encoding
`the N-terminal side was cut with Nde I and Xmn I. The rest of the DNA fragment containing almost all of the T. thermophilus recA gene was
`prepared by cutting pTA3 with Xmn I and Bam HI. The pET3a vector was cut with Nde I and Bam HI. These three DNA fragments were then ligated
`to construct the T. thermophilus recA gene under the regulation of the T7 promoter, and the resulting plasmid was named pEA1. The nucleotide
`sequence of the amplified region in pEA1 was confirmed by dideoxy sequencing using a ‘Taq Dye Terminator Cycle Sequencing System’ with an
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`Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
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`http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00044.x/full
`
`automated DNA sequencer (Applied Biosystems, Perkin Elmer, USA , model 373A). E. coli strain, BL21(DE3) pLysE transformed by pEA1, was
`grown to 4 × 10 cells·mL in LB broth containing 50 µg·mL ampicillin and 20 µg·mL chloramphenicol, and then IPTG was added to a final
`8
`–1
`–1
`–1
`concentration of 50 µg·mL . After additional incubation for 3 h, the cells were harvested and stored at – 80 °C.
`–1
`
`Purification of ttRecA
`
`Frozen cells (20.7 g) were thawed and suspended in 80 mL of buffer I [50 m M Tris/HCl (pH 8.0), 5 m M EDTA, 8.4 m Mβ-mercaptoethanol, 25%
`(w/v) sucrose, 1 M KCl]. The cells were then disrupted by three 90-s sonications with an ultrasonic disrupter (Tomy, UD201) with a standard tip at
`maximum output on ice. Then Brij-58 was added to a final concentration of 0.4% (w/v) and the mixture was held on ice for 30 min. A cleared cell
`lysate was obtained by centrifugation for 90 min at 4 °C (46 000 g , Hitachi R20A2 rotor). Then 100 mL of buffer I was added to the lysate, and the
`sample was heated for 60 min at 65 °C to remove endogenous E. coli proteins. After cooling in ice-water, the heat-treated lysate was centrifuged
`for 15 min at 4 °C (46 000 g , Hitachi R20A2 rotor). The following purification steps were been carried out at room temperature. The supernatant
`was dialyzed against buffer II [20 m M potassium phosphate (pH 6.5), 1 m M EDTA, 5 m Mβ-mercaptoethanol, 10% (v/v) glycerol] and then applied to
`a 160-mL phosphocellulose column equilibrated with the same buffer. The ttRecA was eluted with 1 L of a linear gradient of KCl (0–1.5 M), and the
`collected fractions were combined and dialyzed against buffer III [50 m M Tris/HCl (pH 7.5), 1 m M EDTA, 5 m Mβ-mercaptoethanol, 10% (v/v)
`glycerol]. The sample was applied to a 180-mL DEAE-cellulose column equilibrated with buffer III and eluted with 1 L of a linear gradient of NaCl
`(0–1.5 M). The fractions containing ttRecA were combined. The sample concentrated by an ultrafiltration membrane (Amicon , USA, YM3) was
`dialyzed against buffer IV [50 m M Tris/HCl (pH 7.5), 0.1 m M EDTA, 5 m Mβ-mercaptoethanol, 1.5 M KCl, 10% (v/v) glycerol] and then filtered
`(Millipore , USA, MILLEX-GV 0.22 µm filter). After addition of glycerol to a final concentration of 50% (v/v), it was stored at – 20 °C. The ttRecA was
`stable for at least 6 months under these conditions, checked by measuring its ATPase activity. The amount of the purified ttRecA was 250 mg, and
`its yield was 12 mg per 1 g cells.
`The N-terminal amino acid sequence of the purified protein was determined with a gas-phase protein sequencer (Applied Biosystems, model 473A).
`The absorption spectrum of ttRecA was similar to that of the E. coli protein [23], and had a maximum at 277 nm (data not shown). As ttRecA
`contains one tryptophan and six tyrosine residues [9], the molar absorption coefficient was calculated to be 14 600 M ·cm at 277 nm by the same
`–1
`–1
`procedure as that described previously [24].
`
`ATPase assay
`
`At 37 °C, hydrolysis of ATP by ttRecA was measured by an enzyme coupling method [25, 26] at pH 7.5. A total of 200 µL of reaction mixture
`contained 50 m M Tris/HCl, 10 m M MgCl , 100 m M KCl, 1 m M DTT, 10 m M phosphoenolpyruvate, 2 m M NADH, 25 U·mL pyruvate kinase,
`–1
`2
`25 units·mL lactate dehydrogenase, DNA, ATP and ttRecA as indicated. The activity was measured from the decrease in absorption at 340 nm in
`–1
`a 0.1-cm cell using a Hitachi spectrophotometer, model U-3000. Because the coupling enzymes are heat-inactivated at higher temperature, we used
`a thin-layer chromatography (TLC) method [17] to measure ATPase activity at 65 °C. The procedure was described in the previous paper [27].
`Analysis of the ATPase activity was performed using the Hill equation (28):
`
` are the initial and maximal initial ATPase reaction velocities, [S] is the concentration of substrate (ATP), K is the substrate
`where v and V
`max
`dissociation constant and n is the Hill coefficient. Eqn (1) was rearranged to give
`
`(1)
`
`(2)
`
`Kinetic parameters were determined by linear fitting to Eqn (2) at various concentrations of ATP.
`
`Apparent dissociation constants between ttRecA and DNA
`
`The concentration dependence of various kinds of DNA on the ATPase activity of ttRecA was measured. ATPase activity at 65 °C was measured by
`the TLC method with 2.5 µM of ttRecA. The activity at 37 °C was measured by the enzyme coupling method with 5 µM of ttRecA. To estimate the
`intensity of binding between ttRecA and DNAs, we calculated the apparent dissociation constant from Eqn (3):
`
`where E, L and EL are ttRecA, DNA, and their complex, respectively. K ′ is the dissociation constant defined by
`
`where parentheses represent respective concentrations.
`The total concentrations of ttRecA ([E] ) and DNA ([L] ) are expressed as follows:
`0
`0
`
`The concentration of ttRecA-DNA complex is obtained from Eqns (4)–(6),
`
`The apparent velocity of the reaction, v , is expressed as:
`
`(3)
`
`(4)
`
`(5)
`
`(6)
`
`(7)
`
`(8)
`
`where k is a constant. The kinetic parameters were determined by fitting Eqn (8) to the data. A computer program, Igor (WaveMetrics), was used
`for calculation of the parameters.
`
`Strand exchange assay
`
`A total of 20 µL of reaction mixture contained 10 m M Tris/HCl (pH 7.5), 10 m M MgCl , 30 m M KCl, 20 m M ATP, 1 m M DTT, 60 µM M13mp18
`2
`ssDNA, 30 µM M13mp18 dsDNA linearized by Bam HI digestion, and various concentrations of ttRecA. It also contained an ATP regeneration
`system (10 m M phosphoenolpyruvate, 25 U·mL pyruvate kinase). The mixture was incubated at 37 °C or 65 °C for 30 min, and then the reaction
`–1
`was stopped by addition of a solution to the final concentration of 10 m M EDTA, 1% SDS and 0.2 mg·mL proteinase K. A half of each aliquot was
`–1
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`Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
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`http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00044.x/full
`
`electrophoresed on 0.7% agarose gel, and then the gel was stained with 0.5 µg·mL ethidium bromide.
`–1
`
`Electron microscopy
`
`A total of 20 µL of reaction mixture contained 10 m M Tris/HCl (pH 7.5), 10 m M MgCl , 30 m M KCl, 5 m M ATP, 8 µM ssDNA and 1 µM ttRecA. It was
`2
`incubated at 37 °C for 20 min or at 65 °C for 10 min, and then NaF and Al(NO ) were added to a final concentration of 2.5 m M. Additional
`3 3
`incubation was carried out at 37 °C for 20 min or at 65 °C for 10 min. The sample was then negatively stained with 2% (w/v) uranyl acetate for 2 min
`on carbon-coated copper grids, which had been glow-discharged. A JEOL 1200EXII electron microscope operating at an accelerating voltage of
`80 kV was used for all microscopy.
`
`CD measurement in the vacuum UV region
`
`CD measurements were performed with a Jasco spectropolarimeter, model J-720W. To minimize absorption in the vacuum UV region by H O in the
`2
`sample buffer, a 0.2-mm cell was used. To minimize absorption by O gas in the optical line, the air was replaced by N gas flowing at 10 L·min .
`–1
`2
`2
`The residue molar ellipticity, [ θ ], was defined as 100· θ
`·( lc )
`, where θ
` is the observed molar ellipticity, l is the length of the light path, and
`–1
`c is the residue molar concentration of ssDNAs.
`
`obs
`
`obs
`
`Overproduction and purification of ttRecA
`
`To analyze the biochemical properties of ttRecA, we overproduced and purified it. Details of the purification of ttRecA are described in Experimental
`procedures. Heat treatment at 65 °C for 60 min prior to column chromatography was effective in removing most of the endogenous E. coli proteins.
`After heat treatment, ttRecA was purified by phosphocellulose and DEAE-cellulose column chromatography. To monitor the purification steps,
`SDS/polyacrylamide gel electrophoresis was performed ( Fig. 1). From its relative migration on the gel, ttRecA was estimated to have a molecular
`mass of about 36 kDa, which agreed with that calculated from the amino acid sequence translated from its nucleotide sequence [9]. The N-terminal
`20 residues were M-D-E-S-K-R-K-A-L-E-N-A-L-K-A-I-E-K-E-F, which coincided with the sequence translated from the first ATG of the
`T. thermophilus recA gene. To confirm the accurate molecular mass of ttRecA, the purified protein was analyzed by ion-spray ionization mass
`spectrometry. The determined value, 36 385, agreed well with the calculated one, 36 384.
`
`Figure 1.
`Open in figure viewer
`
`Overproduction and purification of ttRecA. The fractions obtained during the purification of ttRecA were electrophoresed on
`SDS/polyacrylamide (12.5%) gel [46] and stained with Coomassie Brilliant Blue. Molecular mass markers (in kilodaltons) are indicated to
`the left. Lane 1, total cell extract; lane 2, cleared lysate; lane 3, post-heat treatment at 65 °C; lane 4, post-phosphocellulose; lane 5, post-
`DEAE-cellulose. The arrow indicates the ttRecA. About 1–10 µg of protein was loaded into each lane of the gel.
`
`Kinetic parameters for ATPase activity of ttRecA
`
`[29], show ATPase activity. ttRecA also has its
`Including ecRecA, many proteins which have Walker’s A-type nucleotide binding motif, GXXXXGK /
`T
`S
`consensus sequence [9, 10] and ssDNA-dependent ATPase activity [10]. We studied the temperature dependence of ttRecA activity. The ATPase
`activity with poly(dT) as a ssDNA was measured using a TLC method. Its activity appeared maximal at 65 °C. At 37 °C, which is an ordinary
`temperature for many mesophilic organisms, the activity was about half that at the maximum. At over 90 °C and below 15 °C, little activity was
`observed. The optimum temperature of 65 °C for ttRecA activity is consistent with the fact that T. thermophilus can grow between 47 °C and 85 °C
`[8].
`Although some RecA proteins have been isolated from thermophiles [10, 30], no kinetic studies of their activities have been performed. We
`measured the ATPase activity of ttRecA at various concentrations of ATP to determine its kinetic parameters. For measurement of ATPase activity,
`the TLC method is advantageous when the reaction temperature is high, but disadvantageous for following the reaction in real time. On the other
`hand, the enzyme coupling method, which is now used widely for measuring ATPase activity, is useful for analysis of steady-state kinetics because
`the reaction can be followed spectroscopically in real time. However, the enzyme coupling method cannot be used at high temperature as the
`coupling enzymes used here, lactate dehydrogenase (from pig heart) and pyruvate kinase (from rabbit muscle), are not thermostable. Therefore, the
`ATPase activity at 65 °C was measured using the TLC method. In the presence of poly(dT) as a ssDNA, the amount of ATP hydrolyzed by ttRecA
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`Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
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`http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00044.x/full
`
` values
`was increased, and substrate cooperativity for the ATPase activity was observed ( Fig. 2A). As shown in the inset of Fig. 2A, the K and k
`cat
`and the Hill coefficient were determined by Hill plot [28] as described in Experimental procedures. They were 1.4 m M, 0.60·s and 1.9, respectively
`–1
`( Table 1). Such positive cooperativity of ATP binding has also been found in ecRecA [15, 31]. Kinetic parameters for the ATPase of ecRecA protein
`have been well studied at 37 °C. To compare these values for the E. coli and T. thermophilus proteins, we studied the dependence of ATPase
`activity on ATP concentration at 37 °C by the enzyme coupling method. In the presence of poly(dT) or poly(dC) as a ssDNA, ttRecA showed
`cooperativity at 37 °C ( Fig. 2B) as well as at 65 °C ( Fig. 2A). The kinetic parameters at 37 °C were then determined ( Fig. 2B, inset) in the same
` and the Hill coefficient in the presence of poly(dT) were 0.34 m M, 0.28·s and 2.1 at 37 °C ( Table 1).
`way as those at 65 °C. The values of K , k
`–1
`cat
`The K and k
` values were increased with temperature. The cooperativity, n , was almost unaltered with temperature. Thermodynamic
`cat
`parameters for the fast binding step from E + S to ES (∆ G °, ∆ H ° and ∆ S °) and the rate-determining step from ES to ES (∆ G , ∆ H and ∆ S )
`≠
`≠
`≠
`≠
`at 37 °C were obtained from the above kinetic parameters and were combined to construct the energy profiles ( Fig. 3) [32, 33], assuming that ∆ H
`and ∆ H were constant between 37 °C and 65 °C.
`≠
`
`Figure 2.
`Open in figure viewer
`
`ATPase activities of ttRecA under various conditions. (A) ATPase assays were performed by the TLC method at 65 °C as described in
`Experimental procedures. The reaction mixture contained 2.5 µM ttRecA and 100 µM poly(dT). (B) ATPase assays were performed by the
`enzyme coupling method at 37 °C as described in Experimental procedures. The reaction mixture contained 5 µM ttRecA and 200 µM
`poly(dT) (solid circles) or poly(dC) (clear circles). Substrate dissociation constant ( K ), turnover number ( k
`) and Hill coefficient ( n ) for
`hydrolysis of ATP were determined from the insets in the figures (see text for details).
`
`cat
`
`Table 1.  Steady-state kinetic parameters for ATP hydrolysis catalyzed by RecA protein.
`
`Temperature (°C)
`
`ttRecA
`
`ecRecA
`
`65
`
`37
`
`37
`
`37
`
`37
`
`ssDNA
`
`poly(dT)
`
`poly(dT)
`
`poly(dC)
`
`poly(dT)
`
`1.4
`
`0.34
`
`0.29
`
`0.10
`
`φX174 ssDNA
`
`0.038
`
`K (m M)
`a
`
`k
`
`(s )
`−1
`
`cat
`
`b
`
`n
`
`k
`
`cat
`
`/ K (s ·M )
`−1
`−1
`
`References
`
`0.60
`
`0.28
`
`0.34
`
`0.35
`
`0.17
`
`1.9
`
`2.1
`
`2.0
`
`6.6
`
`3.3
`
`4.3 × 10
`
`2
`
`8.2 × 10
`
`2
`
`1.2 × 10
`
`3
`
`3.5 × 10
`
`3
`
`4.5 × 10
`
`3
`
`This work
`
`c
`
`This work
`
`This work
`
`(15)
`
`(31)
`
`a
`
`b
`c
`K, Substrate dissociation constant. n, Hill coefficient. All parameters for ttRecA protein were determined from
`
`Fig. 2
`.
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`Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
`
`http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00044.x/full
`
`Figure 3.
`Open in figure viewer
`
`Reaction profile for ATP hydration reaction by ttRecA at 37 °C. Changes in free energy (A), enthalpy (B) and entropy (C) were
`calculated from the values in Table 1. E, ttRecA; S, ATP; ES, ttRecA-ATP complex; ES , transition state of ES complex. The change of
`≠
`free energy (∆ G °) and enthalpy (∆ H °) for the fast binding step are calculated from – RT ln(1/ K ), and   R d(ln(1/ K ))/d(1/ T ), respectively.
`The changes in the activation free energy (∆ G ) and enthalpy (∆ H ) for the rate-determining step are RT (ln( k T / h ) – ln k
`), and
`≠
`≠
`B
`cat
`– R d(ln k
`)/d(1/ T ) – RT . R is the gas constant; T , the absolute temperature; k , the Boltzmann constant; and h , the Planck
`B
`constant.
`
`cat
`
`Effect of DNA on ATPase activity
`
`As shown above, ttRecA had ssDNA-dependent ATPase activity. To examine the DNA specificity for the activity of the protein, various kinds of
`ssDNA or dsDNA were added to the reaction mixture, and the resulting ATPase activities were measured at pH 7.5. At higher temperature (65 °C),
`all the ssDNAs we tested stimulated the ATPase activity, but dsDNAs did not ( Fig. 4A). The order of stimulating efficiency was:
`poly(dC) > poly(dT) > M13 ssDNA > poly(dA) >> M13 dsDNA, poly(dA)·poly(dT). To clarify whether or not the DNA specificity for the ATPase activity
`would be changed by temperature, an ATPase assay by the enzyme coupling method was performed at 37 °C. As shown in Fig. 4B, the dsDNAs
`also could not stimulate the ATPase activity at this temperature. Among the ssDNAs, poly(dC) and poly(dT) were able to stimulate the ATPase
`activity at 37 °C as well as at 65 °C. The stimulating ability of other ssDNAs, poly(dA) and M13 ssDNA, decreased largely at 37 °C than at 65 °C (
`Fig. 4A,B). The order of stimulating ability was: poly(dC) >> poly(dT) >> M13 ssDNA, poly(dA) > M13 dsDNA, poly(dA)·poly(dT). In the case of
`ecRecA, the observed rate of ATP hydrolysis generally increases linearly until sufficient DNA is present, as the concentration of ssDNA suggests
`tight binding between ecRecA and ssDNA [3, 31]. As shown in Fig. 4, the rate of ATP hydrolysis by ttRecA increases gradually as the concentration
`of ssDNA rises, suggesting loose binding. To quantify the binding intensity between ttRecA and various kinds of DNA, we assume that the velocity of
`ATP hydrolysis is in proportional to the concentration of the ttRecA–DNA complex, and calculate the apparent dissociation constant between them
`by curve fitting, as described in Experimental procedures. The results were summarized in Table 2, and the values were classified into three groups.
`The ttRecA bound strongly to poly(dC) and poly(dT) at both 65 °C and 37 °C. It bound to poly(dA) and M13 ssDNA with lower affinity at 37 °C than
`at 65 °C, and showed little binding to dsDNAs at both temperatures.
`
`Figure 4.
`Open in figure viewer
`
`The concentration dependence of various kinds of DNA on the ATPase activity of ttRecA. The assay procedures were described in
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`Characterization of thermostable RecA protein and analysis of its interaction with single-strand...
`
`http://onlinelibrary.wiley.com/doi/10.1046/j.1432-1327.1999.00044.x/full
`
`Experimental procedures. (A) ATPase activity at 65 °C was measured by the TLC method. The concentration of ttRecA was 2.5 µM. (B)
`ATPase activity at 37 °C was measured by the enzyme coupling method. The concentration of ttRecA was 5 µM. Clear circles, poly(dC);
`solid circles, poly(dT); solid squares, poly(dA); clear squares, M13 ssDNA; clear triangles, poly(dA)· poly(dT); solid triangles, M13 dsDNA.
`
`Table 2.  Apparent dissociation constants between ttRecA protein and DNA. All parameters were determined from Fig. 4.
`ND, not determined.
`
`DNA
`
`poly(dC)
`
`poly(dT)
`
`poly(dA)
`
`M13 ssDNA
`
`poly(dA)·poly(dT)
`
`M13 dsDNA
`
`Temperature (°C)
`
`65
`
`µM
`
`7.6
`
`11
`
`39
`
`26
`
`310
`
`280
`
`37
`
`µM
`
`31
`
`38
`
`ND
`
`ND
`
`ND
`
`ND
`
`Strand exchange activity of ttRecA
`
`As shown above, the ATPase activity of ttRecA in the presence of M13 ssDNA was observed at 65 °C but not at 37 °C ( Fig. 4). It has been reported
`that RecA protein from Thermus aquaticus , which is an extremely thermophilic bacterium closely related to T. thermophilus , promotes homologous
`DNA strand exchange at 65 °C [30]. To study the relationship between the ATPase and homologous DNA strand exchange activities, we carried out
`a strand exchange assay at 37 °C and 65 °C. The substrate DNAs were linearized M13mp18 dsDNA and viral circular M13mp18 ssDNA. As shown
`in Fig. 5A, ttRecA promoted homologous DNA strand exchange at 65 °C like that of T. aquaticus . The strand exchange reaction required ATP
`hydrolysis because it could not progress without an ATP regeneration system (data not shown). On the other hand, the strand exchange product
`was not observed at 37 °C ( Fig. 5B). No strand-transferred product was observed after incubation of the reaction mixture for 90 min. These results
`agree well with the ATPase activity in the presence of M13 ssDNA.
`
`Figure 5.
`Open in figure viewer
`
`Homologous DNA strand exchange by ttRecA. Reaction mixtures contained 60 µM M13mp18 ssDNA, 30 µM M13mp18 dsDNA, 10 m M
`MgCl , 20 m M ATP, ATP-regenerating system and ttRecA. (A) The reactions were carried out at 65 °C for 30 min with various
`2
`concentrations of ttRecA. Lanes 1 and 2 are markers which are circular M13 ssDNA and linear M13 dsDNA, respectively. Lanes 3–7
`contained 0, 1, 2, 5 or 10 µM ttRecA, respectively. (B) The reaction mixtures containing 10 µM ttRecA were incubated with DNAs at 37 °C
`(lane 3) or 65 °C (lane 4) for 30 min. Lanes 1 and 2 are circular M13 ssDNA and linear M13 dsDNA, respectively. Abbreviations: P, nicked
`circular duplex DNA product; ss, circular M13 ssDNA; ds, linearized M13 dsDNA.
`
`Nucleoprotein complex between ttRecA and ssDNA
`
`ecRecA forms nucleoprotein filament with ssDNA and then the ATPase activity appears [1–3]. As indicated above, ttRecA showed poly(dA) or M13
`ssDNA-dependent ATPase activity only at 65 °C but little at 37 °C. We tried to elucidate why the DNA dependency of the ATPase activity changed
`with temperature. One possibility is that ttRecA lost the activity to bind to poly(dA) or M13 ssDNA at lower temperatures. To clarify whether a
`nucleoprotein complex was formed by ttRecA and ssDNA at each temperature, electron microscopy was carried out. As shown in Fig. 6, ttRecA was
`able to form nucleoprotein complex with all the ssDNAs in the presence of ATP at both 37 °C and 65 °C. This indicated that the DNA-binding activity
`of ttRecA was not affected by temperature. A similar nucleoprotein filament was observed using ATP-γ-S instead of ATP (data not shown).
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`Characte