(Received for publication, February 9, 1995; and in revised form, December 23, 1995)
From the
RecA protein promotes a limited DNA strand exchange reaction,
without ATP hydrolysis, that typically results in formation of short
(1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid
DNA is extended in a reaction that can be coupled to ATP hydrolysis.
When ATP is hydrolyzed, the extension phase is progressive and its rate
is 380 ± 20 bp min at 37 °C. A single
RecA nucleoprotein filament can participate in multiple DNA strand
exchange reactions concurrently (involving duplex DNA fragments that
are homologous to different segments of the DNA within a nucleoprotein
filament), with no effect on the observed rate of ATP hydrolysis. The
ATP hydrolytic and hybrid DNA extension activities exhibit a dependence
on temperature between 25 and 45 °C that is, within experimental
error, identical. This provides new evidence that the two processes are
coupled. Arrhenius activation energies derived from the work are 13.3
± 1.1 kcal mole
for DNA strand exchange, and
14.4 ± 1.4 kcal mole
for ATP hydrolysis
during strand exchange. The rate of branch movement in the extension
phase (base pair min
) is related to the k
for ATP hydrolysis during strand exchange
(min
) by a factor equivalent to 18 bp throughout the
temperature range examined. The 18-base pair factor conforms to a
quantitative prediction derived from a model in which ATP hydrolysis is
coupled to a facilitated rotation of the DNA substrates. RecA filaments
possess an intrinsic capacity for DNA strand exchange, mediated by
binding energy rather than ATP hydrolysis, that is augmented by an
ATP-dependent molecular motor.
The RecA protein of Escherichia coli promotes a DNA
strand exchange reaction that mimics key steps in recombinational DNA
repair and homologous recombination. A RecA nucleoprotein filament
forms on a single-stranded DNA circle. The bound single strand is
aligned with a homologous linear duplex DNA, and strand exchange leads
to the formation of a hybrid circular duplex DNA with a nick in one
strand (Cox, 1993, 1994; Kowalczykowski and Eggleston, 1994; Roca and
Cox, 1990; West, 1992). The RecA nucleoprotein filament exhibits an
ATPase activity with a monomer k of about 30
min
when bound to ssDNA (
)(Cox, 1994).
The RecA protein is found in essentially all bacteria (Roca and Cox,
1990). The paradigm extends to eukaryotes, with the RAD51 protein of
yeast now proving to be a true RecA homologue in both structure and
function (Ogawa et al., 1993; Sung, 1994).
NTPases find
cellular employment as motors, timing devices, or recycling functions
(Alberts and Miake-Lye, 1992). The ATPase activity of RecA protein has
come to be viewed largely as a recycling function, involved primarily
in the disassembly of the RecA filament or the recycling of monomers
within a filament (Menetski et al., 1990; Alberts and
Miake-Lye, 1992; West, 1992; Rehrauer and Kowalczykowski, 1993;
Kowalczykowski and Eggleston, 1994; Kowalczykowski and Krupp, 1995).
RecA nucleoprotein filaments have an intrinsic capacity to promote DNA
strand exchange without hydrolyzing ATP, as shown by the limited DNA
strand exchange reactions observed with ATPS (Menetski et
al., 1990), with a RecA mutant protein which binds but does not
hydrolyze ATP (RecA K72R) (Rehrauer and Kowalczykowski, 1993), and in
the presence of ADP-AlF
(Kowalczykowski
and Krupp, 1995).
The recycling function is readily demonstrable,
but provides an incomplete explanation for the RecA ATPase activity.
Reported filament disassembly reactions are filament end-dependent and
rarely account for more than a minute fraction of the ATP hydrolyzed in
a RecA nucleoprotein filament (Lindsley and Cox, 1989, 1990a). When
homologous duplex DNA is added to RecA nucleoprotein filaments to
initiate strand exchange, the monomer k for ATP
hydrolysis drops abruptly to about 20 min
, and then
is invariant during the subsequent strand exchange reaction (Schutte
and Cox, 1987). This ATP hydrolysis has not been accounted for by any
model which assigns the primary role of the ATPase to dissociation of
RecA monomers.
A more complete view arises from a closer examination of the limitations RecA-mediated DNA strand exchange reaction in the absence of ATP hydrolysis. ATP hydrolysis alters the DNA strand exchange reaction fundamentally, conferring properties important to the function of RecA protein in recombinational DNA repair (Clark and Sandler, 1994; Cox, 1993, 1994). When RecA hydrolyzes ATP, the DNA strand exchange reaction becomes unidirectional (Jain et al., 1994; Shan et al., 1996), generates longer hybrid DNA products much more efficiently (Jain et al., 1994), bypasses a range of structural barriers in either DNA substrate (Rosselli and Stasiak, 1991; Kim et al., 1992a; Shan et al., 1996), and accommodates 4 DNA strands (Kim et al., 1992b; Shan et al., 1996). We have suggested that the undirected DNA strand exchange occurring in the absence of ATP hydrolysis constitutes a distinct phase of the normal reaction (Shan et al., 1996). The resulting nascent hybrid DNA, typically 1-2 kilobase pairs in length, is extended in a subsequent reaction phase that is greatly facilitated by ATP hydrolysis.
If the two processes are coupled, the rates of hybrid DNA extension and ATP hydrolysis during strand exchange should be correlated in predictable ways. In particular, the two processes should exhibit a similar dependence on temperature. The effects of temperature have provided evidence for the coupling of NTP hydrolysis to a number of motor functions. An interesting example was reported by Koshland and co-workers, who used coincident Arrhenius plots to relate the myosin ATPase activity to the walking rate of ants (Levy et al., 1959).
To date, there has been no systematic attempt to measure the rate of DNA strand exchange with different substrates or examine the temperature dependence of RecA reactions. The existing data set is probably insufficient even to establish rigorously that there is an intrinsic and characteristic rate of branch movement during DNA strand exchange that can be associated with ATP hydrolysis. In this report, we develop an improved method for estimating the rate of branch movement during DNA strand exchange and use it to establish a closer experimental correlation between RecA-mediated DNA strand exchange and ATP hydrolysis.
Whereas accurate methods are available to monitor
rates of ATP hydrolysis, obtaining a reliable estimate of the rate of
branch movement in DNA strand exchange is problematic. Published
estimates, which vary over at least a 5-fold range, were obtained from
average rates of hybrid DNA formation in a large population of
molecules that initiate the reaction asynchronously (Roca and Cox,
1990). Once initiated, secondary interactions with additional DNA
molecules in solution might interfere with branch movement in some
complexes (coaggregation) (Tsang et al., 1985), exaggerating
the apparent asynchrony. We dealt with these uncertainties by focusing
only on the formation of the circular duplex DNA product (Fig. 1A). In a reaction involving our 8266-bp DNA
substrates, this product first appears after a significant lag and
thereafter accumulates until most of the substrate DNA has been
converted (Fig. 1B). We quantified the bands in
experiments such as that in Fig. 1B, and extrapolated
to the time point when the first product appeared (Fig. 1C, arrow). The time required to
generate the first product should provide the most reliable estimate of
the intrinsic rate of branch movement in a reacting complex, reflecting
an early initiation and a minimization of any subsequent inhibition
from interactions with other DNA molecules. The apparent rate of DNA
branch movement (in bp min) is defined as the length
of the duplex DNA substrate divided by the lag (in minutes) before the
first appearance of products.
Figure 1: Measuring the rate of DNA strand exchange. Reactions were carried out as described under ``Materials and Methods.'' A, the three-strand exchange reaction used in these experiments. The substrates are a single-stranded circle and a linear duplex DNA derived from M13mp8.1037 (8266 bp). The linear duplex DNA was obtained by cleaving supercoiled M13mp8.1037 DNA with AlwNI. B, a typical reaction at 43.5 °C; P, circular duplex DNA products; S, linear duplex DNA substrate; ss, circular single-stranded DNA; I, reaction intermediates. The first lane contains supercoiled and nicked circular duplex M13 mp8.1037 markers. Lanes 2-18 show the reaction at 0, 3, 6, 8, 10, 12, 14, 16, 18, 21, 24, 27, 30, 35, 40, 50, and 60 min, respectively. C, quantitation of the P and S bands. The arrow represents the extrapolated point at which the first products appeared (11 min).
In practice, experiments with relatively few time points were used to roughly characterize the product generation curve, followed by a more detailed time course with time points concentrated near the anticipated lag time to define it as accurately as possible. The error in most lag measurements is about ±1 min. This translates into an error of about ±10% for a typical experiment (the error increases for shorter duplex substrates). Experiments were discarded when excessive scatter in the product generation curve precluded a reliable extrapolation. All of the strand exchange experiments used in this study generated products at levels greater than half of the predicted maximum of 67%.
Two sets of correlations were pursued. In the first, we examined the effect of dividing the duplex DNA substrate into fragments and the capacity of a single RecA filament to carry out multiple strand exchange reactions concurrently. This is done in part to help establish that there is an intrinsic and constant rate of branch movement during DNA strand exchange. In the second, we compare the temperature dependencies of the DNA strand exchange reaction and ATP hydrolysis during strand exchange.
The linear
duplex substrate in these experiments, M13mp8.1037 (8266 bp), was
cleaved to produce one, two, three, or four fragments as shown in Fig. 2. The cleaved substrates were then used in DNA strand
exchange reactions with RecA nucleoprotein filaments bound to
single-stranded M13 mp8.1037 circles. All reactions were done at 37
°C. The results in Fig. 3demonstrate that the appearance of
fully duplex product circles is accelerated as the number of fragments
increases. This experiment was carried out three times with similar
results. Estimates for the rate of branch movement obtained as outlined
in Fig. 1are summarized in Table 1. The rates are fairly
uniform, especially when it is assumed that the rate of product
appearance is limited by the length of the longest DNA fragment
present. The average rate of branch movement obtained by this method is
378 bp min.
Figure 2: DNA strand exchange with multiple branch points. All DNA substrates are derived from M13mp8.1037. Where the linear duplex DNA substrate is divided into fragments, the fragments are nonoverlapping and have the sizes indicated (in bp). Restriction enzymes used to generate the fragments are listed under ``Materials and Methods.'' The fully duplex circular products have a number of nicks equivalent to the number of fragments required to generate them.
Figure 3: Assay for DNA strand exchange with multiple branch points. Reactions were carried out at 37 °C as described under ``Materials and Methods'' with the substrates illustrated in Fig. 2. The lanes marked M contain supercoiled and nicked circular M13mp8.1037 DNA markers. Labels are: P, circular duplex reaction product; I, reaction intermediates; ss, single-stranded circular DNA substrate; S, linear duplex substrates, with brackets where the duplex is divided into fragments. The numbers above each reaction indicate the number of duplex DNA fragments used. Time points for each reaction are, from left to right, 0, 3, 5, 7, 9, 11, 13, 15, 18, 21, 25, 30, 40, and 60 min, respectively.
The results suggest that multiple DNA fragments can react with a single nucleoprotein filament concurrently. To confirm this, some of the reactions were spread and examined by electron microscopy. The molecules shown in Fig. 4, involving two dsDNA fragments, were observed 11 min after the initiation of the reaction. In both cases, the ssDNA is participating in a strand exchange reaction with two fragments, and the strand exchange is progressing in the same direction with both fragments. Although a detailed characterization of the spreads was not undertaken, the results suggested a significant degree of asynchrony in the initiation of strand exchange by the two fragments on a given nucleoprotein filament, since only one-third to one-half of the intermediates observed were reacting with two duplex fragments. Samples were not cross-linked in these trials, and we do not know to what extent the results are affected by loss of DNA joints due to the spontaneous branch migration during sample preparation observed previously (Jain et al., 1994).
Figure 4: Electron microscopy of reactions with multiple duplex DNA fragments. Molecules derived from a reaction with the duplex substrate divided into two fragments (Fig. 2) are shown along with interpretive drawings. These molecules were found 11 min into the reaction.
Rates of ATP hydrolysis were also monitored during these experiments (Table 1). As observed previously (Schutte and Cox, 1987), addition of a homologous linear duplex resulted in a decrease in the rate of ATP hydrolysis of about 30%. This decrease is complete in about 2 min, and its extent is dependent on the length of available homology (with the 30% maximum observed when the homologous duplex substrate was as long as the ssDNA) (Schutte and Cox, 1987). A linear rate was observed during the 30-min span over which data was taken in the present experiments (between 5 and 35 min into the reaction). Rates observed during DNA strand exchange were not affected by dividing the duplex substrate into as many as four fragments (Table 1).
A series of
control experiments were carried out to ensure that any changes
observed reflected temperature-dependent changes in the k for ATP hydrolysis or the rate of branch
movement, as opposed to changes in the amount of bound RecA or the K
for ATP. We established that the K
for ATP was below 300 µM throughout
the temperature range. In RecA protein titrations carried out at five
different temperatures spanning the chosen temperature range, the rate
of ATP hydrolysis saturated in each case at a RecA concentration
corresponding to 3 ± 0.5 nucleotides of DNA per RecA monomer.
The final conditions and methods used to measure rates were affected by our unexpected discovery that ATP regeneration systems based on pyruvate kinase and phosphoenolpyruvate inhibit both DNA strand exchange and ATP hydrolysis in a temperature-dependent fashion. Direct comparisons of DNA strand exchange reactions carried out with the pyruvate kinase ATP regenerating system and the alternative system based on creatine phosphokinase revealed a substantial inhibition of the strand exchange reaction by the former below 33 °C, an effect that increased as the temperature was lowered to 25 °C. A parallel inhibition of the ATPase activity was also observed when experiments using the spectrophotometric coupled assay (which includes pyruvate kinase and phosphoenolpyruvate) were compared to initial rates of ATP hydrolysis measured with the alternative TLC assay (which contains no regenerating system). The basis of the inhibitory effect of the pyruvate kinase/phosphoenolpyruvate system at low temperatures was unclear. At 35 °C and above, the two ATP regeneration systems gave comparable results in strand exchange reactions, and the two methods for monitoring ATP hydrolysis also gave identical results. We do not know which component of the pyruvate kinase/phosphoenolpyruvate system is responsible for the low temperature inhibition. The rates of ATP hydrolysis during strand exchange were monitored with the more accurate spectrophotometric assay above 35 °C, and with the thin layer chromatographic assay at 33 °C and below. Measurements with the latter assay reflect initial rates determined between 2 and 10 min after initiation of DNA strand exchange. Although the two assays for ATP hydrolysis were in good agreement in experiments done at the higher temperatures, the error in the TLC assay is inherently greater. We compensated by increasing the number of experiments done at the lower temperatures. All of the DNA strand exchange experiments were done in the presence of the creatine phosphokinase/phosphocreatine ATP regenerating system.
A final factor that could affect the interpretation of the observed rates of DNA strand exchange is the possibility that exchange occurs in more than one kinetic phase. As described in the Introduction, a phase not requiring ATP hydrolysis may contribute a significant amount of hybrid DNA near the beginning of the reaction. If this phase is as rapid as some studies suggest (Menetski et al., 1990), it could result in a burst of hybrid DNA formation preceding the putative ATP hydrolysis-dependent process we are interested in measuring. This could, in turn, affect the outcome of the temperature dependence study, particularly if the size of the burst phase varied with temperature.
To evaluate the effects of a possible
rapid phase in strand exchange on our overall measurements, we carried
out a series of strand exchange reactions with individual truncated
linear duplex DNA substrates. If a significant rapid phase exists, the
apparent rate of branch movement should increase with shorter DNA
substrates, where the rapid phase would contribute a correspondingly
larger fraction of the hybrid DNA product. Although shorter duplex
substrates were employed in the experiment of Fig. 2, a rapid
phase may have been obscured by any asynchrony in the reaction of the
multiple DNA fragments. The results are shown in Fig. 5. An
increase in the apparent rate of branch movement during DNA strand
exchange is observed as the length of the duplex substrate decreases (Fig. 5B). If we assume that there is a rapid phase
that contributes 1200 bp of hybrid DNA to the reaction, the apparent
rate of branch movement for the remainder of each substrate becomes
independent of length (Fig. 5C). Similar experiments
were carried out at 25, 30, and 42 °C. The effect in Fig. 5B was the largest effect observed. The data in Fig. 5C give an average rate of branch movement of 377
bp min. This compares with a series of uncorrected
rate measurements with the 8266-bp duplex substrate ranging from 360 to
394 bp min
. Since the postulated burst phase appears
to have a minimal effect on the results when long duplex substrates are
used, and there was no evidence that the effects increased in any part
of the temperature range examined, we chose not to apply any
corrections to our rate measurements.
Figure 5:
The apparent rate of DNA strand exchange
as a function of the length of the duplex DNA substrate. A,
substrates used in the reactions. The numbers give the lengths of each
substrate in nucleotides or base pairs. B, apparent rates of
branch movement during DNA strand exchange. Two sets of experiments,
done on different days, are shown. Rates (bp min)
= the length of the duplex DNA substrate in bp, divided by the
time (min) required for the appearance of the first product (see Fig. 1). C, apparent rates of branch movement if it is
assumed that 1200 bp of hybrid DNA is created in a rapid phase
independent of ATP hydrolysis. Rates (bp min
)
= length of the duplex substrate (minus 1200 bp), divided by the
time (min) required for the appearance of the first product. The
1200-bp figure was determined by trial and error as the optimal
correction factor to produce the result in Panel
C.
As shown in Fig. 6,
there is a substantial effect of temperature on the rate of DNA strand
exchange. An Arrhenius plot for RecA-mediated ATP hydrolysis when bound
to ssDNA is shown in Fig. 7A. The plot is linear in the
range of 25 to 45 °C, with no breaks that might signal a change in
rate-limiting step, and yields an Arrhenius activation energy of 11.8
± 0.3 kcal mole. When heterologous dsDNA is
added to this system, there are no measurable changes in the rates of
ATP hydrolysis at 37 or 42 °C (Schutte and Cox, 1987). (
)The rates of ATP hydrolysis decrease when homologous DNA
is added to initiate DNA strand exchange (by 30% at 37 °C), as
reported previously (Schutte and Cox, 1987). Arrhenius plots for
RecA-mediated DNA strand exchange and the ATP hydrolysis that
accompanies it are presented in Fig. 7B, with the data
obtained for ATP hydrolysis included for comparison. The Arrhenius
plots are again linear over this temperature range, and there appears
to be a small increase in Arrhenius activation energy for ATP
hydrolysis. Within experimental error, the slopes of the lines fit to
the data for ATP hydrolysis and DNA branch movement during strand
exchange by unweighted linear regression are identical. The rates of
both processes change by a factor of about 6 over the 25 to 45 °C
temperature range. The slopes of the respective lines yield Arrhenius
activation barriers of 13.3 ± 1.1 kcal mole
for DNA branch movement, and 14.4 ± 1.4 kcal
mole
for ATP hydrolysis during strand exchange. In Fig. 7C, the data for DNA branch movement and ATP
hydrolysis during strand exchange are broken out and compared directly,
with parallel lines drawn through the data replacing the best fit lines
of Fig. 7B.
Figure 6: The temperature dependence of DNA strand exchange. Reactions were carried out as described under ``Materials and Methods'' at the two temperatures indicated. Labels are described in the legend to Fig. 1B. Time points are, from left to right: 0, 5, 10, 15, 20, 23, 26, 29, 33, 37, 45, 60, and 90 min, respectively.
Figure 7:
Arrhenius plots. The rate of DNA strand
exchange (in bp min, square symbols), the k
for ATP hydrolysis in the presence of ssDNA
alone (min
,
's), and the k
for ATP hydrolysis during strand exchange
(min
, circles) are plotted as a function of
temperature. Reactions were carried out under standard conditions
described under ``Materials and Methods.'' In the case of
ssDNA-dependent ATP hydrolysis, data above 37 °C were obtained with
the coupled spectrophotometric assay for ATP hydrolysis, and data below
this temperature were obtained with the thin layer chromatography
assay. Data shown at 37 °C represent multiple experiments carried
out with both assays. In the case of ATP hydrolysis during strand
exchange, the open circles reflect data obtained with the
coupled spectrophotometric assay for ATP hydrolysis (at least two
measurements at each temperature shown). The closed circles reflect data obtained with the thin layer chromatography assay.
For a few of the ATPase measurements carried out below 30 °C, the
RecA, SSB, and DNA concentrations were doubled. This had no discernible
effect on the k
values obtained. A,
rates of RecA protein-mediated ATP hydrolysis in the presence of ssDNA
alone. B, the data of Panel A combined with the data
for both ATP hydrolysis and DNA branch movement during DNA strand
exchange. Each data set in Panels A and B was fitted
separately by unweighted linear regression of ln k on
1/T, using the Minitab statistical software package. C, unfitted parallel lines are drawn through the data for both
ATP hydrolysis and DNA branch movement during strand exchange,
separated by 2.89 units on the y axis. See
``Discussion'' for details.
We have two primary conclusions. First, there is an intrinsic
and constant rate of branch movement in the final phase of DNA strand
exchange. This rate is 380 ± 20 bp min at 37
°C. Second, the rate of branch movement exhibits a dependence on
temperature that parallels the temperature dependence of ATP hydrolysis
during DNA strand exchange. The close correspondence provides a new
piece of evidence that ATP hydrolysis is coupled to the extension phase
of the DNA strand exchange reaction. While the Arrhenius plots cannot
provide rigorous proof for coupling, the correlation fulfills the most
fundamental requirement for a motor protein that a unit of ATP
hydrolysis should provide a definable unit of work under coupled
conditions.
These observations can be placed in the context of an
emerging view encompassing aspects of many different mechanistic
proposals. RecA nucleoprotein filaments formed on single-stranded DNA
have an intrinsic capacity to take up a homologous duplex DNA, using
binding energy to promote a strand exchange reaction independent of ATP
hydrolysis (Menetski et al., 1990; Kim et al., 1992a;
Rehrauer and Kowalczykowski, 1993; Kowalczykowski and Eggleston, 1994;
Shan et al., 1996). This process can account for the limited
exchange seen with ATPS or with the RecA K72R mutant, and the
small burst of exchange observed under some conditions with ATP. There
is no obvious reason why this process should not produce an efficient
and complete DNA strand exchange between long homologous DNA
substrates, but it usually halts or slows greatly before strand
exchange is completed. The extent of the rapid strand exchange observed
without ATP hydrolysis is presumably limited by discontinuities either
in the RecA filament (Menetski et al., 1990) or in a DNA
pairing intermediate (Shan et al., 1996). Extension of this
nascent hybrid DNA can occur without ATP hydrolysis under some
conditions (Shan et al., 1996), but is very slow and
undirected. Under conditions optimal for RecA-mediated DNA strand
exchange in vitro, extension of the hybrid DNA makes use of a
built-in protein machine. This use of ATP can be rationalized in part
by the special requirements of recombinational DNA repair (Clark and
Sandler, 1994; Cox, 1993). An additional rationale for catalyzing a
unidirectional strand exchange can be found in the relatively slow
rates of uncatalyzed branch migration (Panyutin and Hsieh, 1994).
We use the term ``coupling'' in the simplest sense. When ATP is hydrolyzed, the rate of DNA branch movement is determined and limited by the rate of ATP hydrolysis. It might be expected that a coupling between ATP hydrolysis and DNA branch movement could lead to a change in the measured activation energy for ATP hydrolysis when homologous duplex DNA was added. ATP hydrolysis is fully activated when RecA protein is bound to ssDNA, where it is clearly not coupled to work. Addition of homologous duplex DNA results in a measurable and abrupt decrease in the rate of ATP hydrolysis (Schutte and Cox, 1987), but generates only a small apparent change in the measured Arrhenius activation energy for ATP hydrolysis. While changes in the Arrhenius activation energy might reflect a coupling to work, such changes need not occur nor are they an experimental criterion for coupling. For example, myosin heads hydrolyze ATP in the presence of actin filaments under conditions where no work is accomplished, and Arrhenius activation energies for this process have been reported (Levy et al., 1959; Anson, 1992). The Arrhenius activation energy (above 20 °C) for myosin-mediated ATP hydrolysis does not change appreciably when the same reaction is measured under conditions where work is produced, in intact muscle or with tethered filaments in vitro (Levy et al., 1959; Anson, 1992). The Arrhenius activation energies we report for RecA-mediated ATP hydrolysis reflect whatever steps in the ATP hydrolytic cycle are rate-limiting, and cannot be interpreted (beyond the correlation we present here) without a more refined understanding of the cycle. There seems little doubt that the chemical energy (ATP hydrolysis) utilized by a RecA filament is in substantial excess to that required to move a DNA branch between homologous DNA molecules. We presume that this is coupled to some set of conformation changes in individual RecA monomers. When a DNA branch is present, its unidirectional movement is a byproduct of these same conformational changes. A major load may not be placed on the system until the branch encounters a DNA lesion or structural barrier in the course of recombinational DNA repair (Clark and Sandler, 1994; Cox, 1993).
The focus of this work is on the correlations illustrated by the Arrhenius plots rather than the activation energies derived from them. There is no reason why the observed temperature dependence of DNA branch movement should be similar to that for the completely distinct chemical process of ATP hydrolysis unless they are linked in some way. Our interpretation of the strong correlation seen in Fig. 7, that ATP hydrolysis is coupled to the extension of hybrid DNA during strand exchange, does not presuppose any particular coupling mechanism. There are at least two coupling mechanisms in the literature that remain viable. Kowalczykowski and colleagues have proposed that discontinuities in the RecA filament must be rectified by ATP hydrolysis-dependent redistribution of the RecA protein (Menetski et al., 1990). This laboratory has proposed an alternative model that couples ATP hydrolysis to a coordinated rotation of the DNA substrates in order to effect branch movement and resolve discontinuities that seem likely to arise in a key DNA pairing intermediate (Cox, 1994; Shan et al., 1996; Roca and Cox, 1990). Another version of the DNA rotation idea has recently been proposed by Radding and colleagues (Burnett, et al., 1994).
The Arrhenius plots in Fig. 7C are parallel rather
than coincident because the rate of DNA strand exchange and the k for ATP hydrolysis are reported in different
units (bp min
versus min
). The facilitated DNA rotation model makes
a specific prediction about how these two rates should be related (Cox,
1994). DNA bound by RecA protein has a helical periodicity of about 18
bp per turn, so that rotating the DNA by 360° should move a branch
point by 18 bp. The model holds that each RecA monomer hydrolyzes one
ATP for each 360° rotation of the DNA. The DNA branch should
therefore advance at a rate (in bp min
) equivalent
to the ATPase turnover rate (min
), multiplied by a
factor equivalent to the expected 18 bp advance per coupled rotation
(Cox, 1994). In Fig. 7C, the Arrhenius plots for these
processes in Fig. 7B are presented with parallel lines
drawn through the data separated by 2.89 natural log units. The lines
are well within the experimental error of the experiment, and their
separation corresponds to a factor of 18 bp.