©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Spectroscopic Demonstration of a Linkage between the Kinetics of NTP Hydrolysis and the Conformational State of the recA-Single-stranded DNA Complex (*)

(Received for publication, May 2, 1995; and in revised form, June 29, 1995)

Einar Stole Floyd R. Bryant (§)

From the Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We recently constructed a mutant recA protein in which His-163 was replaced by a tryptophan residue; the [H163W]recA protein is functionally identical to the wild-type protein, and the Trp-163 side chain serves as a reporter group for the conformational transitions of the [H163W]recA-single-stranded DNA (ssDNA) complex. We have now examined the fluorescence properties of the [H163W]recA-ssDNA complex in the presence of a series of alternate nucleoside triphosphate cofactors. Under standard conditions (pH 7.5), ATP (S(0.5) = 70 µM) and purine riboside triphosphate (PTP) (S(0.5) = 110 µM) effect a 44% decrease in Trp-163 fluorescence and are active as cofactors for the DNA strand exchange reaction. In contrast, ITP (S(0.5) = 400 µM) elicits only a 20% decrease in Trp-163 fluorescence (a level identical to that observed with the nucleoside diphosphates ADP, PDP, and IDP) and is inactive as a strand exchange cofactor. If the S(0.5) (PTP) is increased to 130 µM (by increasing the pH of the reaction solution), the PTP-mediated quenching of Trp-163 fluorescence decreases to 20%, and PTP becomes inactive as a strand exchange cofactor. These results provide direct evidence for a linkage between the S(0.5) value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S(0.5) of 100-120 µM or lower required for stabilization of the strand exchange-active conformation.


INTRODUCTION

The recA protein of Escherichia coli (M(r) 37,842, 352 amino acids) is essential for homologous genetic recombination and for the postreplicative repair of damaged DNA. The purified recA protein will promote a variety of DNA pairing reactions that presumably reflect in vivo recombination functions. The most extensively investigated DNA pairing activity is the ATP-dependent three-strand exchange reaction, in which a circular ssDNA (^1)molecule and a homologous linear dsDNA molecule are recombined to yield a nicked circular dsDNA molecule and a linear ssDNA molecule. This reaction proceeds in three phases. In the first phase, the circular ssDNA substrate is coated with recA protein to form a presynaptic complex; this complex catalyzes the hydrolysis of ATP to ADP and P(i). In the second phase, the presynaptic complex interacts with a dsDNA molecule, the homologous sequences are brought into register, and pairing between the circular ssDNA and the complementary strand from the dsDNA is initiated. In the third phase, the complementary linear strand is completely transferred to the circular ssDNA by unidirectional branch migration to yield the nicked circular dsDNA and displaced linear ssDNA products (Roca and Cox, 1990; Kowalczykowski, 1991).

The presynaptic complex formed between recA protein and ssDNA is the active recombinational entity in the strand exchange reaction. The recA protein binds cooperatively to ssDNA, forming a right-handed helical protein filament with one recA monomer per four nucleotides of ssDNA and six recA monomers per turn of the filament. In the absence of nucleotide cofactor or in the presence of ADP, the helical filament adopts a ``collapsed'' or ``closed'' conformation (helical pitch 65 Å) that is inactive in strand exchange. In the presence of ATP or the nonhydrolyzable ATP analog, ATPS, however, the filament assumes an ``extended'' or ``open'' conformation (helical pitch 95 Å) that is active in strand exchange (Egelman, 1993).

We have been examining the mechanism of the nucleotide cofactor-mediated isomerization of the recA-ssDNA complex and have identified an apparent dependence of the isomerization on the S(0.5)(^2)value of the cofactor. In studies of the wild-type recA protein with alternate nucleoside triphosphates, we showed that the structurally related nucleoside triphosphates, ATP, purine riboside triphosphate (PTP), ITP, and GTP (Fig. 1), are each hydrolyzed by the recA protein with the same turnover number (18 min). However, only ATP and PTP, which have S(0.5) values of 100 µM or lower, function as cofactors for the strand exchange reaction. ITP and GTP, which have S(0.5) values above 100 µM, are inactive as strand exchange cofactors (Menge and Bryant, 1992). To account for these results, we proposed that nucleoside triphosphates with S(0.5) values greater than 100 µM may be intrinsically unable to stabilize the open conformational state of the recA-ssDNA complex and will therefore be nonfunctional as cofactors for the strand exchange reaction. This proposal was supported by our studies of the mutant [H163A]recA protein. The [H163A]recA protein is able to carry out the ATP-dependent strand exchange reaction at pH 6.0-6.8 but is inactive at higher pH values. The strand exchange activity correlates directly with a pH-dependent change in the S(0.5) value for ATP, with the mutant protein being active in strand exchange (and in isomerization to the open conformational state) at pH values where the S(0.5)(ATP) is 100 µM or lower (Muench and Bryant, 1991; Pinsince et al., 1993; Meah and Bryant, 1993).


Figure 1: Purine ring structure of alternate nucleoside triphosphates. The structures correspond to the purine rings of adenosine triphosphate (A), purine riboside triphosphate (P), inosine triphosphate (I), and guanosine triphosphate (G).



To monitor the conformational transitions of the recA protein directly, we recently constructed a mutant recA protein in which His-163 in the loop 1 region (residues 157-163) of the protein (Story et al., 1992) was replaced by a tryptophan reporter group. The [H163W]recA protein catalyzes ATP hydrolysis with the same turnover number as does the wild-type protein and has a S(0.5)(ATP) value lower than 100 µM at pH 7.5. Accordingly, the [H163W]recA protein is fully functional in the strand exchange reaction under standard reaction conditions. In addition, the fluorescence properties of Trp-163 are very sensitive to the binding of nucleotide cofactors to the [H163W]recA-ssDNA complex. The fluorescence of Trp-163 is modestly quenched by ADP (21%) and strongly quenched by ATPS (70%); since ADP and ATPS stabilize the closed and open conformations of the recA-ssDNA complex, respectively, these levels of fluorescence quenching likely reflect differences in the fluorescence properties of Trp-163 in these two states. ATP effects a level of Trp-163 fluorescence quenching (44%) that is intermediate in intensity between that observed with ADP and ATPS; this level of quenching may represent a mixture of fluorescence signals from a highly quenched ATP state and lesser quenched ADP state, which coexist in the multimeric complex during steady-state ATP hydrolysis (Stole and Bryant, 1994).

The results described above demonstrate that the conformational transitions of the [H163W]recA-ssDNA complex can be followed by monitoring the fluorescence of the Trp-163 reporter group. In this report, we examine the fluorescence properties of the [H163W]recA protein in the presence of a series of alternate nucleoside triphosphate cofactors having a range of S(0.5) values above and below 100 µM. These studies have allowed us to evaluate directly the conformational state of the [H163W]recA-ssDNA complex in the presence of these alternate cofactors.


EXPERIMENTAL PROCEDURES

Materials

Wild-type and [H163W]recA proteins were prepared as previously described (Bryant, 1988; Stole and Bryant, 1994). ATP, ADP, ITP, and IDP were from Sigma. PTP was prepared from purine riboside (Sigma) using the general procedure described by Mishra and Broom(1991). [^3H]ATP and [-P]ATP were from ICN. [-P]ITP and [-P]PTP were prepared from the corresponding nucleoside diphosphates using [-P]ATP and nucleoside diphosphate kinase (Sigma) as previously described (Menge and Bryant, 1992). E. coli SSB was from Pharmacia LKB Biotechnology. Unlabeled circular X ssDNA ((+)strand) and linear X dsDNA were prepared as previously described (Cox and Lehman, 1981). Single- and double-stranded DNA concentrations were determined by absorbance at 260 nm using the conversion factors 36 and 50 µg/ml/A, respectively. All DNA concentrations are expressed as total nucleotides.

Fluorescence Analysis

Fluorescence studies were conducted on a SLM Aminco-Bowman series 2 luminescence spectrometer equipped with a variable temperature holder. The concentrations of stock solutions of the purified wild-type and [H163W]recA proteins were determined by absorbance using the extinction coefficients 0.59 and 1.2 A mg ml, respectively (Stole and Bryant, 1994). Each fluorescence emission spectrum was corrected for background fluorescence by subtracting the corresponding buffer spectrum. Inner filter corrections at high concentrations of DNA or nucleotide were made using the relationship F = F antilog [(A + A)/2] (valid for absorbances leq 0.1 (Lakowicz, 1983)) and were less than 6% in all cases.


RESULTS

ssDNA-dependent Hydrolysis of Alternate Nucleoside Triphosphates (pH 7.5)

The ssDNA-dependent hydrolysis of PTP and ITP by the [H163W]recA protein was analyzed under standard reaction conditions (pH 7.5, 37 °C). The steady-state kinetic parameters for the hydrolysis of each of these nucleotides, as well as those previously determined for ATP hydrolysis (Stole and Bryant, 1994), are presented in Table 1. The kinetic parameters for the hydrolysis of each of the nucleotides by the wild-type recA protein are also included in Table 1for reference (Menge and Bryant, 1992).



Each of the three nucleoside triphosphates was hydrolyzed by the [H163W]recA protein with a turnover number (V(max)/[E]) of 18 min; this value is indistinguishable from that previously obtained for the hydrolysis of these nucleotides by the wild-type recA protein (Menge and Bryant, 1992) and shows that the variations in the structure of the purine ring do not effect the intrinsic rate of NTP hydrolysis by the [H163W]recA protein. The purine ring structure does, however, affect the S(0.5) values for NTP hydrolysis by the [H163W]recA protein, which increase progressively in the order ATP (70 µM), PTP (110 µM), and ITP (400 µM); this pattern is similar to that obtained with the wild-type protein (Table 1, Menge and Bryant(1992)).

Alternate Nucleoside Triphosphates as Cofactors for the Three-strand Exchange Reaction (pH 7.5)

The ability of each of the nucleoside triphosphates to support the [H163W]recA protein-mediated three-strand exchange reaction was evaluated under standard reaction conditions (pH 7.5, 37 °C). In the three-strand exchange assay, a circular X ssDNA molecule and a linear X dsDNA molecule are recombined to form a nicked circular dsDNA molecule and a linear ssDNA molecule; the substrates and products of this reaction are readily monitored by agarose gel electrophoresis (Cox and Lehman, 1981).

As shown in Fig. 2, the [H163W]recA protein exhibited substantial strand exchange activity in the presence of either ATP or PTP (3 mM); as noted above, these nucleoside triphosphates have S(0.5) values of 110 µM or lower. In contrast, no strand exchange was detected in the presence of a saturating concentration of ITP (3 mM); this nucleoside triphosphate has a S(0.5) value that is significantly greater than 110 µM. These results are similar to those previously obtained with the wild-type protein (Menge and Bryant, 1992) and are consistent with our proposal that nucleoside triphosphates must have an S(0.5) value of approximately 100 µM (or now more precisely, 110 µM) or lower to function as cofactors for the strand exchange reaction.


Figure 2: [H163W]recA protein-promoted three-strand exchange reaction (pH 7.5). Three-strand exchange reactions were carried out as described by Cox and Lehman(1981). The reaction solutions contained 25 mM Tris-HCl (pH 7.5), 5% glycerol, 1 mM dithiothreitol, 10 mM MgCl(2), 3.3 µM circular X ssDNA, 6.6 µM linear X dsDNA, 0.3 µM SSB, 2 µM [H163W]recA protein, and 3 mM ATP, PTP, or ITP, as indicated. The reactions were initiated by the simultaneous addition of SSB and NTP after preincubation of all other components for 10 min at 37 °C. After incubation at 37 °C for 60 min, 30-µl aliquots were quenched with 4 µl of SDS (10%) and 1 µl of EDTA (500 mM), and the samples were analyzed by electrophoresis on a 0.8% agarose gel using a Tris acetate-EDTA buffer system. The substrates and products were visualized by ethidium bromide staining. II, circular duplex DNA containing a nick (form II); III, linear duplex DNA (form III).



Effect of Alternate Nucleoside Triphosphates on the Fluorescence of the [H163W]recA-ssDNA Complex

Either wild-type recA or [H163W]recA protein (1 µM) was added to X ssDNA (10 µM) under standard reaction conditions (pH 7.5, 37 °C) to form the corresponding recA-ssDNA complexes. (^3)Fluorescence emission spectra of the complexes were then obtained, using an excitation wavelength of 295 nm so that only tryptophan fluorescence would be measured (Lakowicz, 1984). As shown in Fig. 3, the wild-type recA and [H163W]recA-ssDNA complexes both have an emission maximum at 340 ± 1 nm. The fluorescence intensity of the [H163W]recA protein at the emission maximum is 70% greater than that of the wild-type recA protein, reflecting the additional tryptophan at position 163. This difference in fluorescence intensities is identical to that determined for the free proteins, indicating that the binding of ssDNA does not have a differential effect on the fluorescence intensity of the two proteins (Stole and Bryant, 1994).


Figure 3: Fluorescence emission spectra of the wild-type (WT) and [H163W]recA-ssDNA complexes. The samples contained 25 mM Tris-HCl (pH 7.5), 5% glycerol, 1 mM dithiothreitol, 10 mM MgCl(2), 10 µM X ssDNA, and 1 µM wild-type recA protein or [H163W]recA protein. The excitation wavelength was 295 nm, and the excitation and emission bandwidths were each set at 5 nm. Each fluorescence spectrum was recorded at 37 °C and corrected for background fluorescence and the Raman scattering peak by subtracting the corresponding buffer spectrum.



Various nucleotides (1 mM) were added to the preformed recA-ssDNA complexes, and the effects on the total tryptophan fluorescence of the proteins were measured. In all cases, the emission wavelength maximum of the wild-type and [H163W]recA proteins remained unchanged after the addition of these nucleotides. Furthermore, identical results were obtained with higher nucleotide concentrations, thus indicating that the fluorescence results reported below are representative of recA-ssDNA complexes that are saturated with each nucleotide. We have shown previously that Trp-163 fluorescence quenching and ssDNA-dependent ATP hydrolysis have a similar dependence on ATP concentration, indicating that the quenching of Trp-163 fluorescence by ATP is directly related to the saturation of the nucleotide binding sites in the polymeric [H163W]recA-ssDNA complex (Stole and Bryant, 1994).

As shown in Table 2, the addition (1 mM) of ATP, PTP, ITP, or any of the corresponding nucleoside diphosphates (ADP, PDP, IDP) resulted in only minor changes in the tryptophan fluorescence of the wild-type recA-ssDNA complex. This indicates that the fluorescence properties of the two intrinsic tryptophan residues of the wild-type recA protein (Trp-290 and Trp-308) are relatively insensitive to nucleotide cofactor-dependent changes in the conformation of the recA-ssDNA complex, consistent with our previous results (Stole and Bryant, 1994). (^4)In the fluorescence measurements described below, the minor effects of the various nucleotides on the fluorescence of the intrinsic tryptophan residues of the recA protein were substracted from the total change in fluorescence of the [H163W]recA protein, and only the changes in the fluorescence of the Trp-163 reporter group are discussed. There is no overlap between the fluorescence emission spectrum of the [H163W]recA protein and the absorption spectra of any of the nucleotides or the ssDNA (data not shown). Thus, the changes in Trp-163 fluorescence that are reported below are likely due to conformational changes of the protein, which alters the local environment of Trp-163, rather than to energy transfer from Trp-163 to the nucleotide cofactor or ssDNA (Lakowicz, 1984).



The effects of the various nucleoside diphosphates (1 mM) on the fluorescence of the [H163W]recA-ssDNA complex are shown in Fig. 4A and are tabulated in Table 2. In contrast to the minor effects that were observed on the intrinsic tryptophan residues, there was a 20% reduction in Trp-163 fluorescence when either PDP or IDP was added to the [H163W]recA-ssDNA complex. These values are indistinguishable from the 21% decrease in Trp-163 fluorescence that occurs when ADP is added to this complex (Table 2, Stole and Bryant(1994)), indicating that the three nucleoside diphosphates have similar effects on the recA-ssDNA complex.


Figure 4: Effect of nucleotide cofactors on the tryptophan fluorescence of the [H163W]recA-ssDNA complex. Fluorescence spectra of the [H163W]recA-ssDNA complex in the presence of either 1 mM IDP, ADP, and PDP (panelA) or 1 mM ITP, ATP, and PTP (panelB) were obtained as described in the legend to Table 2.



The effects of the various nucleoside triphosphates (1 mM) on the fluorescence of the [H163W]recA-ssDNA complex are shown in Fig. 4B and are tabulated in Table 2. A 42% decrease in Trp-163 fluorescence was observed when PTP was added to the [H163W]recA-ssDNA complex; this value is similar to the 44% decrease in Trp-163 fluorescence that occurs with ATP and indicates that PTP and ATP have similar effects on the [H163W]recA-ssDNA complex. In contrast, there was only a 20% reduction in Trp-163 fluorescence when ITP was added to the [H163W]recA-ssDNA complex; this value is identical to that obtained with each of the nucleoside diphosphates and is significantly lower than that obtained with either ATP or PTP. This suggests that in terms of the conformational state of the [H163W]recA-ssDNA complex, the effect of ITP is functionally similar to that of a nucleoside diphosphate.

Nonhydrolyzable NTP Analogs

The fluorescence results reported above suggest that when the S(0.5) value for a nucleoside triphosphate is greater than 100-120 µM (i.e. ITP, GTP), the conformational state of the [H163W]recA-ssDNA complex (as judged by Trp-163 fluorescence) is dominated by the nucleoside diphosphates that are generated by the NTP hydrolysis reaction. To explore this possibility further, the effects of two nonhydrolyzable nucleoside triphosphate analogs on the fluorescence of the [H163W]recA-ssDNA complex were examined; ATPS was selected to represent those NTPs with S(0.5) values below 100 µM, and (since ITPS is not available commercially) GTPS was chosen to represent those NTPs with S(0.5) values above 100 µM. (^5)

As shown in Table 2, the addition of ATPS resulted in a 69% decrease in the Trp-163 fluorescence of the [H163W]recA-ssDNA complex, consistent with our previous results (Stole and Bryant, 1994). An identical level of Trp-163 fluorescence quenching was obtained when GTPS was added to the [H163W]recA-ssDNA complex. Furthermore, both nonhydrolyzable analogs were able to support a limited [H163W]recA protein-mediated strand exchange reaction (data not shown), as previously reported for the wild-type protein (Menge and Bryant, 1992). These results indicate that ATPS and GTPS have similar effects on the [H163W]recA-ssDNA complex.

A pH-mediated Conformational Switch

In previous studies, we showed that the S(0.5)(ATP) for both the wild-type recA protein and the [H163A]recA protein increases with pH over the range of pH 6.0-8.4. For the wild-type protein, the S(0.5)(ATP) remains below 100 µM over this pH range, and correspondingly, the wild-type protein exhibits ATP-dependent strand exchange activity over this pH range. In the case of the [H163A]recA protein, however, the S(0.5)(ATP) increases from 40 µM at pH 6.0 to 150 µM at pH 7.5, and the pH at which S(0.5)(ATP) increases above 100 µM (pH 6.7) correlates precisely with the induction of an isomerization defect of the [H163A]recA-ssDNA complex and a loss of strand exchange activity (Muench and Bryant, 1991; Pinsince et al., 1993; Meah and Bryant, 1993).

As reported above (Table 1), the S(0.5)(PTP) for the [H163W]recA protein at pH 7.5 is 110 µM and is presumably just at the threshold for viability as a strand exchange cofactor. If our general premise is correct, we reasoned that we would be able to increase the S(0.5)(PTP) by increasing the pH of the reaction solution and that this would, in turn, lead to a collapse of the active conformational state of the [H163W]recA-ssDNA complex (as judged by Trp-163 fluorescence) and a loss of strand exchange activity. The following series of experiments was performed to test this idea.

First, the effect of pH on kinetic parameters for ssDNA-dependent hydrolysis of both ATP and PTP by the [H163W]recA protein was determined (Fig. 5A). As predicted, the S(0.5) values varied considerably with pH; the S(0.5)(PTP) increased from 85 µM at pH 6.5 to 130 µM at pH 8.2, and the S(0.5)(ATP) increased from 60 µM at pH 6.5 to 80 µM at pH 8.2. The turnover numbers for PTP and ATP hydrolysis were relatively insensitive to pH, fluctuating between 16 and 18 min over this pH range (data not shown).


Figure 5: Dependence of [H163W]recA protein functions on pH. Reaction solutions at different pH values were adjusted to a constant ionic strength by adding the appropriate concentration of NaCl; the final pH of each reaction solution was measured at 37 °C. PanelA, [H163W]recA protein-catalyzed ssDNA-dependent ATP and PTP hydrolysis. The reaction solutions contained either 25 mM Tris-HCl (pH 7.0-8.2) or 25 mM BisTris-HCl (pH 6.0-6.9), 30 µM X ssDNA, 10 mM MgCl(2), 1.0 µM [H163W]recA protein, and various concentrations of ATP (closedcircles) or PTP (opensquares). The reactions were initiated by addition of protein and were carried out at 37 °C. The S(0.5)(NTP) values at each pH were determined from plots of the dependence of the initial rates of NTP hydrolysis on NTP concentration. NTP hydrolysis was measured using a thin-layer chromatography method as previously described (Weinstock et al., 1979). PanelB, ATP and PTP-mediated quenching of Trp-163 fluorescence. The reaction solutions contained 25 mM Tris-HCl (pH 7.5-8.3), 10 mM MgCl(2), 10 µM X ssDNA, 1.0 µM [H163W]recA protein, and 1.0 mM ATP (closedcircles) or PTP (opensquares). Fluorescence emission spectra were obtained as described in the legend to Table 2. PanelC, [H163W]recA protein-promoted three-strand exchange reaction (pH 8.2). Strand exchange reactions were carried out as described in the legend to Fig. 1, except that the reaction buffer consisted of Tris-HCl (pH 8.2).



Next, the effect of ATP and PTP on the fluorescence of the [H163W]recA-ssDNA complex was measured as a function of pH. As shown in Fig. 5B, the addition of PTP resulted in a 40% decrease in Trp-163 fluorescence over the range of pH 7.5-7.9. At pH 8.0 and above, however, the fluorescence quenching by PTP decreased abruptly to 20%, a value indistinguishable from that of PDP (or any of the other nucleoside diphosphates). These results are consistent with the idea that the modest elevation of the S(0.5)(PTP) at higher pH leads to an inability of PTP to stabilize the strand exchange-active conformation of the [H163W]recA-ssDNA complex. In contrast to the results obtained with PTP, the addition of ATP resulted in a 42-44% decrease in Trp-163 fluorescence at all pH values between 7.5 and 8.3, consistent with the observation that the S(0.5)(ATP) remains below 100 µM across this pH range (Fig. 5B).

Finally, the three-strand exchange activity of the [H163W] recA protein was examined at pH 8.2 in the presence of either ATP or PTP. As shown in Fig. 5C, ATP supported a level of [H163W]recA protein-mediated strand exchange at pH 8.2 that was comparable to that observed at pH 7.5; this result is again consistent with the fact that the S(0.5)(ATP) remains below 100 µM at pH 8.2. In contrast, the [H163W]recA protein exhibited no detectable strand exchange activity in the presence of PTP at pH 8.2. These results are consistent with the idea that the pH-mediated elevation of the S(0.5)(PTP) to 130 µM at pH 8.2 leads to an inability of PTP to function as a cofactor for the strand exchange reaction.


DISCUSSION

The structurally related nucleoside triphosphates ATP, PTP, and ITP are each hydrolyzed by the [H163W]recA protein with the same turnover number. However, only ATP and PTP support the [H163W]recA protein-promoted three-strand exchange reaction; ITP is ineffective as a cofactor for this reaction. The fluorescence properties of the [H163W]recA protein clearly reveal differences in the conformation of [H163W]recA-ssDNA complex in presence of these alternate nucleoside triphosphates. PTP effects a level of Trp-163 fluorescence quenching (42%) that is indistinguishable from that observed with ATP; this level is presumably indicative of the strand exchange-active, open conformation of the recA-ssDNA complex. ITP, in contrast, elicits only a 21% decrease in Trp-163 fluorescence; this level is identical to that observed with all nucleoside diphosphates (ADP, PDP, IDP), and is characteristic of the strand exchange-inactive, closed conformation of the recA-ssDNA complex.

These results provide direct spectroscopic evidence to support our earlier proposal that nucleoside triphosphates with S(0.5) values greater than 100 µM (and now more precisely, 110-120 µM) will be unable to support the strand exchange-active conformational state of the recA-ssDNA complex and will therefore be inactive as cofactors for the strand exchange reaction; both ATP and PTP, which have S(0.5) values of 110 µM or lower, support the active conformational state of the complex, whereas ITP, with an S(0.5) value greater than 110 µM, does not. This idea is reinforced by the demonstration here of a pH-mediated conformational switch when PTP is employed as a cofactor. When the S(0.5)(PTP) is increased from 110 to 130 µM (by increasing the pH of the reaction solution from 7.5 to 8.2), the PTP-mediated quenching of Trp-163 fluorescence decreases from 40 to 20% (a value equivalent to that of the nucleoside diphosphates), and PTP becomes inactive as a strand exchange cofactor. Under the same conditions, the S(0.5)(ATP) increases to only 80 µM, the ATP-mediated quenching remains at 44%, and ATP continues to function as a strand exchange cofactor. These results clearly demonstrate the linkage between the S(0.5) value of a nucleoside triphosphate and the conformational state of the recA-ssDNA complex, with an S(0.5) value of 100-120 µM or less being required for stabilization of the active conformation.

Our fluorescence results indicate further that the effect of nucleoside triphosphates with S(0.5) values greater than 100-120 µM on the recA-ssDNA complex is similar to that of a nucleoside diphosphate. This suggests that for these nucleoside triphosphates, the conformational state of the [H163W]recA-ssDNA complex is determined by the nucleoside diphosphates that are generated by the NTP hydrolysis reaction. Since the effects are apparent before a significant concentration of nucleoside diphosphate has accumulated in solution, it is likely that they arise from the nascent nucleoside diphosphates as they are generated in the individual active sites of the multimeric recA-ssDNA complex by the ongoing NTP hydrolysis reaction.

In considering the influence of nascent NDP molecules on the conformational state of the recA-ssDNA complex, it is significant that the complex is able to maintain the open conformation in the presence of ATP, even though some of the recA monomers in the complex at any instant must be bound to ADP molecules that are being generated in the NTP hydrolysis active sites. To account for this observation, it has been suggested that the subset of recA monomers that are bound to unhydrolyzed ATP molecules are able to stabilize the entire complex in the ATP-induced conformational state (Lee and Cox, 1990a, 1990b). Our spectroscopic results indicate, more generally, that when the S(0.5) value of a nucleoside triphosphate is 100-120 µM or less, the recA-ssDNA complex will be able to sustain the strand exchange-active, open conformation during ongoing NTP hydrolysis. When the S(0.5) value is greater than 120 µM, however, our results indicate that the recA-ssDNA complex adopts a conformation that is functionally equivalent to the NDP-stabilized closed conformation. Although NTP hydrolysis continues from this complex (demonstrating that a sustained open conformation is not required for NTP hydrolysis), the complex is inactive in the strand exchange reaction.

The proposal that nucleoside triphosphates with high S(0.5) values are unable to allosterically stabilize the active conformation of the recA-ssDNA complex against destabilization by the NDP molecules produced by the NTP hydrolysis reaction is consistent with the results that were obtained with nonhydrolyzable NTP analogs. In contrast to differential effects that were observed with the various alternate hydrolyzable nucleoside triphosphates, the nonhydrolyzable analogs have similar effects on the conformation of the [H163W]recA-ssDNA complex, with GTPS able to induce a level of Trp-163 fluorescence quenching identical to that obtained with ATPS. This result suggests that each of the alternate hydrolyzable nucleoside triphosphates may actually induce isomerization of the recA-ssDNA complex to the active conformational state when they initially bind to the complex. In this case, if the S(0.5) value of the nucleoside triphosphate is 100-120 µM or lower, the complex remains in the active conformational state during ongoing NTP hydrolysis, whereas if the S(0.5) value is greater than 120 µM, the complex collapses to a state resembling the inactive, closed conformation. An alternate possibility, however, is that a nucleoside triphosphate must bind with high affinity to induce isomerization of the recA-ssDNA complex to the active conformational state. In this case, nucleoside triphosphates with S(0.5) values of 100-120 µM or lower (as well as the nonhydrolyzable NTP analogs) bind tightly enough to the complex to induce isomerization, whereas those with S(0.5) values greater than 120 µM do not bind with sufficient affinity to induce isomerization. Experiments to distinguish between these mechanistic alternatives are in progress.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1 GM 36516 (to F. R. B.) and Postdoctoral Grant F32 GM16284 (to E. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. 410-955-3895.

(^1)
The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; X, bacteriophage X174; SSB, E. coli SSB protein; PTP, purine riboside triphosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate; ATPS, adenosine 5`-O-(thiotriphosphate); ITPS, inosine 5`-O-(thiotriphosphate).

(^2)
S(0.5) is the substrate concentration required for half-maximal velocity.

(^3)
We previously showed that 10 µM is the minimum concentration of X ssDNA that is able to fully complex 1 µM [H163W]recA protein (Stole and Bryant, 1994).

(^4)
Both Trp-290 and Trp-308 are located in a small carboxyl-terminal domain (amino acids 270-352) that protrudes from the surface of the recA protein filament. This domain may not be affected appreciably by conformational changes of the recA-ssDNA complex.

(^5)
We previously showed that GTP and ITP have similar effects on the properties of the recA-ssDNA complex (Menge and Bryant, 1992). However, the S(0.5) value for GTP is even higher than that for ITP, and thus, very high concentrations of GTP are required to saturate the [H163W]recA-ssDNA complex. Because this leads to prohibitively high inner filter effects, the effects of GTP on the fluorescence properties of the [H163W]recA protein are not included in this manuscript.


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