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INTRODUCTION |
Homologous recombination plays an essential role in chromosomal
segregation and DNA repair. Recombinase proteins act in a filamentous
nucleoprotein structure that stretches the DNA and hydrolyzes ATP.
Essential features of the structure and function of this
recombinase-DNA filament have been conserved from phage to man (1, 2).
The most widely studied recombinase is the RecA protein of
Escherichia coli. Major steps of the strand exchange reaction can be reproduced by RecA protein in vitro in the
presence of ATP or a stable analog such as
ATP
S1 (3-5).
Early characterization of this reaction led to the functional
definition of two DNA binding sites on the RecA protein (6). ssDNA
forms a complex with RecA at site I known as a presynaptic filament.
dsDNA then binds at a second site, site II. If the DNA sequences at
site I and II are complementary, synapsis can occur, leading to strand
exchange. ssDNA can also bind at site II (1, 5, 7-9). The location of
sites I and II within the RecA molecule have been investigated by x-ray
crystallography (10), cross-linking experiments (11-14), limited
protease digestion (15), and properties of mutant RecA proteins
(16-21).
Although an equilibrium constant for binding of dsDNA at site II has
been reported (22, 23), binding constants for the reaction of ssDNA
with site II of RecA protein are unknown. We have recently (24)
measured the equilibrium binding parameters for the reaction of ICP8,
the ssDNA-binding protein of herpes simplex virus type I, with ssDNA
using an approach that combines fluorescence anisotropy and
macromolecular binding density function analysis (25). Here we employ
this method to study the reaction between site II of the RecA
nucleoprotein filament and a 5'-fluorescein-labeled ss 32-mer oligonucleotide.
One of the reasons that equilibrium constants for this reaction have
not previously been measured is that ss polynucleotides bind tightly to
RecA in the presence of cofactor (26, 27). Use of an oligonucleotide,
which may bind less to RecA than do polynucleotides, was
expected to overcome this difficulty. We chose an oligonucleotide known
to be a substrate for RecA-catalyzed DNA strand exchange (8, 9) and
labeled the 5'-extremity with fluorescein. It was necessary to 1)
demonstrate that the protein formed a functional complex with the
fluorescent oligonucleotide, 2) show that the anisotropy reflected
protein binding rather than local effects near the fluorescein label,
and 3) experimentally determine the relation between the anisotropy
signal and fractional saturation for the reaction. These conditions
were met, and we have characterized the equilibrium binding of ssDNA
with site II of the RecA nucleoprotein filament in the presence of
ATP
S.
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EXPERIMENTAL PROCEDURES |
RecA protein was purified from E. coli as previously
described (19) and was free of nuclease activity. Oligonucleotides 1 (5'-F CCA TCC GCA AAA ATG ACC TCT TAT CAA AAG GA-3', where
F is fluorescein), 2 (5'-CCA TCC GCA AAA ATG ACC TCT TAT CAA
AAG GA-3'), and 3 (5'-TCC TTT TGA TAA GAG GTC ATT TTT GCG
GAT GG-3') were synthesized by Genosys. To prepare double-stranded
oligonucleotides 1/3 and
2/3, an equimolar mixture of complementary
oligonucleotides in 10 mM Tris acetate, pH 7.4, was heated
to 90 °C and slowly cooled to ensure hybridization. Protein and
oligonucleotide concentrations were determined by UV spectroscopy with
280 = 2.17 × 104
M
1 cm
1
and
260 = 8.64 × 103
M
1 cm
1,
respectively. Concentrations of ss oligonucleotides are
M nt, and ds oligonucleotides are M base pairs.
Strand exchange in vitro was carried out following published
methods (8). Presynaptic complexes were formed by reacting 15 µM RecA, 1 mM ATP
s, and 45 µM nt ss oligonucleotide 15 min at 37 °C in TMDG
buffer (20 mM Tris acetate, 2 mM
Mg(CH3C00)2, 1 mM dithiothreitol,
and 5% glycerol, pH 7.5). The Mg(CH3C00)2 concentration was then increased to 12 mM, and after 5 min
of incubation, strand exchange was initiated by the addition of ds oligonucleotide (45 µM base pairs). 10-µl aliquots were
removed from the reaction at various times and deproteinized at room
temperature by adding 50 mM EDTA, 1% SDS. After 5 min, 5 µl of glycerol/bromphenol blue were added, and samples were stored on
ice prior to 20% polyacrylamide gel electrophoresis (0.1 M
Tris, 90 mM boric acid, 1 mM EDTA, pH 8.4) for
75 min at 150 V. Strand exchange was quantified from fluorescence
intensities on wet gels using a PhosphorImager Storm 840 apparatus
(Molecular Dynamics) in the Fluorescence Blue operating mode. The
intensity of the signal from ds oligonucleotide was corrected for
quenching (see "Results").
Fluorescence experiments were performed by a model MOS-400
spectrophotometer from Bio-Logic (Claix, France). Samples were excited
at 490 nm and stirred continuously at various temperatures ± 1 °C in a 1.0 × 0.4-cm temperature-controlled cuvette
with the 4-mm path oriented in the direction of the exciting light.
Inner filter effects were negligible. The pH was measured at the
beginning and end of control experiments to assure that it remained
constant during titrations. Addition of RecA protein led to a 3-nm red shift, with no change in the shape of the fluorescence emission spectrum of oligonucleotide 1. Therefore, to optimize the sensitivity of the experiments, total spectral intensity was
subsequently measured using a 515-nm cutoff filter. Fluorescence and
fluorescence anisotropy were measured using a photoelectric modulator
(Hinds) in
/2 mode. Results are reported as either
Aobs or A = Aobs
A0(1
'); Aobs is the observed anisotropy,
A0 is the signal of the oligonucleotide alone,
and
' is the estimated fraction of bound oligonucleotide,
(Aobs
A0)/(Amax
A0), where Amax is the
anisotropy of oligonucleotide complexed to RecA.
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RESULTS |
Functional Characterization of RecA Filament with Fluorescent
Oligonucleotide--
We first determined whether RecA protein
catalyzed the strand exchange reaction with the fluorescein-labeled
oligonucleotide. When the spontaneous background reaction was
subtracted from the data, 47 ± 7% strand transfer was observed
(Fig. 1), which required protein,
ATP
S, and sequence homology (data not shown). The extent of this
reaction was independent of the placement of the fluorescein label in
the ss or ds substrate and identical for reactions carried out at
37 °C and 25 °C within experimental uncertainty (Fig.
1b). These results are consistent with the extent of strand
exchange previously reported for the oligonucleotide without
fluorescein label (8, 9). Hence, the fluorescein label did not appear to perturb the strand exchange reaction, and the subsequent
fluorescence spectroscopy studies were carried out in reaction
conditions where strand exchange takes place.

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Fig. 1.
DNA strand exchange of fluorescent labeled ss
oligonucleotide with unlabeled ds oligonucleotide. a,
the presynaptic complex formed between RecA (15 µM) and
oligonucleotide 1 (45 µM nt) was subsequently
reacted with unlabeled double-stranded oligonucleotide 1/3
(45 µM base pairs) for the indicated times at 37 °C in
the presence or absence of RecA protein and ATP S. Migration of ss
and ds oligonucleotide during SDS-polyacrylamide gel electrophoresis is
indicated to the left of the gel. Circles
are RecA protein, lines are DNA, and F is
fluorescein. b, kinetics of strand exchange at 25 °C
( ) and 37 °C (x). ds DNA formed in the absence of
protein was subtracted.
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The stoichiometry of the reaction was determined from the spectral
changes observed during the titration of fluorescent ss oligonucleotide
by RecA protein (Fig. 2). RecA quenched
fluorescent oligonucleotide 1 by 30 ± 4%. The initial
anisotropy of oligonucleotide, 0.027 ± 0.005, increased to
0.19 ± 0.01, indicating a decreased mobility of the protein-DNA
complex. The extent of spectral changes was independent of ATP
S.
Stoichiometry, determined from the ratio of oligonucleotide:protein
necessary to saturate the signal, was 5.8 ± 0.3 nt/RecA. This
value is in agreement with the results of previous studies showing that
RecA protein has two sites with binding site sizes of 3 nt/RecA
molecule (1, 7).

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Fig. 2.
Fluorescence ( ) and
anisotropy ( ) changes when RecA protein binds to
fluorescent oligonucleotide in the presence (c and
d) or absence (a and
b) of 100 µM
ATP S. Left panels, titration
of 5 µM oligonucleotide 1 with RecA (TMDG, pH
7.5; 25 °C); right panels, titration of complexes in
left panels by NaCl. Fl, fluorescence;
Aobs, Aobs × 10.
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Addition of NaCl to the complex formed in the absence of cofactor
increased the fluorescence and decreased the anisotropy (Fig.
2b). In both cases the final signal was equal to that of free oligonucleotide at these concentrations of NaCl, and the salt
titration midpoint (STMP) was 115 ± 10 mM NaCl. In
the presence of ATP
S (Fig. 2d), addition of NaCl
increased the fluorescence signal by 16 ± 5% (which can be
accounted for by increased oligonucleotide fluorescence in the presence
of salt (24)) and decreased the anisotropy by 11 ± 6%. These
results show that in the absence, but not in the presence, of ATP
S,
NaCl can dissociate oligonucleotide from the complex formed with excess
RecA. A similar effect of cofactor on the stability of complexes with
polynucleotides has been reported, although STMP values are greater for
polynucleotides than for oligonucleotides (26, 27).
Determination of Reaction Conditions for Equilibrium
Binding--
The RecA protein has two DNA binding sites. In the
previous experiments, oligonucleotide bound stoichiometrically to
protein, and the complex formed in the presence of excess RecA,
presumably binding at site I, was stable in the presence of high
concentrations of NaCl. We wished to confirm this result and, if
possible, to find equilibrium conditions for binding to the individual
sites. To this end we attempted to make complexes with fluorescent
oligonucleotide in either site I or site II and titrated them with
various salts to find conditions where oligonucleotide bound
incompletely to protein (Fig. 3). The
anisotropy of the oligonucleotide alone increased with salt
concentration, and this phenomenon was taken into account in the
determination of the STMP.

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Fig. 3.
Salt-dependent fluorescence
anisotropy of fluorescent oligonucleotide 1 in site I and site II.
1 µM RecA and 100 µM ATP S was
equilibrated with 0 ( ), 1.5 µM
( ), 3 µM ( ), 6 µM (x), or 9 µM ( ) unfluorescent
oligonucleotide 2 prior to addition of 3 µM
oligonucleotide 1, a fluorescent oligonucleotide that
has the same sequence. The resulting complex was titrated with
(a) NaCl or (b) MgCl2. Reaction
conditions were as described in the legend to Fig. 2.
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Reacting 1 µM RecA with 3 µM fluorescent
oligonucleotide 1 formed a complex between oligonucleotide
1 and site I (1, 7). Anisotropy of this complex was 0.18 and
decreased only slightly on addition of 800 mM NaCl (Fig.
3a). This result shows that ssDNA binding at site I was
insensitive to NaCl, as suggested by the results in Fig. 2d.
However, when 6 or 9 µM oligonucleotide 2 was
equilibrated with RecA prior to adding oligonucleotide 1, the fluorescence anisotropy decreased with addition of NaCl; the final
anisotropy was the same as for oligonucleotide alone at these salt
concentrations, indicating that all fluorescent oligonucleotide was
displaced (STMP = 170 mM). Equilibration of 1 µM RecA with 1.5 or 3 µM nonfluorescent
oligonucleotide 2 prior to addition of oligonucleotide
1 gave comparable results, although fluorescent
oligonucleotide was not completely displaced. The STMP was the same in
all cases, indicating a second mode of binding to RecA, with different
salt sensitivity than the complex with site I. In the case where 3 µM oligonucleotide 2 was equilibrated prior to
addition of equimolar oligonucleotide 1, the fluorescent
oligonucleotide could not be entirely removed by NaCl, showing that
some oligonucleotide 1 was bound in site I; these results
indicate that although stoichiometric binding at site I is insensitive
to salt, it may be kinetically labile, at least at higher salt concentrations.
Similar experiments were carried out using other salts (Table
I). The STMP value of KCl, 170 mM, was the same as NaCl. Na(CH3C00) was less
efficient (STMP = 240 mM). MgCl2 readily
dissociated these complexes (STMP = 55 mM). High
concentrations of MgCl2 also displaced oligonucleotide
1 from site I (STMP = 450 mM) (Fig.
3b). To confirm this latter observation, 5 µM
fluorescent oligonucleotide 1 was titrated with RecA as in
Fig. 2; at the end of the titration, oligonucleotide was in the
presence of excess RecA and therefore bound primarily at site I. This
complex, which was insensitive to NaCl (Figs. 2d and
3a), was nevertheless dissociated by MgCl2
(STMP = 485 mM) (data not shown). Finally, MgCl2 disrupted a complex of 1 µM RecA, 1.5 µM oligonucleotide 2, and 3 µM
oligonucleotide 1 by a two-step process (Fig.
3b).
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Table I
Salt concentrations required for 50% dissociation of RecA-ss
oligonucleotide complex (100 µM ATP S, TMDG buffer;
25 °C)
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These results demonstrate the existence of two types of oligonucleotide
binding to RecA, with different sensitivities to salts. Oligonucleotide
binding at site I (3 nt/RecA protein) was stable in the presence of
both monovalent cations studied. The second mode of binding, observed
when site I was occupied by unlabeled oligonucleotide, was disrupted by
salt (STMP = 170 mM NaCl). Stoichiometric analysis of
the data is not possible because binding was not quantitative at high
salt concentrations. It nevertheless seems reasonable to suppose that
the second mode of binding corresponds to the reaction of
oligonucleotide with site II.
Equilibrium Constant for the Reaction of Oligonucleotide with Site
II of RecA--
Based on these results, the equilibrium constant for
binding of single-stranded oligonucleotide to site II of the RecA
filament was determined at 25 °C in 150 mM NaCl, 100 µM ATP
S, pH 7.5. The formation of the presynaptic
complex is quantitative in these reaction conditions (see above), and
the concentration of unreacted RecA can be neglected at protein and DNA
concentrations where bound and free oligonucleotides are present.
Consequently, the titration can be described by the equilibrium (Fig.
4a)

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Fig. 4.
a, two-state equilibrium for binding ss
oligonucleotide to site II of the RecA nucleoprotein filament.
Circles are RecA protein monomers; lines are
oligonucleotides; and site I and site II are the bottom and
top of RecA, respectively. b, fluorescence
anisotropy as a function of protein concentration. 3 µM
( ), 6 µM ( ), 9 µM ( ), 12 µM
( ), or 15 µM ( ) fluorescent
oligonucleotide 1 was titrated with RecA protein, 100 µM ATP S, 150 mM NaCl, TMDG, pH 7.5 at
25 °C. Theoretical binding isotherms were calculated using
n = 3 nt/RecA, K = 1.5 ± 0.5 × 106 M 1 (see
"Results").
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where RN1 is the presynaptic complex, RN2 is the complex with ss
oligonucleotides in both site I and site II, and N is the free
nucleotide concentration (M nt). RT is equal to RN1 + RN2, which is the total protein concentration (M
protein monomer). Because RN2 has two DNA binding sites, this complex
enters once into the equation of mass for the protein and twice in the
expression for concentration of nucleotides
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(Eq. 1)
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where NT is the total nucleotide concentration, Nb is the
bound nucleotide concentration (M nt), and n is
the binding site size (nt/protein). The equilibrium constant,
K = [RN2]/[RN1][N], was determined using the
macromolecular binding density function method (25). This approach is
based on the principle that all reaction conditions that produce the
same fractional saturation,
= Nb/NT = n(RN1 + 2·RN2)/NT, will have the same spectroscopic signal. Application of
this approach to the RecA nucleoprotein filament assumes that the
anisotropy signal is the same when fluorescent oligonucleotide is bound
in either site (see "Discussion"). Combining the expressions for
and RT yields Equation 2.
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(Eq. 2)
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We titrated five concentrations of fluorescent oligonucleotide
1 with RecA protein (Fig. 4b). The observed
signal was corrected for the anisotropy of unbound oligonucleotide (see "Experimental Procedures"). Each value of A gave five
pairs of concentrations, {RT, NT}, which have the same fractional
saturation. For anisotropy values in the range of 0.02 to 0.12, plots
of RT as a function of NT for the sets {RT, NT} were linear (data
not shown), and
and RN1 were determined for each plot using
Equation 2.
Each value of
calculated by this method corresponds to a unique
anisotropy, and in this way the dependence of A on
fractional saturation can be determined (Fig.
5a). Results for
A < 0.1 were reproducible in three separate
titrations. The variations observed above this value of A
were not a consequence of nonlinear plots of Equation 2. The plot of
A versus
showed small significant positive
curvature, indicating that the mobility of the fluorophore decreased to
a greater extent at high levels of binding than at low levels, as was
previously observed for ICP8 protein (24). The graph of
as a
function of presynaptic filament concentration RN1 (Fig. 5b)
is a model-free binding isotherm that is independent of the
relationship between A and
(25).

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Fig. 5.
a, fluorescence anisotropy as a function
of fractional saturation. was calculated from protein titration
curves of Fig. 4 using the method of macromolecular binding density
function analysis (25), with binding site size n = 3 nt/protein. b, model-free binding isotherm for
protein titration used in a. The calculated isotherm
( ) is presented with theoretical fits using a
simple two-state thermodynamic model (n = 3 nt/RecA
protein (see "Results")). Upper curve,
K = 1.5 × 106
M 1; middle curve,
K = 1.25 × 106
M 1; lower curve,
K = 1.0 × 106
M 1.
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The model-free binding isotherm was fit using the simple two-state
equilibrium described by REACTION I. For this model,
= (C + 1)/(C + 1 +(1/(2n·K·RN1))), where C = RN1/(2·RN2). We first assumed that C could be ignored
(C
1; (1/K)
N) and fit the isotherm
using Equation 3.
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(Eq. 3)
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Theoretical curves for K = 1.5 × 106 M
1,
K = 1.25 × 106
M
1, and K = 1.0 × 106 M
1
are shown in Fig. 5b. Using these values, C
1 as long as the free nucleotide concentration N
1 µM, which appears to be the case at least for the
titrations of 6, 9, 12, and 15 µM oligonucleotide by low
concentrations of protein.
To confirm this binding constant, we fit the titration curves (Fig.
4b). The calculated fractional saturation (Equation 3) was
transformed into anisotropy using results in Fig. 5a and a value for the plateau of the titration curve,
= 1, of
A = 0.18. RecA concentration was determined using
Equation 2, taking into account dilution of total nucleotide, NT,
during titration. Theoretical curves using n = 3 nt/RecA protein and K = 1.5 ± 0.5 × 106 M
1 fit
experimental data well (Fig. 4b).
Reverse titrations of various concentrations of RecA protein with
fluorescent oligonucleotide were also carried out, and the binding
constant was determined. Binding isotherms (Fig.
6) exhibited a plateau of
Aobs = 0.175 ± 0.01 during the initial
part of the titration curve. Plots of {RT, NT} corresponding to a
constant anisotropy were linear for Aobs between
0.05 and 0.16 (data not shown), and the analysis of the reverse
titration revealed the same relation between anisotropy and fractional
saturation,
, as that in Fig. 5a. Values of RN1
calculated from the intercepts of these plots were small, as expected
for high concentrations of oligonucleotide, and their uncertainty was
too large to determine a model-free binding isotherm.

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Fig. 6.
Fluorescence anisotropy as a function of
oligonucleotide concentration. 0.5 µM
( ), 1 µM ( ), 1.5 µM ( ), 3 µM ( ),
or 5 µM ( ) RecA protein was titrated with
fluorescent oligonucleotide 1; reaction conditions were as
described in the legend to Fig. 4. Experimental isotherm
(symbols) were fit as in Fig. 5 using K = 2.3 ± 0.7 × 106 M 1,
n = 2.8 ± 0.2 nt/RecA, and assuming an anisotropy
for oligonucleotide alone of 0.025.
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However experimental curves for the reverse titration could be directly
fit by the equilibrium used to describe the forward titration by RecA
protein. The calculated fractional saturation was determined using
Equation 3 and transformed into anisotropy as above; oligonucleotide
concentration, NT, was calculated by Equation 2. It is worth noting
that NT is proportional to binding site size, n (Equation 1). Simulated titrations showed that K intervened primarily
in the shape of the curve, whereas n displaced it to lower
or higher oligonucleotide concentrations (data not shown). Hence both
parameters were varied to fit experimental data. Satisfactory agreement
was obtained by n = 2.8 ± 0.2 nt/RecA and
K = 2.3 ± 0.7 × 106
M
1, where uncertainties are the
range of values used to obtain the best visual fits (Fig. 6). Hence
forward and reverse titrations could be described by a simple two-state
model (Fig. 4a; REACTION I and Equations 2 and
3) (K = 1.5 ± 0.5 × 106
M
1); the binding site size
determined by the reverse titration provided an independent
confirmation of the results of stoichiometric binding experiments (Fig.
2).
The NaCl dependence of the equilibrium constant was estimated from the
data for 6 and 9 µM oligonucleotide 2 in Fig. 3a. The signal at various salt concentrations was corrected
for the anisotropy of unbound oligonucleotides using
A0 = 0.03 (see "Experimental Procedures"),
and the resulting anisotropy was converted into fractional saturation
using Fig. 5a. The binding constant was calculated by
assuming that the resulting anisotropy was due to oligonucleotide
binding at site II only (K = (
/(1
))(1/n(RT
(
/n)NT)))). The slope of
the linear parts of the plots of log(K) versus
log([NaCl]),
3.5 ± 0.3, is a function of the electrostatic interactions per protein monomer contributing to the stability of the
nucleoprotein filament (28). A similar calculation for Na(CH3C00) gave
log(K)/
log(Na(CH3C00)) =
2.7 ± 0.1, confirming the influence of anion on the equilibrium
constant, which was suggested by the STMP (Table I).
Finally, we investigated the effect of temperature on the reaction. The
binding constant at 37 °C was determined using the macromolecular
binding density function analysis (25) as above. Four concentrations of
oligonucleotide (3-12 µM) were titrated with RecA
protein in TMDG buffer, 150 mM NaCl, pH 7.5. A slightly positively curved relation between anisotropy and
was observed at
37 °C, similar to results at 25 °C (Fig. 5a)
(Amax = 0.18). This relationship was used to fit
the experimental binding isotherms using the same method as in Fig. 4
(K = 0.45 ± 0.15 × 106
M
1). Binding of oligonucleotide
to site II of the nucleoprotein filament was inhibited by NaCl, and
log(K)/
log([NaCl]) was the same at 25 and 37 °C.
Because the relationship between A and
appeared to be
independent of temperature, titrations of 5 µM oligonucleotide 1 with RecA protein were carried out at various temperatures, and binding constants were determined by fitting
the isotherms (Table II). In all cases,
temperature was measured in the reaction solution before and after
titration. Enthalpy and entropy were calculated from a van't Hoff plot
of these data that was linear (Fig. 7)
(
H =
22.4 ± 2.0 kcal/M;
S =
23.6 ± 3.3 cal/(K·M/ (K is
degrees Kelvin).
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Table II
Equilibrium constants for the titration of RecA protein with
oligonucleotide 1 (100 µM ATP S, 150 mM NaCl, TMDG buffer, pH 7.5) at various temperatures
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Fig. 7.
van't Hoff analysis of equilibrium constant
as a function of temperature. Vertical lines are the
range of values found at 25 and 37 °C (Table II).
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DISCUSSION |
Fluorescence anisotropy of 5'-fluorescein-labeled oligonucleotides
appears to be a useful method to investigate the reaction of DNA with
RecA protein under conditions where DNA strand exchange can occur.
Using this technique we found that the stoichiometry of the
nucleoprotein complex formed between fluorescent oligonucleotide 1 and RecA protein was 5.8 ± 0.3 nt/RecA, independent
of cofactor (Fig. 2), in agreement with previous results for
polynucleotides (1, 3-5, 7) and oligonucleotides (8); the same
stoichiometry was observed in experiments using titration by RecA (Fig.
2) and titration by oligonucleotide (Fig. 6). In addition, the
oligonucleotide-RecA complex formed with excess protein was disrupted
by NaCl in the absence of cofactor, whereas it was stable in the
presence of ATP
S (Fig. 2), in agreement with experiments with
polynucleotide-RecA complexes (26, 27). These results show that the
observed fluorescence anisotropy changes reflect DNA binding in the
nucleoprotein filament. Finally, the oligonucleotide with fluorescein
at its 5'-extremity was a substrate for RecA-catalyzed strand exchange
in vitro (Fig. 1). The reaction required protein, ATP
S,
and sequence homology, as expected. RecA protein carried out strand
exchange with fluorescein-labeled oligonucleotide with an efficiency
comparable with that with unlabeled oligonucleotides (8, 9). Hence the
fluorescein label had no significant effect on protein-DNA interactions
necessary for strand exchange. These results show that fluorescence
anisotropy of this oligonucleotide can be used to study protein-DNA
interactions in a productive RecA nucleoprotein filament.
Fluorescence anisotropy is not proportional to fluorophore
concentration (29), and it was important to experimentally determine the relationship between spectroscopic signal and fractional
saturation,
. Macromolecular binding density function analysis (25)
is a useful method to address this question (24). The starting point of
this approach is that the signal is a function of fractional saturation. RecA has two binding sites, and straightforward application of this method requires that the anisotropies of fluorescent
oligonucleotide in sites I and II be the same. The following
observation suggested that this was the case. The filament with two
fluorescent oligonucleotides, RN2, (which is present when the
anisotropy signal saturates in tight binding conditions) rearranged
upon further addition of RecA to produce a filament with a single
oligonucleotide in site I, RN1 (Fig. 2); the identification of this
species as RN1 was consistent with its stability in NaCl and
MgCl2 (Figs. 2d and 3). The constant signal
above saturating RecA concentrations indicates that fluorescent
oligonucleotide had the same anisotropy in both filaments RN1 and RN2.
Macromolecular binding density function analysis showed that the slight
sigmoidal shape in the experimental binding isotherms (Fig.
4b) was a result of the nonlinear relation between
A and
(Fig. 5a) and did not reflect a
property of the binding process, such as cooperativity (30).
During homologous recombination, RecA protein reacts with two DNA
molecules, and it was proposed many years ago that the protein therefore has two functional DNA binding sites. Early studies showed
that site I binds ssDNA to form a presynaptic filament and that site II
binds complementary dsDNA (6). In this model, site II would contain
ssDNA after strand exchange, and tight binding of ss product DNA at
this site may drive the reaction (9). The amino acids corresponding to
site II have been investigated. We recently observed that mutant RecA
protein, RecAE207Q, with a single amino acid substitution
in the L2 loop, lacked one of the two ssDNA binding sites of wild type
protein; the remaining site was functional, and biochemical activities
indicated that the mutant protein possessed an active primary site
(20). Taken together with structural studies, cross-linking
experiments, and properties of mutant RecA proteins (10-21), these
results suggest that the L2 loop may be at or near the secondary
binding site. The reaction between DNA and peptides representing the L2
loop has been studied (31, 32). Here we report a thermodynamic analysis
of ssDNA binding at site II of the protein.
Site II can be distinguished from site I by its weaker DNA binding
properties. It has previously been reported that, in the presence of
ATP
S, ss oligonucleotides and E. coli ssDNA
binding protein, SSB, displace ssDNA from site II of the filament but not from site I (8, 9). We report here evidence that ss oligonucleotide
binding at site II is also more sensitive to salt than is binding at
site I. Titration of RecA-oligonucleotide complexes with various salts
revealed two binding modes, which differed by their stability at high
salt concentrations (Figs. 2 and 3; Table I). Oligonucleotide binding
at site I was shown to be insensitive to monovalent salts. Because RecA
has two binding sites, 3 nt/protein monomer (Fig. 2; Refs. 1 and 7),
the salt-sensitive reaction corresponds to binding at site II.
Quantitative binding of oligonucleotide to RecA at site I in the
presence of ATP
S was not disrupted by 150 mM NaCl (Figs. 2 and 3a; Table I); therefore, in these solution conditions, when oligonucleotide concentration is sufficiently high so that free
oligonucleotide is present, the concentration of unreacted RecA can be
ignored, and equilibrium DNA binding can be described as a reaction
between ssDNA and the presynaptic complex (Fig. 4a).
Fractional saturation was calculated from a two-state model for this
equilibrium (Equation 3), assuming (1/K)
N).
Most of the titrations occurred in these limits, and a value for the
binding constant of K = 1.5 ± 0.5 × 106 M
1 gave a good
fit for 3-15 µM oligonucleotide titrated with RecA at
25 °C (Figs. 4b and 5b). A similar binding
constant was found for the reverse titration of RecA with
oligonucleotide (Fig. 6).
This reaction was favored by enthalpy and opposed by entropy.
Similar results are reported for the reaction of single-stranded DNA-binding protein gp32 with DNA (34). In contrast, both entropy and
enthalpy contribute to the reaction of ssDNA with the herpes ssDNA-binding protein ICP8 (24). Displacement of positively charged
counter-ions from the polynucleotide by positively charged proteins
increases entropy and can stabilize protein-DNA interactions (28).
Negative entropy indicates that this mechanism does not contribute
significantly to our reaction. In support of this hypothesis, it is
worth noting that the binding of ssDNA with the L2 loop peptide has
only a small electrostatic contribution and is stabilized primarily by
nonelectrostatic interactions (31).
Likewise, the different capacities of Cl
and
CH3COO
anions to inhibit the reaction cannot
be explained by displacement of positively charged counter-ions.
Rather, these salt effects probably involve displacement of anions from
the protein (33, 34). These results imply that, in the intact
presynaptic complex, anion-sensitive protein conformations or
protein-protein interactions may be important for tight binding of
ssDNA at site II.
The techniques developed in this study should be useful for
investigating the thermodynamic basis of homologous recombination. The
simplest model of this reaction would be an equilibrium between a
reactive filament (with ssDNA at site I and dsDNA at site II) and a
product filament (with dsDNA at site I and ssDNA at site II). We report
here the equilibrium constant for the reaction of ssDNA at site II of a
nucleoprotein filament in which ssDNA also occupied site I. Work is in
progress to extend this approach to RecA nucleoprotein filaments that
are potential intermediates of the strand exchange reaction.