From the Division of Biological Sciences, Sections of Microbiology and of Molecular and Cell Biology, University of California, Davis, California 95616-8665
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ABSTRACT |
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Saccharomyces cerevisiae Rad51
protein is the paradigm for eukaryotic ATP-dependent DNA
strand exchange proteins. To explain some of the unique characteristics
of DNA strand exchange promoted by Rad51 protein, when compared with
its prokaryotic homologue the Escherichia coli RecA
protein, we analyzed the DNA binding properties of the Rad51 protein.
Rad51 protein binds both single-stranded DNA (ssDNA) and
double-stranded DNA (dsDNA) in an ATP- and
Mg2+-dependent manner, over a wide range of pH,
with an apparent binding stoichiometry of approximately 1 protein
monomer per 4 (±1) nucleotides or base pairs, respectively. Only dATP
and adenosine 5'- Homologous recombination is a ubiquitous biological process.
Elucidation of this process has come mostly from detailed analysis of
Escherichia coli RecA protein, a key enzyme in both
homologous genetic recombination and recombinational repair of damaged
DNA in the eubacteria. RecA-like proteins have been found in a variety of eukaryotes, from yeast to human. The first identified and the most
extensively studied eukaryotic RecA protein-analog is the Rad51 protein
of Saccharomyces cerevisiae, which is establishing the
paradigm for RecA protein-like functions in the eukaryotes. The
RAD51 gene of S. cerevisiae is required for
mitotic and meiotic recombination (1) and for the repair of
double-strand DNA breaks caused by ionizing irradiation (2).
Biochemical studies of Rad51 protein demonstrated that it shares
similar properties to RecA protein, including
ssDNA1-dependent
ATPase activity, homologous pairing, and DNA strand exchange activity
(3-5), and the formation of helical filaments on both double-stranded
DNA (dsDNA) and single-stranded DNA (4, 6) (for review, see Ref. 7).
In vitro, the most extensively investigated DNA pairing
reaction is the three-strand exchange reaction, in which the pairing of
circular ssDNA and homologous dsDNA yields nicked circular dsDNA and
linear ssDNA. During the formation of a presynaptic complex in the
first phase of the reaction, the RecA protein polymerizes on ssDNA in
the 5' All DNA strand exchange proteins follow this general scheme for DNA
strand exchange; however, the eukaryotic proteins (Rad51 protein for
S. cerevisiae and human) display some unique characteristics (3-5, 10, 11). The DNA strand exchange reaction is very sensitive to
Rad51 protein concentration, and it occurs only in a narrow range of
protein concentration, which corresponds to a stoichiometry of 3-4
nucleotides of ssDNA per Rad51 protein monomer; an excess of Rad51
protein severely inhibits DNA strand exchange (4). Another important
distinction is that, in contrast to the RecA protein-promoted reaction,
Rad51 protein-promoted DNA strand exchange is almost totally dependent
on the presence of a ssDNA-binding protein, either eukaryotic
replication protein-A (RPA) or prokaryotic ssDNA-binding protein (SSB)
(4, 5). Furthermore, in the presence of ATP In this paper, we describe the DNA binding properties of Rad51 protein
from S. cerevisiae. These properties offer insight into the
specific requirements for the DNA strand exchange reaction that were
described above.
Reagents--
All chemicals were reagent grade; solutions were
made using Barnstead NANOpure water. ATP, ADP, CTP, UTP, GTP, dATP,
TTP, dCTP, and dGTP were purchased from Sigma. ATP DNA--
Both ssDNA and dsDNA from M13 mp7 phage (viral (+)
strand and replicative form I) were purified as described (14). M13
dsDNA was linearized by digestion with EcoRI. Etheno M13 DNA
was prepared as described (15).
Construction of the Rad51 Protein-overproducing Plasmid--
For
overproduction of the Rad51 protein in E. coli, the
RAD51 gene was cloned under the control of the T7 promoter
in the pET3a vector to form the plasmid pEZ5139. The
Nde-BamHI fragment that contains the RAD51 gene
was isolated from plasmid YEP13/Rad51-23 (16). Because the
RAD51 gene contains several sites for NdeI restriction endonuclease, the desired Nde-BamHI fragment
(1470 bp) was reconstituted from two independently purified fragments, NdeI-Bsu36I (230 bp) and
Bsu36I-BamHI (1240 bp). Both fragments were
ligated with the NdeI-BamHI-digested vector,
pET3a (17). After transformation of E. coli strain
DK1( Proteins--
RecA protein was purified from E. coli
JC12772 (18) using a preparative protocol based on spermidine acetate
precipitation (19). SSB protein was purified as described (20), and RPA was purified as described (5, 21). S. cerevisiae Rad51
protein was purified from E. coli strain ENZ21 (a
Rad51 protein was also purified from S. cerevisiae strain
LP2749-B, harboring pR51.3 (kindly supplied by Dr. P. Sung) (3) using
the same protocol described above for the isolation of Rad51 protein
from E. coli. Rad51 protein was eluted from MonoQ column in
two separate peaks: 70% of the protein eluted at about 400 mM KCl (analogously to Rad51 protein from E. coli) and 30% of the protein eluted at about 500 mM
KCl. We believe, but have not investigated the possibility further,
that the protein in the second peak could have been
post-translationally modified (e.g. perhaps phosphorylated).
The proteins from both peaks displayed similar DNA binding and DNA
strand exchange activities. The yield of Rad51 protein purified from
the yeast cells was about 3 mg from 80 g cells.
Etheno M13 DNA Binding Assay--
Etheno M13 DNA fluorescence
was measured by exciting at 300 nm and monitoring at 405 nm using an
SLM-Aminco 8000 fluorescence spectrophotometer (15). Protein titrations
of etheno M13 DNA and salt titrations of the protein-etheno M13 DNA
complexes were performed by adding aliquots of protein or concentrated
salt (NaCl) solutions, respectively, to samples in reaction buffer (30 mM Tris acetate (pH 7.5) or Na-MES (pH 6.2), 0.1 mM DTT, 10 mM magnesium acetate). The
concentration of etheno M13 DNA was 3 µM nucleotides (or
as indicated), and the concentration of nucleotide cofactors was 2 mM, unless otherwise indicated. An ATP-regenerating system (10 units/ml pyruvate kinase and 3 mM phosphoenolpyruvate)
was added to reactions with RecA protein but was omitted for Rad51 protein. Reactions were carried out at 37 °C with continuous
stirring. The relative fluorescence increase (RFI) of etheno M13 DNA
due to protein binding was measured as the ratio of the fluorescence increase upon the protein binding to the fluorescence of the free etheno M13 DNA and free protein. For salt titration experiments, the
percentage of remaining protein-ssDNA complexes was determined by
dividing the fluorescence of the protein-DNA complex, before salt
titration, by the fluorescence of this complex after the salt titration
(with corrections for dilution).
dsDNA Binding Assay--
The fluorescence of dsDNA-DAPI or
dsDNA-EtBr complexes was measured by exciting at 359 nm and monitoring
at 461 nm for DAPI, and 510 and 595 nm for EtBr, respectively
(22).3 The binding of Rad51
protein or RecA protein to the dsDNA-DAPI (or dsDNA-EtBr) complexes
causes the displacement of dye from the dsDNA, resulting in a
fluorescence decrease. Reactions were performed in buffer containing
either 30 mM Tris acetate (pH 7.5) or 30 mM
Na-MES (pH 6.2) and 0.1 mM DTT; magnesium acetate and ATP
concentrations were as indicated. The concentration of DAPI was 200 nM and that of EtBr was 2 µM. The
concentration of M13 dsDNA was 6 µM (nucleotides). The
experiments were conducted at 37 °C with continuous stirring. The
extent of Rad51 (RecA) protein binding to dsDNA was determined from the
ratio of the observed fluorescence signal upon the addition of the
protein, relative to the initial fluorescence of the DAPI-dsDNA complex.
Agarose Gel Mobility Shift Assay--
M13 DNA was mixed with
varying concentrations of Rad51 protein in the indicated buffers in a
total volume of 20 µl and incubated at 37 °C for 30 min. Depending
on the experiment, magnesium acetate and ATP either were present in the
buffer or were added subsequently. When added separately, magnesium
acetate or ATP were added, and incubation was continued for an
additional 30 min. Sample loading buffer (3 µl of 40 mM
Tris acetate (pH 7.5), 50% glycerol, and a trace of bromphenol blue)
was added to each sample, and the complexes were separated by
electrophoresis through 0.8% agarose gel in TAE buffer (40 mM Tris acetate, 2 mM EDTA (pH 8.0)) at 120 V
for 2 h and were visualized by EtBr staining.
Binding of Rad51 Protein to Etheno M13 ssDNA--
In the presence
of ATP and Mg2+, the binding of Rad51 protein to etheno M13
DNA results in an increase in the fluorescence of this modified M13 DNA
(2). This increase reaches a maximum when the stoichiometric ratio
between Rad51 protein and ssDNA corresponds to 1 monomer of Rad51
protein to approximately 4 (±1) nucleotides of ssDNA (Fig.
1). The relative fluorescence increase (RFI) value for Rad51 protein is 3.6-3.8, a value that is slightly lower than that for RecA protein (4.2), with this preparation of etheno
M13 DNA, and under the same experimental conditions.
In the absence of ATP at pH 7.5, Rad51 protein does not show any
significant increase in etheno M13 DNA fluorescence; this is unlike the
behavior of RecA protein, which produces an RFI value in the absence of
ATP of about 2 (not shown). The slight apparent increase in
fluorescence upon addition of Rad51 protein in the absence of ATP shown
in Fig. 1 is probably due to light scattering from aggregation of Rad51 protein.
We discovered that preincubation of Rad51 protein in Tris acetate
buffer at pH values higher than 7.0, containing 10 mM
magnesium acetate but without ATP, inactivates the protein with regard
to DNA binding (Fig. 2). The subsequent
addition of ATP to such inactivated protein failed to restore binding
activity to Rad51 protein. However, the prior presence of ATP under
these conditions prevents the inactivation of Rad51 protein; the
fluorescence increase after addition of etheno M13 DNA is the same as
in the protein titration experiment (see Fig. 1).
Sensitivity of Rad51 Protein-Etheno M13 DNA Complexes to
Dissociation by NaCl--
The sensitivity of protein-DNA complexes to
disruption by NaCl is a relative measure of the affinity of a protein
for ssDNA (24). After formation of the Rad51 protein-etheno M13 DNA
complex, its stability to dissociation by an increasing concentration
of NaCl was examined. A decrease in fluorescence is observed upon the
addition of NaCl until the complete dissociation of the protein-DNA complex occurs (Fig. 3). The salt
titration midpoint for dissociation of the Rad51 protein in the
presence of ATP and 10 mM magnesium acetate is about 550 mM NaCl; for comparison, the salt titration midpoint for
RecA protein under identical conditions is about 700 mM
NaCl (25) (data not shown).
Dependence of Rad51 Protein-Etheno M13 DNA Complex Formation on NTP
Cofactors--
The effects of different nucleotide cofactors on the
binding of Rad51 protein to etheno M13 DNA was investigated (Table
I). In addition to ATP, for which the RFI
value for the complex was 3.6-3.8 (Fig. 1 and Table I), only dATP and
ATP
Unlike RecA protein (15), ATP pH Dependence of Rad51 Protein Binding to ssDNA--
In the
presence of ATP and Mg2+, no significant changes in the
binding activity of Rad51 protein to etheno M13 DNA were observed upon
changing the pH values over the range of 6.0-8.5 (Fig.
4). However, in the absence of ATP, an
increase in etheno M13 DNA fluorescence upon Rad51 protein addition was
detected only at pH values below 6.8. Furthermore, at these acidic pH
values, the increase in etheno M13 DNA fluorescence upon the binding of
Rad51 protein occurred in the absence of both nucleotide cofactor and magnesium ion. Without ATP and magnesium acetate, the maximum increase
in etheno M13 DNA fluorescence is observed at pH 6.2, and no increase
is detected at any pH values above 7.0. At a pH lower than 5.0, Rad51
protein seems to inactivate; an initial increase in fluorescence is
seen immediately after Rad51 protein addition to the cuvette, but it
gradually decreases to the fluorescence of free etheno M13 DNA (data
not shown). The chemical nature of buffers did not affect the binding
activity of Rad51 protein, because buffers with overlapping pH values
(sodium citrate (pH 5.0-6.2), Na-MES (pH 5.8-7.2), and Tris acetate
(pH 6.8-8.5)) showed no difference in RFI.
Titrations of etheno M13 DNA with Rad51 protein at pH 7.5, pH 6.6, and
pH 6.2 (Fig. 5) revealed that both the
RFI value and the apparent binding stoichiometry are affected by pH and
by the presence of both ATP and magnesium acetate. In the presence of ATP and magnesium ion, Rad51 protein binds to etheno M13 DNA over a
wide range of pH (the RFI value is 3.8 over the pH range of 6.0-8.0
(Fig. 4)), with an apparent binding stoichiometry of 1 protein monomer
per
The increase in RFI value with decreasing pH suggests either that the
binding affinity of Rad51 protein to etheno M13 DNA is increasing or
that the structure of the protein-DNA complex is changing. Since the
salt titration midpoint is a relative measure of DNA binding affinity
(15), the stability of Rad51 protein-etheno M13 DNA complexes, formed
at different pH values, to disruption by NaCl was examined (Fig.
6). The salt titration midpoint values are 60 mM NaCl and 110 mM NaCl at pH 6.6 and pH
6.2, respectively, indicating that the interaction of Rad51 protein
with ssDNA in the absence of ATP and magnesium acetate is stronger at
reduced pH. When compared with the results that were presented in Fig. 3, it is evident that the stability of complexes formed in the presence
of both ATP and magnesium acetate is higher than those formed in the
absence of ATP cofactor and magnesium acetate (midpoints of 550 mM NaCl and 110 mM NaCl, respectively), which
correspond to the behavior of RecA protein (15).
To verify these fluorescence measurements, Rad51 protein-native M13
ssDNA complexes were examined using a gel mobility shift assay (Fig.
7). The mobility of Rad51 protein-ssDNA
complexes, formed with increasing concentrations of Rad51 protein in
the absence of ATP and magnesium acetate at pH 6.2, was different than
the mobility of free DNA; this change in mobility paralleled the
fluorescence titration shown in Fig. 5. Also in agreement with the
fluorescence data, only a slight change in mobility of M13 ssDNA was
observed upon addition of Rad51 protein at pH 7.5 in the absence of ATP
and magnesium acetate, showing that the absence of an increase in
fluorescence of etheno M13 DNA was due to an absence of complex
formation and not just to a failure to induce a change in
fluorescence.
Effect of Mg2+ on the Formation of Rad51 Protein-ssDNA
Complexes--
The requirement for Mg2+ in Rad51
protein-etheno M13 DNA complex formation shows a dependence on both the
presence of ATP and the pH value (Fig.
8). In the presence of ATP, at neutral pH
(pH 7.5), no binding was detected until the Mg2+
concentration reached 2 mM; however, at an acidic pH (pH
6.2), Rad51 protein bound to etheno M13 DNA in the absence of magnesium acetate to yield an RFI value of 3.2. The RFI displayed the curious trend of initially decreasing from the RFI of 3.2 at 0 mM
Mg2+ to 2.6 at 4 mM Mg2+ but then
increasing at higher Mg2+ concentrations and finally
saturating at 7-10 mM magnesium acetate. Titrations of
etheno M13 DNA with Rad51 protein in the presence of ATP (at pH 6.2)
also revealed a change in the apparent binding stoichiometry that
depends on Mg2+ concentration; the apparent binding
stoichiometry changes from 6:1 (nucleotides of ssDNA per protein
monomer) at 1-2 mM magnesium acetate to a stoichiometry of
about 4:1, at 7-10 mM magnesium acetate (data not
shown).
In the absence of ATP, Rad51 protein does not change the etheno M13 DNA
fluorescence at pH 7.5, regardless of the magnesium ion concentration,
but, at pH 6.2, the maximum RFI value is seen at 0 mM
magnesium acetate (Fig. 8). Increasing the magnesium acetate concentration in the absence of ATP at pH 6.2 reduces the RFI (compare
the RFI value of 3.2 at 0 mM and 1.8 at 10 mM
magnesium acetate) and, by inference, the binding affinity of Rad51
protein to etheno M13 DNA.
Finally, a mobility shift assay (Fig. 9)
shows that the migration of the Rad51 protein-ssDNA complexes formed at
these experimental conditions is different. The Rad51 protein-ssDNA
complexes formed in the presence of magnesium acetate and ATP
(lanes 8-10) migrated faster than ones formed in the
presence of ATP alone (lane 5) and in the absence of
magnesium acetate and ATP (lane 1). In the presence of ATP,
the Rad51 protein-ssDNA complexes formed at 4 mM magnesium
acetate had a mobility faster than the complexes formed at 1 and 10 mM magnesium acetate (compare lane 9 to
lanes 8 and 10). This complicated dependence of
Rad51 protein-ssDNA complex mobility on magnesium ion concentration
parallels the dependence of RFI (see Fig. 8) on Mg2+
concentration; the lowest RFI is seen at 4 mM magnesium
acetate. This might indicate that a
Mg2+-dependent transition between two different
modes of Rad51 protein binding to ssDNA occurs at about 4 mM magnesium acetate.
Two Different Binding Modes of Rad51 Protein to ssDNA Are Not
Interconvertible--
The previous binding data (Figs. 5-8) suggest
that Rad51 protein exhibits two different modes of binding to ssDNA,
which depend on ATP and Mg2+ concentration. These two
complexes have different apparent binding stoichiometries and different
mobilities through agarose gels. To test whether one binding mode can
be converted to the other, the Rad51 protein-ssDNA complexes that form
in the absence of ATP and Mg2+ were incubated with
subsequently added ATP alone (Fig. 9, lane 6), magnesium
acetate alone (lanes 2 and 3), or both ATP and
magnesium acetate (lane 7). Fig. 9 shows that these
complexes are not simply interconvertible; the mobility of the existing
complexes does not change after addition of ATP and
Mg2+.
Binding of Rad51 Protein to dsDNA--
To investigate the binding
of Rad51 protein to dsDNA, we employed both a dye-displacement assay,
and a gel mobility shift assay. The dyes used were DAPI and EtBr. DAPI
interacts with dsDNA in the minor groove and EtBr intercalates between
bases of dsDNA to produce dye-dsDNA complexes with increased
fluorescence (26, 27). These fluorescent dye-dsDNA complexes were used
previously to study RecA protein binding to dsDNA by monitoring the
decrease in fluorescence upon protein binding (22).3 The
binding of Rad51 protein to dye-dsDNA complexes results in the
displacement of the dye from dsDNA and, consequently, the fluorescent
signal decreases (Fig. 10, A
and B).
The binding of Rad51 protein to dsDNA is also magnesium
ion-dependent. As can be seen in Fig.
11, in the presence of ATP, Rad51 protein binds dsDNA poorly at 1-3 mM magnesium acetate. An
increase in the magnesium ion concentration beyond 4 mM
magnesium acetate results in an increase in the apparent affinity of
Rad51 protein for dsDNA, with apparent saturation occurring above 10 mM magnesium acetate. At these saturating concentrations of
magnesium acetate, the extent of Rad51 protein binding is the same for
both the DAPI and EtBr assays (Fig. 11, A and B),
and the apparent binding stoichiometry is unchanged at approximately 1 monomer per 4.5 (±0.5) bp, whereas at the lower concentrations of
magnesium acetate, the binding of Rad51 protein becomes progressively
more sigmoid (Fig. 10B and other data not shown). The
efficiency of EtBr displacement from dsDNA by Rad51 protein at low
Mg2+ concentrations (1-3 mM magnesium acetate)
is higher than the displacement of DAPI (Fig. 11, A and
B); this may reflect differing affinities and modes of
binding for each of these dyes and is consistent with DAPI being a more
effective competitor than EtBr of Rad51 protein-dsDNA complex
formation. Furthermore, in the presence of ATP, the binding of Rad51
protein to dsDNA is equally effective at pH 6.2 and at pH 7.5 (Fig. 11,
A and B).
In the absence of ATP, the DAPI displacement assay shows that Rad51
protein does not eject DAPI from dsDNA, regardless of pH (Fig. 11,
A and B and Fig.
12). However, displacement of EtBr from
dsDNA is observed at pH 6.2 but not at pH 7.5 (Fig. 11B and Fig. 12). This is also consistent with the idea that EtBr is more readily displaced or actually facilitates Rad51 protein binding, as it
does for RecA protein (28). The extent of EtBr displacement upon Rad51
protein binding is about 50%, and the apparent binding stoichiometry
is different than in the presence of ATP (1 Rad51 protein monomer per 6 bp of DNA versus 1 per 4.5 bp, respectively). In the absence
of ATP, the maximum extent of EtBr displacement occurs in the absence
of magnesium acetate and decreases with increasing magnesium ion
concentration (Fig. 11B), presumably due to a reduction in
binding affinity of Rad51 protein to dsDNA. Interestingly, in the
absence of ATP and magnesium ions, RecA protein binds dsDNA ejecting
EtBr but not DAPI, exhibiting a behavior similar to Rad51 protein (Fig.
12). Analogous results were obtained using the mobility shift assay
(see Fig. 13); complexes of Rad51 protein and M13 dsDNA formed in the presence of ATP at pH 7.5 and at pH
6.2 exhibit the same mobility through agarose gels (Fig. 13,
lanes 4 and 5). However, in the absence of ATP
and magnesium acetate, the binding of Rad51 protein to dsDNA is
pH-dependent. The formation of Rad51-dsDNA complexes in the
absence of ATP and magnesium acetate occurred only at reduced pH and
was not detected at pH 7.5 (Fig. 13, lanes 2 and
3). This difference suggests the existence of two different
modes of Rad51 protein binding to dsDNA, and these modes are dependent
on the presence or absence of ATP.
In this study, we examined the binding of Rad51 protein to ssDNA
and dsDNA using both spectrofluorimetric and gel assays. The speed and
convenience of the fluorimetric etheno M13 DNA binding assay permitted
a rapid survey of binding behavior, and although indirect, the
fluorimetric assay produced results that coincided with the more direct
but tedious gel mobility shift assay. This correspondence suggests
that, for Rad51 protein, an increased fluorescence signal in the etheno
M13 DNA binding assay generally coincides with an increased amount of
protein-ssDNA complex. Based on these two assays, we make several
conclusions about the DNA binding properties of Rad51 protein. At pH
7.5, the protein binds to ssDNA in an ATP- and
Mg2+-dependent manner, with an apparent binding
stoichiometry of about 1 Rad51 protein monomer per 4 nucleotides of
DNA. However, in the absence of ATP, Rad51 protein shows a unique pH
dependence of ssDNA binding. In contrast to RecA protein, Rad51 protein
does not bind ssDNA in the absence of ATP at pH 7.5. Instead, it
irreversibly loses DNA binding activity when magnesium acetate is
present, probably due to aggregation, since the subsequent addition of ATP to Rad51 protein does not restore DNA binding activity (Fig. 2).
Strangely, this inactivation does not occur at pH 6.2; rather, in the
absence of ATP, Rad51 protein binds to ssDNA at acidic pH values,
saturating at an apparent binding stoichiometry of 1 protein monomer
per 7-9 nucleotides of DNA. Thus, Rad51 protein exhibits two different
ssDNA-binding modes, one ATP-dependent and the other
ATP-independent, with the transition controlled by the bound nucleotide
cofactor. Moreover, these ssDNA binding modes are not inter-convertible
(Fig. 9), a behavior similar to that observed for RecA protein
(29-31).
The stability of Rad51 protein-ssDNA complexes was examined using salt
titration experiments. The salt titration midpoint for the dissociation
of Rad51 protein- and RecA protein-etheno M13 DNA complexes was found
to be 550 and 700 mM NaCl, respectively. Without knowledge
of the salt concentration dependence of the affinity constant, it is
not possible to compare the apparent affinities at these two different
salt concentrations. However, the data do show that ATP induces a high
affinity, high RFI (i.e. extended) Rad51 protein-ssDNA
complex with characteristics similar to those of RecA protein (9).
The most notable difference between RecA and Rad51 proteins is in their
dsDNA binding activities. To bind dsDNA, RecA protein must overcome a
kinetic barrier that limits nucleation on dsDNA. Therefore, at neutral
pH values, the binding of RecA protein to dsDNA is so slow that it is
practically negligible (32). Reduced pH (pH 6.2) greatly facilitates
the nucleation step and, consequently, the binding to dsDNA (33, 34).
In contrast to RecA protein, Rad51 protein much more easily circumvents
the slow nucleation step and binds to dsDNA in a pH-independent manner.
Interestingly, Rad51 protein binds dsDNA poorly at low Mg2+
concentrations (1-3 mM), conditions that facilitate dsDNA
binding for RecA protein. In the absence of ATP, both proteins bind to dsDNA only at reduced pH 6.2, with an apparent binding stoichiometry of
approximately 1 protein monomer per 4.5 bp of DNA. Binding to dsDNA at
this pH for both Rad51 and RecA proteins is accompanied by displacement
of EtBr but not DAPI. In contrast, the ATP-dependent binding of RecA and Rad51 proteins results in the displacement of both
EtBr and DAPI. The ATP-dependent binding is likely to occur
through the minor groove of dsDNA, given that DAPI is a minor groove
binder. Therefore, as for ssDNA, Rad51 protein displays two different
modes of dsDNA binding that are modulated by ATP binding, and these two
modes differ from each other in binding stoichiometry.
Although the intracellular pH of S. cerevisiae is in the
range of 6.15-6.6 (35, 36), the ability of Rad51 protein to bind DNA
in the absence of ATP at low pH conditions is unlikely to be
physiologically significant. However, in vitro, this
characteristic may be useful; for example, we successfully used a
change in pH from 6.2 to pH 7.5 to elute Rad51 protein from
ssDNA-cellulose as a useful purification step.2
Interestingly, both Rad51 and RecA proteins show significant similarities in DNA binding properties at reduced pH conditions; conversely, at neutral pH, both the efficient binding of Rad51 protein
to dsDNA in the presence of ATP and the inability to bind ssDNA in the
absence of ATP differentiate Rad51 protein from RecA protein.
The DNA binding properties of Rad51 protein explain some features of
the DNA strand exchange mediated by this protein. The formation of an
active and contiguous nucleoprotein filament on ssDNA is the first step
of recombination. In contrast to the RecA protein-dependent
reaction, DNA strand exchange mediated by Rad51 protein is nearly
absolutely dependent on RPA (or SSB protein) function (3, 5), a protein
whose role is to remove DNA secondary structure from ssDNA (37-39). In
the absence of RPA, it is likely that Rad51 protein binds to and
stabilizes dsDNA within the regions of ssDNA containing secondary
structure. The Rad51 protein-dsDNA complexes formed within regions of
secondary structure are probably stable since Rad51 protein displays
both a high affinity for dsDNA and a low rate of redistribution. This
conclusion is based on the fact that the fluorescence of either the
etheno M13 DNA-Rad51 protein or the DAPI-dsDNA-Rad51 protein complexes
does not change upon the subsequent addition of a 3-fold excess of
competitor DNA (either dsDNA or ssDNA, respectively) during the time
scale of a typical DNA strand exchange reaction.2 In
addition, a low rate of Rad51 protein monomer redistribution is
expected due to its low ATPase activity; hence, the Rad51 protein-dsDNA complex formed on DNA secondary structure is more stable than the ssDNA
complex because of a 2.5-10-fold lower rate of
dsDNA-dependent ATP hydrolysis compared with the
ssDNA-dependent rate (3, 5). Therefore, the secondary
structure within ssDNA that is stabilized by Rad51 protein contributes
to the low efficiency of DNA strand exchange promoted by Rad51 protein
in the absence of a ssDNA-binding protein. The same considerations
potentially explain the higher activity of DNA strand exchange mediated by Rad51 protein is very sensitive to an
excess of protein (4). The binding of Rad51 protein to dsDNA inhibits
DNA strand exchange by forming nucleoprotein filaments on both DNA
partners in the reaction. Our DNA binding data explain why presynaptic
complex formation is optimal at 3-4 mM Mg2+
ion concentrations (4, 12); at these conditions, the inhibitory effect
of the DNA secondary structure is minimized because the stability of
DNA secondary structure is low and the binding of Rad51 protein to
dsDNA is minimal (Fig. 8 and Fig. 11).
In a recent paper, the binding stoichiometry of Rad51 protein to etheno
M13 DNA was reported as 1 protein monomer per 5.5 to 7 nucleotides
(41), values which, considering experimental error, may overlap with
our own. In addition, there might be some systematic differences
arising from the different Rad51 proteins used. In that work, Rad51
protein was purified from a baculovirus expression system, and this
protein lacked 22 amino acid residues from the N terminus due to an
alternative site of translation initiation (40).
In sum, we have demonstrated the existence of two different DNA binding
modes for Rad51 protein; the mode utilized depends on a complex
interplay of pH and of ATP and Mg2+ concentration. The DNA
binding properties of yeast Rad51 protein, as discussed above, explain
some of the known differences in the requirements of, and the
conditions for, DNA strand exchange promoted by Rad51 protein when
compared with the RecA protein-mediated reaction. The limitations of
DNA strand exchange mediated by Rad51 protein clearly suggest the
requirements of auxiliary proteins needed to enhance the efficiency of
this reaction. The studies reported here may provide a basis for
understanding the mechanism of the stimulatory effects of Rad55/Rad57
proteins (42), Rad52 protein (43-45), and Rad54 protein (23) and,
hence, represent a first step in obtaining the complete picture of
yeast homologous recombination.
-(thiotriphosphate) (ATP
S) can substitute for
ATP, but binding in the presence of ATP
S requires more than a 5-fold
stoichiometric excess of protein. Without nucleotide cofactor, Rad51
protein binds both ssDNA and dsDNA but only at pH values lower than
6.8; in this case, the apparent binding stoichiometry covers the range
of 1 protein monomer per 6-9 nucleotides or base pairs. Therefore,
Rad51 protein displays two distinct modes of DNA binding. These binding
modes are not inter-convertible; however, their initial selection is
governed by ATP binding. On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA
strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and
synaptic phases of the DNA strand exchange reaction as follows: during
presynapsis, Rad51 protein interacts with and stabilizes secondary
structures in ssDNA thereby inhibiting formation of a contiguous
nucleoprotein filament; during synapsis, Rad51 protein inactivates the
homologous dsDNA partner by directly binding to it.
INTRODUCTION
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Abstract
Introduction
References
3' direction to form a right-handed helical structure in
which the DNA is extended to 1.5 times its original length (8). Pairing
with homologous duplex DNA in the second phase of the reaction results
in the rapid uptake of the dsDNA into homologous register and the
associated exchange of DNA strands. Then, as a final step of the
reaction, unidirectional branch migration completes the exchange of DNA
strands (9).
S, a non-hydrolyzable ATP
analogue, the reaction requires a 6-fold excess of Rad51 protein above
the aforementioned stoichiometric amount (3, 12).
EXPERIMENTAL PROCEDURES
S and AMP-PNP were purchased from Boehringer Mannheim. Dyes were obtained from Molecular Probes, and their concentrations were determined using the following extinction coefficients: 4',6-diamidino-2-phenylindole (DAPI), 33 × 103 M
1 cm
1 at
345 nm and ethidium bromide (EtBr), 5.5 × 103
M
1 cm
1 at 546 nm (13).
recA), the authenticity of recombinant plasmid was
confirmed by sequencing the entire RAD51 gene. Attempts to
clone RAD51 gene under T7 promoter using any other
expression vectors were unsuccessful, presumably due to both the high
uninduced level of expression and the toxicity of Rad51 protein to
E. coli.2
recA derivative of BL21(DE3)), carrying both plasmids
pEZ5139 and pLysS. The cells (80 g) from a 10-liter culture that was
induced with isopropyl-1-thio-
-D-galactopyranoside for
4 h were resuspended in 200 ml of 50 mM Tris-HCl (pH
7.5), 600 mM KCl, 5 mM EDTA, 10% sucrose, 10 mM
-mercaptoethanol, 5 µg/ml
4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 0.1 mM benzamidine. Cells were lysed by two cycles of freeze-thawing and by
passage through a French pressure cell (Aminco Inc.). The insoluble
material was removed by centrifugation at 40,000 rpm for 2 h at
4 °C. The proteins were precipitated from the supernatant with
ammonium sulfate (0.24 g/ml) and resuspended in 120 ml of P buffer (20 mM potassium phosphate (pH 7.5), 10% glycerol, 0.5 mM EDTA, 0.5 mM DTT), containing 50 mM KCl, 5 µg/ml AEBSF, 0.1 mM benzamidine.
This solution was loaded at a flow rate of 120 ml/h onto an 80-ml
Cybacron Blue-agarose column, equilibrated with P buffer. The column
was washed with P buffer + 100 mM KCl until the
A280 approached zero, and then 200 ml of a
0.2-1 M KCl gradient was applied. Fractions containing
Rad51 protein were combined and dialyzed against P buffer + 100 mM KCl, 5 µg/ml AEBSF, and 0.1 mM
benzamidine, and then loaded onto an 80-ml Q-Sepharose column,
equilibrated with P buffer + 100 mM KCl. Proteins were fractionated with a 600-ml KCl gradient (200-800 mM KCl);
Rad51 protein eluted at about 400 mM KCl. Pooled fractions
were dialyzed against H buffer (5 mM potassium phosphate
(pH 6.5), 0.5 mM DTT, 10% glycerol, 40 mM KCl)
containing 5 µg/ml AEBSF and 0.1 mM benzamidine, and then
loaded onto a 50-ml Bio-Gel hydroxyapatite column that was equilibrated
with H buffer. Bound proteins were eluted with a 300-ml linear gradient
of 20-200 mM potassium phosphate (pH 6.5) at a flow rate
of 60 ml/h. Rad51 protein eluted at about 50 mM potassium
phosphate. These fractions were pooled and diluted to bring the
phosphate concentration to about 25 mM and then loaded onto
a MonoQ (HR 10/10) column. A linear gradient (160 ml) of 200-600
mM KCl in P buffer was used to elute Rad51 protein (at about 400 mM KCl). Fractions containing purified Rad51
protein were dialyzed against storage buffer (20 mM
Tris-HCl (pH 7.5), 1 mM DTT, 40% glycerol, 0.5 mM EDTA, 1 µg/ml AEBSF, 0.05 mM benzamidine), aliquoted, and stored at
80 °C. The final yield of Rad51 protein was about 15 mg from 10 liters of culture. The concentration of Rad51
protein was calculated using an extinction coefficient (determined from
amino acid composition) of 1.29 × 104
M
1 cm
1 at 280 nm. The purity of
Rad51 protein was >99% based on SDS-polyacrylamide gel electrophoresis.
RESULTS
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Fig. 1.
Binding of Rad51 and RecA proteins to etheno
M13 DNA. Reactions were performed in 30 mM Tris
acetate buffer (pH 7.5), 10 mM magnesium acetate, 2 mM ATP at 37 °C, containing 3 µM
(nucleotides) of etheno M13 DNA. Titrations in the presence of ATP and
magnesium ion with RecA protein ( ) and Rad51 protein (
). The
titration with Rad51 protein in the absence of ATP (
).
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Fig. 2.
ATP prevents a magnesium
ion-dependent irreversible inactivation of Rad51
protein. Rad51 protein (1 µM) was incubated in 30 mM Tris acetate buffer (pH 7.5) and 10 mM
magnesium acetate, either with 1 mM ATP ( ) or without
ATP (
), for 15 min at 37 °C; ATP (1 mM) was then
added to the mixture that did not have ATP. Components were added at
the times indicated on the graph. The fluorescence was
measured in arbitrary units. The concentration of etheno M13 DNA was 3 µM (nucleotides).
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Fig. 3.
Salt titration of Rad51 protein-etheno M13
DNA complexes assembled in the presence of ATP. After the Rad51
protein-etheno M13 DNA complex was formed, the change in fluorescence
upon the addition of indicated amount of NaCl was monitored. The
initial concentrations of etheno M13 DNA and Rad51 protein were 3 and
1.5 µM, respectively. Reactions were performed in 30 mM Tris acetate buffer (pH 7.5) in the presence of 2 mM ATP and 10 mM magnesium acetate.
S support binding. In the presence of dATP, the RFI value is
2.6-2.8, but the apparent binding stoichiometry is the same as that
obtained with ATP (data not shown). No change in the fluorescence of
etheno M13 DNA upon addition of Rad51 protein was detected for any of
the other NTPs (Table I), implying that either Rad51 protein does not
bind ssDNA under these conditions or if binding does occur but there is
no extension of the etheno M13 DNA to produce a fluorescence change. Etheno M13 DNA complex formation by Rad51 protein was sensitive to the
ratio of ATP to ADP; in the presence of a mixture containing 1 mM ADP and 1 mM ATP, the RFI decreased by 25%,
in the presence of 3 mM ADP and 1 mM ATP, the
RFI decreased by 80% (Table I).
Binding of Rad51 protein to etheno M13 DNA depends on nucleotide
cofactor
S barely supported Rad51 protein
binding to etheno M13 DNA, as indicated by the slight increase in
fluorescence of etheno M13 DNA when a stoichiometric amount of Rad51
protein was present (RFI value of 1.3). However, ATP
S apparently
binds to Rad51 protein and prevents ATP binding, since upon subsequent
addition of ATP to the Rad51 protein/etheno M13 mixture that had been
preincubated with ATP
S, no change in fluorescence was detected.
However, addition of ATP
S to a preformed ATP-Rad51 protein-etheno
M13 DNA complex resulted in a decrease in fluorescence (data not
shown), suggesting that ATP
S could bind after ATP turnover. Increasing the Rad51 protein concentration above the stoichiometric ratio produced a substantial fluorescence signal in the presence of
ATP
S; we observed RFI values of 1.3, 1.9, 3.0, and 3.3 at Rad51
protein concentrations that are 1-, 2-, 3-, and 5-fold higher than the
stoichiometric ratio, respectively.
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Fig. 4.
Dependence of Rad51 protein-etheno M13 DNA
complex formation on pH. The RFI values obtained upon addition of
Rad51 protein (2 µM) to etheno M13 DNA (3 µM) were measured as described under "Experimental
Procedures." Buffers containing 30 mM sodium citrate,
Na-MES, or Tris acetate were employed for the pH intervals 5.0-6.0,
5.5-7.2, and 6.8- 8.5, respectively. Reactions were performed in the
presence of 2 mM ATP and 10 mM magnesium
acetate ( ); in the presence of 10 mM magnesium acetate
but without ATP (
); and neither magnesium acetate nor ATP
(
).
4-5 nucleotides of etheno M13 DNA (Fig. 5). However, in the
absence of both nucleotide cofactor and magnesium ion, the RFI value
increases with decreasing pH (the RFI values are 1.3 at pH 7.5, 2 at pH
6.6, and 3.2 at pH 6.2 (see Fig. 4)), and the apparent binding
stoichiometry of Rad51 protein changes from no obvious plateau, to 6:1,
to 9:1 nucleotides of ssDNA per protein monomer (Fig. 5).
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Fig. 5.
Complexes with two distinct stoichiometries
are formed between Rad51 protein and ssDNA. Etheno M13 DNA (3 µM) was titrated with Rad51 protein in standard buffers
at pH values of 7.5, 6.6, and 6.2, either in the presence or the
absence of both ATP (2 mM) and magnesium acetate (10 mM). The filled symbols represent experiments in
the presence of ATP and magnesium acetate, and the open
symbols represent experiments without ATP or magnesium acetate: 30 mM Na-MES, (pH 6.2) ( ); 30 mM Na-MES (pH
6.2), 2 mM ATP, 10 mM magnesium acetate (
);
30 mM Tris acetate (pH 7.5) (
); 30 mM Tris
acetate (pH 7.5), 2 mM ATP, 10 mM magnesium
acetate (
); 30 mM Na-MES (pH 6.6) (
).
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Fig. 6.
Rad51 protein-etheno M13 DNA complexes
assembled in the absence of nucleotide cofactor and magnesium ion have
a greater stability at lower pH values. Salt titrations were
performed with complexes that were formed in the absence of ATP and
magnesium acetate in 30 mM Na-MES buffer at pH 6.2 ( )
and pH 6.6 (
). Both reactions contained 3 µM
(nucleotides) of etheno M13 DNA and 2 µM Rad51
protein.
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Fig. 7.
The binding of Rad51 protein to M13 ssDNA
detected by a gel mobility shift assay parallels the fluorescent
assays. Rad51 protein was bound to DNA at the indicated ratio
(given in protein monomer per nucleotides) in Tris acetate (pH 7.5)
(lanes 2-7) or Na-MES (pH 6.2) (lanes 9-11) for
30 min at 37 °C, and the complexes were analyzed by electrophoresis
through a native 0.8% agarose gel as described under "Experimental
Procedures." Lanes 1 and 8 contain free DNA.
The concentration of M13 ssDNA was 30 µM.
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Fig. 8.
Magnesium ion dependence of Rad51 protein
binding to etheno M13 DNA. Each point represents the relative
fluorescence increase value for etheno M13 DNA obtained upon
addition of Rad51 protein (2 µM) at different
concentrations of magnesium acetate. Filled symbols
represent experiments in the presence of 2 mM ATP;
open symbols represent experiments without ATP: Na-MES (pH
6.2) ( ), Na-MES (pH 6.2), ATP (
); Tris acetate (pH 7.5) (
);
Tris acetate (pH 7.5), ATP (
). Concentration of etheno M13 DNA was 3 µM (nucleotides).
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Fig. 9.
The binding of Rad51 protein to ssDNA in the
absence of both ATP and magnesium ion is not interconvertible.
Complexes of Rad51 protein and M13 ssDNA were formed in Na-MES buffer
(pH 6.2) at the indicated initial concentrations of ATP and magnesium
acetate and were incubated for 30 min at 37 °C. Then, as indicated,
ATP and magnesium acetate were added, and the incubation was continued
for 30 min more. Sample loading buffer was added, and the reaction
products were analyzed by gel electrophoresis as described under
"Experimental Procedures." Lane 4 contains free DNA. The
concentrations of Rad51 protein and M13 ssDNA were 15 and 30 µM nucleotides, respectively.
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Fig. 10.
Rad51 protein binding to dsDNA monitored by
the dye displacement assay. Binding of Rad51 protein to dsDNA was
measured by observing the displacement of a DNA-bound fluorescent dye
upon protein-DNA complex formation. A, decrease in
fluorescence of dsDNA-DAPI complexes upon addition of indicated
concentrations of Rad51 protein. B, dependence of DAPI and
EtBr displacement from dsDNA on the concentration of Rad51 and RecA
proteins. Reactions contained 30 mM Tris acetate (pH 7.5),
10 mM magnesium acetate, 2 mM ATP, 200 nM DAPI (or 2 µM EtBr), and 6 µM (nucleotides) dsDNA. Filled symbols
represent experiments with Rad51 protein, and open symbols
represent experiments with RecA protein:DAPI displacement by Rad51
protein ( ), EtBr displacement by Rad51 protein (
), DAPI
displacement by RecA protein (
), and EtBr displacement by RecA
protein (
).
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Fig. 11.
Dependence of Rad51 protein binding to dsDNA
on magnesium ion concentration. The extent of DAPI and EtBr
displacement from dsDNA was determined upon the binding of Rad51
protein to dye-dsDNA complexes at different concentrations of magnesium
acetate. A, reactions were performed in Tris acetate buffer
(pH 7.5). B, reactions were performed in Na-MES buffer (pH
6.2). Filled symbols represent experiments in the presence
of 2 mM ATP, and open symbols are in the absence
of ATP: EtBr displacement ( ,
) and DAPI displacement (
,
).
The concentration of dsDNA was 6 µM nucleotides. The
concentration of Rad51 protein was 2 µM.
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Fig. 12.
Binding of Rad51 protein and RecA protein to
dsDNA in the absence of nucleotide cofactor and magnesium ion.
Titrations with Rad51 protein and RecA protein were performed using
either dsDNA-DAPI or dsDNA-EtBr complexes in Na-MES buffer (pH 6.2) in
the absence of magnesium acetate and ATP. The concentration of dsDNA
was 6 µM (nucleotides). Filled symbols
represent EtBr displacement experiments, and open symbols
represent DAPI displacement experiments: EtBr displacement by Rad51
protein ( ), EtBr displacement by RecA protein (
), DAPI
displacement by Rad51 protein (
), and DAPI displacement by RecA
protein (
).
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Fig. 13.
Binding of Rad51 protein to dsDNA detected
by a gel mobility shift assay parallels the EtBr displacement
assay. Rad51 protein was bound to M13 dsDNA at a stoichiometry of
1 protein monomer to 3 base pairs (6 nucleotides) of DNA (lanes
2-5), at the indicated conditions, for 15 min at 37 °C;
complexes were analyzed as described in Fig. 7. Lane 1 contains free dsDNA. The concentration of dsDNA was 30 µM
(nucleotides). The concentration of Rad51 protein was 5 µM.
DISCUSSION
X174 ssDNA
versus M13 ssDNA in DNA strand exchange promoted by Rad51
protein2 (3, 40), since M13 ssDNA has a higher level of
secondary structure compared with
X174 ssDNA.
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ACKNOWLEDGEMENTS |
---|
We thank the following members of this laboratory for their critical reading of this manuscript: Dan Anderson, Deana Arnold, Carole Barnes, Frederic Chedin, Jason Churchill, Frank Harmon, Alex Mazin, and Jim New.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant AI-18987.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 530-752-5938;
Fax: 530-752-5939; E-mail: sckowalczykowski{at}ucdavis.edu.
The abbreviations used are:
ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; DAPI, 4',6-diamidino-2-phenylindole; ATPS, adenosine
5'-
-(thiotriphosphate); etheno M13 DNA, modified M13 ssDNA
containing 1,N6-ethenoadenosine and
3,N4ethenocytidine residues; RFI, relative
fluorescence increase; EtBr, ethidium bromide; bp, base pair; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; RPA, replication protein-A; SSB protein, single-stranded DNA-binding protein; MES, 4-morpholineethanesulfonic acid.
2 E. M. Zaitseva, E. N. Zaitsev, and S. C. Kowalczykowski, unpublished observations.
3 E. N. Zaitsev and S. C. Kowalczykowski, submitted for publication.
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REFERENCES |
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