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INTRODUCTION |
Homologous recombination of DNA is a universal biochemical process
required for chromosomal segregation and certain mechanisms of DNA
replication and repair (1). Recombinases act in nucleoprotein filaments
whose essential features have been conserved from phage to man (2, 3).
The most thoroughly studied recombinase, Escherichia coli
RecA protein, carries out the principle reactions of homologous
recombination in vitro (2, 4). RecA protein has two
functional DNA binding sites (5). In the first step of the reaction,
multiple RecA monomers bind
ssDNA1 at one site of the
protein to form a right-handed helical polymer, 3 nt per RecA monomer,
known as a presynaptic complex. In the presence of ATP, this filament
binds dsDNA, and if the DNA sequences are complementary, strand
exchange occurs.
RecA protein catalyzes DNA-dependent ATP hydrolysis
uniformly throughout the nucleoprotein filament (6). The presynaptic complex is necessary and sufficient for ATPase activity, even though
this filament can bind additional ssDNA or dsDNA (7). The effect of ATP
hydrolysis on the DNA strand exchange reaction carried out by RecA
protein has been investigated using stable ATP analogs such as ATP
S
(8, 9) or the mutant RecA protein K72R (10, 11). In these conditions
the presynaptic filament binds but does not hydrolyze cofactor. This
filament nevertheless carries out limited DNA strand exchange and is
considered to reproduce some of the early steps of homologous
recombination. In particular, binding of ATP or ATP
S produces a
filament in which DNA is extended (5.1 Å per base pair) and underwound
(18 base pair/turn) (12-14). Concomitantly ATP or ATP
S increase the
stability of the nucleoprotein filament, while ADP destabilizes the
filament relative to no cofactor (15).
The mechanism by which RecA protein forms the presynaptic complex is
poorly understood. Equilibrium binding studies have been carried out
for the reaction of RecA with ssDNA in the absence of cofactor (15,
16). However, equilibrium constants and rate constants for formation of
the presynaptic complex with ATP or ATP
S have not been investigated
in part because of the high stability of the filament in the presence
of cofactor. It has been reported that addition of nucleoside
triphosphate cofactor to a 3/1 nt/RecA filament may induce transient
dissociation of the filament (17-19). Furthermore the use of
oligonucleotide substrates can destabilize nucleoprotein filaments by
reducing the number of protein-protein interactions. Here we use the
dissociation-reassociation reaction and oligonucleotide nucleoprotein
filaments to investigate the mechanism of presynaptic complex formation.
We have recently developed fluorescence anisotropy methods to study the
binding of oligonucleotide to the presynaptic filament in the presence
of ATP
S (20). The anisotropy of a 5'-fluorescein-labeled oligonucleotide increases as protein binds to DNA. Control experiments show that the 5'-fluorescein label does not affect the DNA strand exchange reaction and that the spectroscopic signal reflects the properties of the nucleoprotein filament rather than the local behavior
of the fluorophore. We apply this method to study the presynaptic complex.
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EXPERIMENTAL PROCEDURES |
Fluorescein-labeled oligonucleotide, 5'-F CCA TCC GCA AAA ATG
ACC TCT TAT CAA AAG GA where F is fluorescein, was synthesized by
Genosys. RecA protein and M13 DNA were purified, and concentrations were determined as described (20, 21). RecA concentration units are M
protein monomer; oligonucleotide concentrations are M nt.
TMDG buffer is 20 mM Tris acetate, 2 mM
Mg(CH3COO)2, 1 mM dithiothreitol,
and 5% glycerol, pH 7.5.
Unless otherwise stated, nucleotide filaments were prepared with
stoichiometry of 3/1 nt/RecA protein in TMDG buffer at 25 °C ± 1 °C; concentration units of the filament are M nt. In a typical experiment, nucleoprotein complex in the absence of cofactor was prepared and equilibrated in the cuvette, cofactor was added at the
beginning of the reaction, t = 0 s, and
fluorescence signal followed for 1000 s. Manual mixing time for
addition of cofactor was 15 s.
Fluorescence experiments were performed using a model MOS-400
spectrophotometer from Bio-Logic (Claix, France). Measurements were
taken of samples in a 1.0 × 0.4-cm cuvette stirred continuously at the indicated temperature ± 1 °C. To maximize sensitivity, a single polarizer configuration was used to determine fluorescence anisotropy, A, and fluorescence intensity, Fl (V) (22). Fluorescence is
reported as quenching, Q = 1
(Fl/Flo) where
Flo is the signal from oligonucleotide alone. Samples were
excited at 490 nm, and total emission intensity was measured at 1-s
intervals using a 515-nm cutoff filter.
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RESULTS |
Dissociation of Nucleoprotein Filament by ATP
S--
We first
wished to show that dissociation and reassociation of the RecA
nucleoprotein filament can be studied by fluorescence anisotropy. The
fluorescence signals of various complexes of 1.0 µM
5'-fluorescein 32-mer oligonucleotide and 0.33 µM RecA
protein are shown in Fig. 1. Fluorescence
anisotropy of free oligonucleotide in the absence of RecA was A = 0.05 ± 0.005. Anisotropy of the 3/1 nt/RecA complex without
cofactor was A = 0.19 ± 0.005 and fluorescence quenching was
0.3 ± 0.05. Addition of ADP to this complex decreased the
anisotropy to 0.05, the same value as free oligonucleotide;
fluorescence quenching decreased to 0.025, and these spectroscopic
signals did not change with time. NaCl decreased the anisotropy and
fluorescence quenching of the 3/1 complex of oligonucleotide and RecA
protein in absence of cofactor, while the signal of the corresponding
filament in the presence of ATP
S was stable in 800 mM
salt (not shown). ADP is known to destabilize the RecA protein-DNA
complex, while ATP
S stabilizes it with respect to salt denaturation
(15). Hence these fluorescence signals measure known effects of
cofactor and salt on the formation and stability of the 3/1 nt/RecA
nucleoprotein filament.

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Fig. 1.
Spectroscopic changes caused by addition of
nucleotide cofactor to the nucleoprotein filament formed by RecA
protein and fluorescein-labeled oligonucleotide. 1.0 µM nt and 0.33 µM RecA protein were
incubated at 25 °C in TMDG buffer. 100 µM nucleotide
cofactor or buffer was added immediately before the time designated as
t = 0 s, and the signal followed as a function of
time. a and b, upper curve, buffer;
lower curve, ADP. c and d, upper
curve, ATP S; lower curve, ATP added to 1.0 µM nt and 0.33 µM RecA protein;
middle curve, ATP added to 4.8 µM nt and 1.6 µM RecA protein. a and c,
fluorescence anisotropy; b and d, fluorescence
quenching.
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When ATP
S was added to the filament without cofactor, the anisotropy
signal decreased. In this experiment, the intensity at
t = 0 s, which is defined as the beginning of
measurement after mixing, was A = 0.12 and signal gradually
increased over 15 min to A = 0.19 (Fig. 1c). In the
same experiment quenching decreased immediately after addition of
ATP
S to 0.2; signal then increased for about 50 s to Q = 0.26 and thereafter slowly decreased to Q = 0.22. After addition
of ATP, the anisotropy and fluorescence signals initially decreased;
signals then increased for about 50 s in these experiments and
subsequently decreased. The time-dependent spectroscopic
signals observed for the reaction with ATP are consistent with a
combination of cofactor binding followed by hydrolysis to ADP.
To further understand the kinetic effects brought about by nucleoside
triphosphate binding (separate from its hydrolysis), we investigated
the reaction with ATP
S. If the decrease in fluorescence anisotropy
produced by the addition of ATP
S (Fig. 1c) was caused by
cofactor-induced dissociation, the resulting free RecA protein should
react with competitive nonfluorescent DNA. To test this hypothesis,
various concentrations of M13 phage ssDNA were added with ATP
S at
the beginning of the reaction (Fig. 2).
The decrease in the anisotropy and fluorescence quenching signals at
t = 0 s caused by addition of ATP
S was not
influenced by 0-4 µM competitor DNA. However, the
recovery of fluorescence signals was inhibited in a
concentration-dependent manner. Adding 10 µM
nonfluorescent ssDNA at the end of the reaction caused a small decrease
in the anisotropy and no change in the quenching (not shown),
indicating that once it is formed the ATP
S-RecA nucleoprotein
filament was not significantly perturbed by competitor DNA. These
results support the hypothesis that addition of ATP
S dissociated
protein molecules from the 3/1 oligonucleotide/RecA complex.

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Fig. 2.
Competitive inhibition of the reassociation
reaction. 1.0 µM nt and 0.33 µM RecA
protein were incubated at 25 °C. 100 µM ATP S was
added immediately before the time designated t = 0 s. Various concentrations of M13 ssDNA were added with ATP S. From
top to bottom: 0 µM, 2 µM, 4 µM, or 10 µM M13
DNA.
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The anisotropy increase following this dissociation (designated
t > 0 s, Fig. 1c) could be caused by
reassociation of RecA protein with DNA. To test this possibility, we
compared two reactions (Fig. 3). First,
addition of cofactor to a 3/1 nt/RecA complex led to a decrease in the
anisotropy followed by recovery of signal as in Fig. 1c.
Second, for the same concentrations of reactants, RecA protein and
ATP
S were added to fluorescent oligonucleotide. For this reaction,
the anisotropy of the oligonucleotide alone was 0.05 and signal
increased after addition of RecA as protein bound to oligonucleotide.
The increased anisotropy and fluorescence quenching changes were the
same for the two reactions. These results were confirmed in eight
independent experiments using 3/1 nt/RecA complexes made with 0.5-10
µM nt. Hence the increase in fluorescence anisotropy
observed after addition of cofactor corresponds to the reaction of
RecA-ATP
S with fluorescent oligonucleotide.

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Fig. 3.
Reassociation of RecA protein and
oligonucleotide. Dark curve, ATP S was added to a
mixture of 0.5 µM oligonucleotide 0.167 µM
RecA at t = 0 s. Light curve, a mixture
of 0.167 µM RecA and ATP S was added to a 0.5 µM oligonucleotide at t = 0 s.
Reaction conditions are as in Fig. 1.
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Dependence of the Reaction on ATP
S Concentration--
The
kinetics of the dissociation-reassociation reaction were unchanged for
ATP
S concentrations above 15 µM. To study the requirement of the reaction for ATP
S, 0-10 µM ATP
S
were added to a 3/1-nt/RecA complex, 1 µM nt (Fig
4). The anisotropy at t = 0 s decreased from A = 0.2 to A = 0.15 as ATP
S
concentrations increased from 0 to 2 µM. Binding of RecA
protein to oligonucleotide was greater without ATP
S or for ATP
S
concentrations > 10 µM than for intermediate
concentrations.

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Fig. 4.
Effect of ATP S
concentration on reassociation kinetics. 1 µM
oligonucleotide and 0.33 µM RecA protein were incubated
at 25 °C in TMDG buffer. The indicated concentrations of ATP S
were added at 0 s, and anisotropy followed as a function of
time.
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Low concentrations of ATP
S would be expected to produce a mixture of
RecA molecules with and without ATP
S. These results show that
binding of such a mixed population of RecA molecules to single-stranded
DNA was qualitatively different than for either species by itself (see
"Discussion").
Dependence of the Reaction on Filament
Concentration--
Complexes of 3/1 nt/RecA containing from 0.5 µM to 10 µM oligonucleotide were prepared,
ATP
S was added, and the anisotropy and fluorescence quenching were
observed as a function of time (Fig. 5).
The effect of filament concentration on several features of the
anisotropy kinetic curves is shown in Fig.
6a. In absence of ATP
S, 3/1
RecA/nt formed a complex at high filament concentrations with A = 0.21 ± 0.005 and Q = 0.35 ± 0.05 (designated
t < 0 s, Fig. 5; circles, Fig.
6a). These spectroscopic values correspond to a complex in
which fluorescent oligonucleotide is saturated by RecA protein (20).
Spectroscopic signals of this complex decreased for oligonucleotide
concentrations below 2.0 µM. The simplest explanation of
this observation would be that oligonucleotide and protein dissociate
at low concentrations of filament.

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Fig. 5.
Effect of filament concentration on
fluorescence anisotropy. Complexes of (i) 0.5 µM,
(ii) 1 µM, (iii) 2 µM (light
curve), or (iv) 3 µM fluorescent oligonucleotide
were equilibrated with RecA protein, 3 nt/protein molecule; 100 µM ATP S was added at t = 0 s.
Reaction conditions as in Fig. 1. a, fluorescence
anisotropy; b, fluorescence quenching.
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Fig. 6.
a, anisotropy values of different parts
of the kinetic curves in Fig. 5a. Circles, prior
to addition of ATP S, t < 0 s, Fig.
5a; squares, immediately after addition of
ATP S, t = 0 s; triangles,
t = 1000 s. b, dissociation of the
presynaptic filament in presence of ATP S. , dilution of 4.8 µM nt 3/1 nt/RecA complex, 100 µM ATP S,
into TMDG buffer with cofactor; , titration of buffer and cofactor
with 3/1 nt/RecA complex, 4.8 µM nt.
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The intensity of the signals immediately after addition of ATP
S,
designated t = 0 s in Fig. 5, increased with
filament concentration and remained constant above 2.5 µM
complex: A = 0.17 ± 0.01, Q = 0.30 ± 0.05 (Fig.
6a, squares). At all concentrations the value of
anisotropy was significantly less than the signal observed for the
reactant or the product complexes. These results show that
reassociation after addition of ATP
S occurred in two kinetically distinct steps. A rapid reaction formed a stable intermediate. Kinetics
of this reaction could be observed at sufficiently low filament
concentration. At concentrations above 2.5 µM this
reaction was completed during the mixing time of the experiment. This
intermediate evolved slowly to give the final product that had the same
anisotropy as the initial filament (Fig. 6a,
triangles). Spectroscopic properties of the various stages
of the reaction are summarized in Table I.
The anisotropy of the product filament decreased below 2.5 µM complex in a manner similar to the filament without
cofactor (compare circles and triangles, Fig.
6a). These results suggest that the filament with ATP
S
dissociated at low concentrations. To test this hypothesis, we prepared
4.8 µM 3/1 nt/RecA complex in the presence of 100 µM ATP
S and either i) diluted this complex into buffer
with 100 µM ATP
S or ii) titrated this buffer with the
complex (Fig. 6b). In both cases, fluorescence anisotropy decreased at low filament concentrations and results were
superimposable on data in Fig. 6a. Hence the equilibrium
constant for binding of RecA protein to ssDNA can be determined in the
presence of ATP
S in these reaction conditions. Equilibrium constants
for the reaction between RecA protein and ssDNA have not been measured in the presence of ATP
S because significant concentrations of free
protein were not previously observed. Dissociation was apparently enhanced in our experiments by dilution and by using short
oligonucleotide substrate that presumably would have less cooperative
interactions between protein monomers than a filament on longer ssDNA.
The binding constant can be estimated from the filament concentration at which 50% of the oligonucleotide is bound, [F] (M
nt), by the relation (23),
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(Eq. 1)
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where K is the intrinsic binding constant of a protein
monomer and
is the cooperativity factor. Data in Fig. 6 indicate that 1/K
= 10
7 (M nt) at
25 °C in TMDG buffer.
Dependence of the Reaction on Temperature and Salt
Concentration--
In the absence of cofactor, the anisotropy and the
fluorescence quenching of the 3/1 nt/RecA protein complex, 1 µM oligonucleotide, decreased slowly over 15 min at
37 °C. This reaction was inhibited by lower temperatures (Fig.
7a) and by higher filament
concentration (not shown). It is likely due, at least in part, to
dissociation of oligonucleotide and aggregation of free protein (see
below). After addition of ATP
S to this complex, RecA protein
reassociated with oligonucleotide in a biphasic reaction similar to
that observed at 25 °C.

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Fig. 7.
Temperature and salt dependence of the
reaction. 1.0 µM fluorescent oligonucleotide and
0.33 µM RecA protein were incubated in TMDG buffer prior
to addition of 100 µM ATP S at t = 0 s. Reaction conditions as in Fig.1. a, temperature
dependence; upper curve, 25 °C; middle curve,
31 °C; lower curve, 37 °C. b, effect of
NaCl concentration, 25 °C; curves from top to
bottom 0, 125 mM, 250 mM, 500 mM, 1 M NaCl. NaCl was added with
ATP S.
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The nucleoprotein complex formed in the absence of ATP
S appeared to
be unstable at low Mg2+ concentrations. Addition of 0.167 µM RecA protein to 0.5 µM fluorescent oligonucleotide at 25 °C in 20 mM Tris buffer, pH 7.4, and no Mg2+ initially produced a filament with A = 0.16 and Q = 0.3. The anisotropy and fluorescence quenching
subsequently decreased during 15 min to A = 0.07, Q = 0.12. The decrease was less apparent at higher filament concentration;
e.g. for a 2 µM complex, spectroscopy signals
decreased less than 10%. Similar instability was also apparent under
certain conditions in standard buffer. For example, a slow decrease of
anisotropy from A = 0.18 to A = 0.1 was observed in TMDG
buffer for 3/1 nt/RecA complexes with 1 µM nt at 37 °C (Fig. 7a) but not at 25 °C (Fig. 1a). The
effect of low Mg2+ concentration was reversible. Addition
of 15 mM Mg(CH3COO)2 20 min after
incubation of the complex at 25 °C in the absence of Mg2+ restored the 3/1 complex as well as the
ATP
S-induced dissociation and reassociation reactions. The
instability of the 3/1 complex in the absence of cofactor and
Mg2+ may be due to slow aggregation of free protein into a
nonproductive complex as previously reported (24). It is worth noting
that high Mg2+ concentrations dissociate the presynaptic
complex (20).
Addition of NaCl favored the ATP
S-induced dissociation reaction
(Fig. 7b, t = 0 s) as expected from
known salt effects on filament stability (15, 20). NaCl also inhibited
the reassociation reaction with 50% inhibition observed for 250 mM NaCl. In contrast, addition of these concentrations of
NaCl at the end of the reaction did not greatly decrease the anisotropy
of product filament, as previously reported (15); for example, addition
of 1 M NaCl to the product filament with 1 µM
nt decreased the anisotropy from 0.18 to 0.14. Hence salt
concentrations that inhibited formation of the
ATP
S-RecA-oligonucleotide complex did not destabilize the complex
once it was formed. Similar results were observed using etheno-modified
M13 DNA (not shown).
Kinetic Analysis--
The preceding results indicate that a
mechanism for the reaction of ATP
S with the 3/1 nt/RecA filament
would include at least the following elements (Fig.
8). i) Prior to addition of ATP
S, free
RecA protein (R) and fluorescent oligonucleotide (F) are in equilibrium
with the nucleoprotein complex (RF) (reaction 1). ii) The slow
dissociation of RF, which was observed in the absence of
Mg2+, indicates a competitive reaction in which free RecA
formed aggregates (24) that can not bind to the oligonucleotide
(reaction 2). iii) Reaction of ATP
S with free RecA dissociates the
3/1 nt/RecA filament (reaction 3, see "Discussion"). iv)
Subsequently RecA-ATP
S (Rg) binds to the oligonucleotide to form a
stable reaction intermediate RgF (reaction 4). v) The product of
reaction 4 is slowly transformed into the final product RgF' (reaction
5). At low filament concentrations, an equilibrium was also observed
between free RecA protein and product filament in the presence of
ATP
S (Fig. 6b).

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Fig. 8.
Proposed reaction mechanism for the
cofactor-induced dissociation and reassociation of the 3/1 nt/RecA
filament. R, RecA; F, fluorescent
oligonucleotide; RF, 3/1 nt/RecA complex in absence of
cofactor; Rg, RecA-ATP S complex; RgF and
RgF', 3/1 nt/RecA complexes in presence of cofactor.
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Time-dependent spectroscopic signals were analyzed in an
attempt to determine the rate constants for the association reactions 4 and 5. Kinetics were studied for addition of ATP
S to 1 µM filament. Reaction 5 was approximated as a first order
process (see below). Rate constants were determined for the mechanism
shown in Fig. 9a, which
further assumes that the binding of RecA is a two-step reaction between
free protein, Rg, and oligonucleotide, F. Kinetic equations were solved
numerically (25)
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(Eq. 2)
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where A(t) and F(t) are the
time-dependent anisotropy and fluorescence.
ci(t) are the concentrations of fluorescent
species i (µM nt) calculated from the equation in Fig.
9A. Ai and qi are the anisotropy and
effective quantum yields of each species; the latter was approximated
by fluorescence intensity (25). Calculations were carried out as
follows. i) Initial values of Ai and qi were determined from steady state parameters, Table I; the spectroscopic signals of free oligonucleotide were kept constant, A = 0.05, Fl = 0.375 V. ii) Rate constants for the forward and reverse steps of reaction 4, k4f and
k4r, and for the first order reaction 5 were
adjusted to visually fit the experimental data (Fig. 9B, Table II).

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Fig. 9.
Kinetic analysis. A,
two-state mechanism for the association reaction. Abbreviations are as
in Fig. 8. B, kinetic analysis of the association reaction.
Anisotropy (a) and fluorescence (b) changes after
addition of ATP S to 1 µM fluorescent oligonucleotide
with 0.33 µM RecA protein, reaction conditions are as in
Fig. 1; symbols, average values of from 11 independent experiments;
lines, theoretical curves for parameters Table II (see
"Results"). Residuals for the fit of the anisotropy
(c) and fluorescence data (d).
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Good agreement between experimental and theoretical curves was observed
for data t > 150 s, k5 = 0.36 min
1. The rate constant for the second step of the
reaction was confirmed by nine additional experiments in which ATP
S
was added to 3/1 nt/RecA complexes containing 2-8 µM
filament, conditions where only this reaction was observed (Fig.
6a); anisotropy kinetic curves in these experiments were
identical, indicating a reaction mechanism that was independent of
filament concentration. These curves could be fit by a single
exponential with average rate ± S.D., k5 = 0.4 ± 0.1 min
1. A concentration-independent rate
constant indicates that neither protein binding nor aggregation
contributes significantly to the time-dependent
spectroscopic signals. Hence the increased anisotropy and fluorescence
that occur during the second step of the reaction (Fig. 9) appear to be
caused by a process other than protein binding, such as a
conformational change (see "Discussion"). The rate constant for the
second step of the reaction was independent of temperature between
25 °C and 37 °C (Table III).
Fit of the early part of the reaction was poor, and rate constants were
not consistent with K
obtained from the dissociation of
the filament at low concentrations (Fig. 6b). The DNA
binding reaction 4 probably needs to be represented by another
mechanism than a simple two-state process. For example, the reaction of RecA protein with ssDNA can be analyzed in the general context of
cooperative binding between a ligand with a finite binding site size
and a one-dimensional lattice (26, 27). This formalism shows that
binding is a function of the size of the binding site, cooperative
interactions, and effects of oligonucleotide extremities as well as the
association constant. Equilibrium binding parameters for the reaction
of RecA protein with ssDNA have been previously determined by this
method (15, 16), but a kinetic formalism has not been developed to our knowledge.
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DISCUSSION |
E. coli RecA protein forms a nucleoprotein filament
with ssDNA in the presence of ATP
S, 3 nt/RecA monomer, which is able to carry out the initial steps of homologous recombination in vitro and is considered to be a good model for the presynaptic complex (8, 9). We have investigated this RecA nucleoprotein filament
using the fluorescence anisotropy of a 5'-fluorescein-labeled 32-mer
oligonucleotide. This method is sensitive to hydrodynamic properties of
the fluorophore (28), and consequently the fluorescence anisotropy of
the oligonucleotide increases with RecA binding (20). Here we
demonstrate that addition of nucleoside triphosphate to the
nucleoprotein filament can cause its dissociation under some conditions
as previously suggested from biochemical experiments (18, 19).
Furthermore, the spectroscopic measurements showed a two-step
association reaction between RecA and ssDNA. The most important result
of this study is the observation of a previously unknown intermediate
in the polymerization of RecA protein on ssDNA that we will attempt to
define in this Discussion.
Polymerization of RecA protein on the oligonucleotide in the presence
of ATP
S took place in two steps. In the first, fluorescence was
quenched by 0.30 ± 0.05 and fluorescence anisotropy increased from 0.05 to 0.17 ± 0.005 (Fig. 6a, Table I). Kinetics
of this reaction could be observed below filament concentrations of 2 µM nt; at higher concentrations the reaction was complete
in the mixing time of the experiments, 15 s (Figs. 5,
6a). In the second step of the reaction the anisotropy
increased to 0.21 ± 0.005 and fluorescence quenching decreased to
0.23 ± 0.05. The rate of this reaction was independent of
filament concentration (Fig. 5) and temperature (Table III),
k5 = 0.3 ± 0.1 min
1.
Concentration-independent kinetics indicate that neither aggregation nor protein binding contribute significantly to the reaction.
The fluorescence measurements give some information about the relative
physical properties of the reaction intermediate and the product. Both
filaments appear to have quantitatively bound RecA protein at filament
concentrations above 2 µM (Fig. 6a). However,
the intermediate is more hydrodynamically mobile judging from its lower
anisotropy (28). RecA protein is known to extend the nucleoprotein
filament (12-14). An increased mobility of the intermediate could be
explained if it had a smaller persistence length than the product filament.
Low concentrations of ATP
S decreased the anisotropy of the
nucleoprotein filament, while higher concentrations of ATP
S restored anisotropy (Fig. 4). The maximum decrease was observed for 2 µM ATP
S. In these conditions the anisotropy of the
complex was reduced to A = 0.15 ± 0.01, which corresponds to
the anisotropy of the polymerization intermediate described above (Fig.
6a). This filament was stable for at least 15 min. Although
the equilibrium constants for the reaction between RecA protein and
nucleotide cofactors are not known, sufficiently low concentrations of
the nucleoside triphosphate should give two populations of RecA
protein, with and without ATP
S. Hence, these results suggest that
the intermediate filament formed during the polymerization reaction may
have a conformation similar to a mixed filament containing RecA
molecules with and without ATP
S. The low anisotropy of this latter
filament could be explained if relatively rigid segments of polymerized RecA (R) and RecA-ATP
S (Rg) were separated by flexible "hinges" caused by the incompatible protein-protein interactions between R and
Rg monomers (29, 30). At high ATP
S concentrations, cofactor would
bind to all protein and the more rigid product filament would form via
reaction 5 (Figs. 4 and 8). This reaction is apparently inhibited in a
filament containing a mixed population of RecA monomers with and
without cofactor.
These considerations suggest that a rearrangement takes place in the
slow step of the polymerization reaction that increases the persistence
length of the filament (reaction 5 in Fig. 8). Reported kinetics of ATP
hydrolysis suggest a possible mechanism for this process. Using a
combination of intrinsic protein fluorescence and ATP hydrolysis
measurements, Paulus and Bryant demonstrated a two step mechanism for
ATP hydrolysis in similar reaction conditions to those used in our
experiments (31). Rapid binding of ATP is followed by rate-limiting
"isomerization" of the filament. When ATP
S is used in the
reaction, isomerization is observed without hydrolysis. ATP
S
hydrolyzes in these conditions but the rate, k = 0.01 min
1, is too slow to contribute to our reaction 5. Pre-steady state kinetic analysis of ATP hydrolysis and its inhibition
by ATP
S showed that the forward rate constant for the isomerization
reaction is the same as that for ATP hydrolysis, k = 20 min
1 at 37 °C. Hence in our reaction, this
isomerization has likely occurred in the intermediate complex. The
reverse rate for the isomerization, k' = 0.1-0.2 min
1
corresponds well to the rate constant for the slow reaction observed in
our experiments, k5 (Tables II and III). These
results suggest that reversal of isomerization is required for the
transition from the flexible intermediate to the more rigid product.
To summarize, we present spectroscopic evidence for a previously
unknown intermediate in the reaction that forms the RecA nucleoprotein
filament in the presence of ATP
S. Fluorescent anisotropy measurements indicate that the intermediate is less rigid than the
final product. It has anisotropy similar to mixed filaments of extended
and condensed RecA monomers observed at low ATP
S concentrations.
However, the intermediate is observed in the presence of a large excess
of nucleotide cofactor, and presumably all RecA monomers are in the
extended conformation. The greater flexibility of the intermediate
filament might result from some sort of incorrect binding between
relatively rigid segments of polymerized RgF (Fig. 8). This defect
could be due to faulty protein-protein or protein-DNA interactions at
the site between the segments. Our preferred hypothesis is that
polymerization may initiate from multiple sites on the oligonucleotide
and leave gaps of 1 or 2 nt between adjacent polymerization events,
which are too small to react with RecA, whose binding site size is 3 nt
(2, 4). Whatever the mechanism responsible for this flexibility, it
disappears by the slow second step of the reaction. Comparing the rate
of this process with published data (31) suggests that reversal of the
nucleoside triphosphate dependent isomerization of the RecA filament is
required to reshuffle RecA monomers in the filament to form a more
rigid structure.
Taken together these results also show that the 32-mer
oligonucleotide-RecA complex studied here can exist in two forms with different hydrodynamic properties. The more rigid form was observed (i)
in filaments without cofactor and (ii) for the product filament in
presence of ATP
S. The more flexible structure was observed (iii)
during the polymerization reaction in presence of ATP
S and (iv) in
mixed filaments of RecA protein with and without ATP
S. We argue that
flexibility is caused by a break between segments of polymerized RecA
protein. In the reaction intermediate (iii), the break is probably due
to a discontinuity in the polymer, perhaps the result of multiple
polymerization events. In the mixed filament (iv), the break likely
comes from the inability of extended and condensed RecA monomers to
form adequate protein-protein interactions for polymerization (29).
A transition between filament conformations with different mobilities
such these could be coupled to ATP hydrolysis. RecA monomers are
released from the nucleoprotein filament during ATP hydrolysis, albeit
with a low efficiency compared with the hydrolysis rate (32).
Furthermore, ATP hydrolysis can introduce conformational changes that
alter protein-protein interactions between adjacent RecA monomers
(29.30). Either of these effects could introduce a flexible hinge in
the filament at the site of ATP hydrolysis thereby introducing local
segmental mobility, which could play a role in homologous recombination.