(Received for publication, May 2, 1995; and in revised form, June 29, 1995)
From the
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 = 70 µM) and purine
riboside triphosphate (PTP) (S
= 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
= 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
(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
value
of a nucleoside triphosphate and the conformational state of the
recA-ssDNA complex, with an S
of 100-120 µM or lower required for stabilization of the strand exchange-active
conformation.
The recA protein of Escherichia coli (M 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 (
)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
. 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(
)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
values of 100 µM or lower, function as
cofactors for the strand exchange reaction. ITP and GTP, which have
S
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
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
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
(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(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 ATP
S
(70%); since ADP and ATP
S 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 ATP
S; 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 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.
Each of the three nucleoside triphosphates was
hydrolyzed by the [H163W]recA protein with a turnover number (V/[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
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)).
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 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
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
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, 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).
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, 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). ()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.
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 GTP
S 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 ATP
S and GTP
S have similar
effects on the [H163W]recA-ssDNA complex.
As reported above (Table 1), the S(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
(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 values varied considerably with pH; the S
(PTP)
increased from 85 µM at pH 6.5 to 130 µM at
pH 8.2, and the S
(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
, 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
(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
, 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
(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
(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(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
(PTP) to 130 µM at pH 8.2 leads to an
inability of PTP to function as a cofactor for the strand exchange
reaction.
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 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
values of 110
µM or lower, support the active conformational state of
the complex, whereas ITP, with an S
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
(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
(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
value of a nucleoside triphosphate and the
conformational state of the recA-ssDNA complex, with an S
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 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 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
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 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 GTP
S able to induce a
level of Trp-163 fluorescence quenching identical to that obtained with
ATP
S. 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
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
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
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
values greater than 120 µM do not bind with
sufficient affinity to induce isomerization. Experiments to distinguish
between these mechanistic alternatives are in progress.