From the Department of Biochemistry, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5425
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Like RecA, Saccharomyces cerevisiae
Rad51p promotes strand exchange between circular single-stranded DNA
(ssDNA) and linear double-stranded DNA (dsDNA). We have investigated
several parameters characteristic of the interaction of Rad51p with
ssDNA and dsDNA, particularly the effects of the nucleotide cofactors
ATP and ADP and the analogs adenosine
5'-O-(thiotriphosphate) (ATPS) and adenylyl-imidodiphosphate (AMP-PNP). Rad51p binding to both
1-N6-ethenoadenosine and
3-N4-ethenocytidine ssDNA (
DNA) and
dsDNA requires the presence of Mg2+ and ATP; no binding
occurs in the presence of ADP, AMP-PNP, or ATP
S. Binding of Rad51p
to dsDNA also requires ATP; ADP is ineffective, whereas ATP
S and
AMP-PNP are considerably less able to promote binding and only at
elevated concentrations of Rad51p. ATP binding, not ATP hydrolysis, is
required for Rad51p binding to DNA. The Kd values
for ATP for promoting binding of Rad51p to ssDNA and dsDNA are 1 and 3 µM, respectively. Rad51p binding occurs with a
stoichiometry of one monomer of Rad51p per ~6.3 nucleotides of
DNA
and ~3.3 base pairs of dsDNA. Once formed, Rad51p·ssDNA complexes
are stable so long as sufficient ATP levels are maintained. ATP
hydrolysis causes dissociation of Rad51p from DNA. Moreover, the
preformed complex is stable in the presence of a 10-fold excess of ADP
or AMP-PNP over ATP. ATP
S, however, in the same -fold excess over
ATP causes dissociation of the Rad51p·ssDNA complex.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Saccharomyces cerevisiae RAD51 gene is a member of the RAD52 epistasis group, that is required for efficient DNA double strand break repair and genetic recombination (1). The amino acid sequence of RAD51 strongly resembles that of the Escherichia coli RecA protein (2-4), and like RecA, Rad51p catalyzes joint molecule formation and strand exchange between circular ssDNA1 and linear dsDNA (5-8). The reaction promoted by Rad51p also requires Mg2+, ATP, and a single-stranded DNA-binding protein, yeast replication protein A, being the most efficient (6-8). However, Rad51p-promoted strand exchange has certain striking differences compared with RecA. For example, RecA has the unique ability to pair homologous ssDNA and dsDNA to form three-stranded joint molecules, which are then resolved into heteroduplex DNA; this pairing can occur even between DNAs without ends (9-11). In the reaction promoted by Rad51p, joint molecules are formed only between ssDNA and linear dsDNA having either 3'- or 5'-overhanging complementary ends; joint molecules are not formed with linear DNA having blunt or recessed complementary ends (8). As a consequence of being able to use either a 3'- or 5'-overhanging complementary strand to initiate joint molecule formation, the direction of strand exchange depends on which end initiates the reaction (8).
The interactions of RecA with ssDNA and dsDNA are important
determinants for strand exchange (12-14). To understand the molecular mechanism of Rad51p-promoted strand exchange, we have examined the
interactions between Rad51p and single- and double-stranded DNA,
particularly the role of nucleotides and salt in these interactions. Rad51p binding to ssDNA was followed by measuring the increased fluorescence of chemically modified
1-N6-ethenoadenosine and
3-N4-ethenocytidine ssDNA (etheno-DNA,
DNA) that accompanies protein binding (13, 15); binding to dsDNA was
followed by protection of the DNA against DNase I degradation (14) and,
in some cases, by nitrocellulose filter binding (16). Our data indicate
that ATP and Mg2+ are essential for Rad51p binding to both
kinds of DNA and that ADP, adenosine 5'-O-thiotriphosphate
(ATP
S), and adenylyl imidodiphosphate (AMP-PNP) do not satisfy this
requirement. The ATP analog ATP
S, which is unable to promote Rad51p
binding to DNA, nevertheless causes dissociation of the Rad51p·
DNA
complex. The results of dissociation of the Rad51p·
DNA complex at
different temperatures indicate that ATP binding, not ATP hydrolysis,
is required for Rad51p binding to DNA.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins, Buffers, and Reagents--
Yeast Rad51p was expressed
in and purified from insect cells as described previously (8). The
concentration of Rad51p was determined using an extinction coefficient
(determined from amino acid composition) of 1.26 × 104 at 280 nm. Unless otherwise noted, the standard
reaction buffer used in all experiments contained 40 mM
K-Mes (pH 6.5), 4 mM MgCl2, 1 mM
dithiothreitol, and 5% glycerol. In experiments testing pH dependence,
K-Mes was replaced by 25 mM Tris-HCl (pH 7.5 or 8.5). ATP
and ADP were obtained from Pharmacia Biotech Inc., and ATPS and
AMP-PNP were from Boehringer Mannheim. All reactions were carried out
at 30 and 37 °C.
DNA--
The concentrations of the DNA substrates are expressed
as nucleotide equivalents. Etheno-M13 DNA (DNA) was prepared as
described (17), and the concentration was determined using an
extinction coefficient of 7000 M
1
cm
1 (18). Uniform 3H-labeled pBluescript
SK+ DNA was prepared with SssI methylase and
S-[methyl-3H]adenosyl-L-methionine.
Linear DNA was prepared by digestion of 3H-labeled
pBluescript SK(+) dsDNA with PstI restriction
endonuclease.
ssDNA Binding Assay--
The binding of Rad51p to ssDNA was
monitored by measuring the change in fluorescence of DNA at 410 nm
following excitation at 305 nm using an AMINCO-Bowman luminescence
spectrometer. Sample volumes were normally 0.6 ml, and additions did
not exceed 40 µl. The titrant was either Rad51p in "forward"
titrations or concentrated sodium chloride solution in the
"salt-back" titration. When ATP, ADP, ATP
S, and AMP-PNP were
included in the titrations, their concentrations were 100 µM each. Following the addition of each titrant, the
fluorescence was measured until the value reached a stable plateau. In
experiments measuring the stability of the Rad51p·
DNA complex, the
complex was formed in the presence of 100 µM ATP, and the
change in fluorescence was monitored after the addition of 1000 µM ADP, ATP
S, or AMP-PNP. Measurements were performed
at a constant temperature, maintained by a circulating water bath.
Corrections for fluorescence of the protein were made from the values
obtained after adding Rad51p in the absence of
DNA. The fluorescence
values observed in these control titrations were subtracted from the
measured values made in the presence of
DNA.
Nuclease Protection Assay--
3H-Labeled linear
dsDNA (10 µM) and variable amounts of Rad51p were
incubated in the standard reaction buffer containing 0.5 mM
ATP, ADP, ATPS, or AMP-PNP at 30 °C for 10 min. Aliquots (20 µl) of the reaction mixtures were incubated with 1 µl (12 units) of
pancreatic DNase I (Boehringer Mannheim) under standard conditions for
1 min. The reaction was stopped by adding 10 µl of a solution containing 2 mg/ml salmon sperm DNA (heat-denatured) and 0.375 M EDTA followed by 0.9 ml of ice-cold 10% trichloroacetic
acid. The samples remained on ice for 30 min and then were filtered through Whatman GF/C filter discs. The filters were washed twice with 1 ml of 10% trichloroacetic acid followed by 2 ml of 95% ethanol. The
filters were dried and assayed for radioactivity in a liquid
scintillation counter. For each experiment, complete protection was
equivalent to the value obtained without DNase I digestion. Complete
digestion was equivalent to the value obtained after DNase I digestion
when Rad51p was omitted from the binding reaction. The degree of
protection was obtained by dividing the amount of acid-precipitable
3H for each sample by the value obtained without DNase I
digestion after the background (the value without addition of Rad51p)
was subtracted from both. This background was ~10% of the value for 100% protection.
Nitrocellulose Filter Binding Assay-- Rad51p binding to DNA was also measured by the protein-dependent retention of DNA on nitrocellulose filters (HAWP, 0.45 µm, Millipore Corp.) (19). Aliquots (20 µl) of the reaction mixtures were diluted in 1 ml of the same reaction buffer and filtered through prewashed filter discs. The filters were washed two times with 1 ml of same buffer and dried, and the radioactivity was measured in a liquid scintillation counter.
Effect of ATP and Mg2+ on Rad51p Binding to
DNA--
The ATP dependence of Rad51p binding to ssDNA was determined
by measuring the increased fluorescence of DNA in the standard reaction buffer containing 5 µM
DNA, 1 µM Rad51p, and varying levels of ATP with an
ATP-regenerating system. The effect of ATP concentration on the binding
of Rad51p to dsDNA was studied with the nuclease protection assay using
the same reaction conditions except that the mixture contained 10 µM 3H-labeled linear dsDNA and 2 µM Rad51p.
ATP Hydrolysis Assay--
ATPase activity was measured as
described (20). 1 µM Rad51p was added to 5 µM DNA in a 90-µl reaction mixture containing 2.5 µM ATP, 10 units/ml pyruvate kinase, 10 units/ml lactate
dehydrogenase, 0.3 mM phosphoenolpyruvate, and 100 µM NADH in the standard reaction buffer. Oxidation of
NADH was measured as a decrease in absorbance at 340 nm.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Binding of Rad51p to ssDNA--
Rad51p binding to ssDNA was
followed by measuring the increase in fluorescence associated with
binding of the protein to chemically modified ssDNA (DNA) (21). The
increase in fluorescence is presumed to be a consequence of stretching
the DNA and unstacking the bases in the Rad51p·ssDNA complex (13,
15).
|
Binding of Rad51p to dsDNA--
Rad51p forms a helical filament
with dsDNA that is nearly identical to the structure seen by electron
microscopy when RecA interacts with dsDNA (23). To characterize several
physical parameters governing the interaction of Rad51p with dsDNA, two different measurements were made: protection of 3H-labeled
dsDNA against degradation by DNase I and nitrocellulose filter binding.
Unlike RecA (14), Rad51p binding to dsDNA rendered the DNA insensitive
to degradation by DNase I; protection was complete even after 1 min of
incubation, and the complex was stable for at least 1 h (Fig.
2A). As in the case of ssDNA,
binding of Rad51p to dsDNA was most effective in the presence of ATP.
With 1 µM Rad51p, ADP, ATPS, and AMP-PNP did not
promote binding, but at higher concentrations of Rad51p, these
nucleotides promoted some binding (Fig. 2B). ATP was also
required for Rad51p binding to dsDNA in the nitrocellulose filter
binding assay (Fig. 2C). Rad51p-mediated protection of the
dsDNA against DNase I action in the presence of ATP reached a limiting
value of ~3.3 base pairs/Rad51p. Unlike RecA, which binds to dsDNA
most effectively at low pH (14, 16, 24, 25), Rad51p bound dsDNA with
the same efficiency at pH 6.5-8.5 in the presence of ATP (data not
shown). Here, too, as in the case of ssDNA, we estimate an association
constant for Rad51p binding to dsDNA of ~8 × 105
M
1, which is close to the value for Rad51p
binding to ssDNA.
|
Effect of ATP and Mg2+ Concentration on Binding of
Rad51p to DNA--
Fig. 3 demonstrates
the dependence of Rad51p binding to DNA on ATP concentration. The
apparent Kd value for ATP for Rad51p binding to
DNA is ~1 µM, and that for binding to dsDNA is ~2
µM. However, because binding to ssDNA and dsDNA was
measured in the presence of 1 and 2 µM Rad51p,
respectively, these values may reflect the stoichiometry of binding of
Rad51p and ATP rather than a Kd, which could be
considerably different.
|
|
Stability of the Rad51p·DNA Complex in the Presence of
Different Nucleotides--
Once formed in the presence of 100 µM ATP and maintained in this medium, the Rad51p·
DNA
complex was stable for an extended period of time (Fig.
5A). However, if the complex
was formed with 2.5 µM ATP, its half-life was ~12-13
min at 30 °C. This is most likely due to loss of ATP by hydrolysis
and not to the accumulation of ADP because even a 10-fold excess of ADP
over ATP did not inhibit the binding (Fig. 5A). Similarly,
AMP-PNP, which was also unable to support complex formation (Fig. 1),
also failed to destabilize the Rad51p·
DNA complex formed with ATP
(Fig. 5B).
|
|
Effect of Salt on Rad51p Binding to DNA--
The formation of
the Rad51p·
DNA complex was inhibited by sodium chloride
concentrations exceeding 100 mM (Fig.
7A). It appears that both the
rate and extent of Rad51p·
DNA complex formation are affected by
salt. About 20 s were required for the formation of 95% of the
complex without salt, whereas >250 s were required to form the same
amount of complex in the presence of 200 mM of NaCl (Fig.
7A). Once formed, however, the Rad51p·
-DNA complex was
more stable at particular salt concentrations than would be expected
from their effects on complex formation (Fig. 7B). Note that
the time scales are different in Fig. 7 (A and
B), indicating that in 400-500 mM sodium
chloride, little or no complex was formed by 10 min, but ~70% of the
complex remained 10 min after the addition of 400 mM salt
to the preformed complex and somewhat more than 40% after 10 min in
500 mM salt. Thus, it is clearly more difficult to
dissociate the Rad51p·
DNA complex at high salt concentrations than
it is to inhibit the binding.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The strong sequence similarity between S. cerevisiae Rad51p and bacterial RecA proteins suggested early on that they perform similar or related functions in recombination (2-4). Indeed, both proteins promote strand exchange between a linear duplex DNA and a homologous circular single-stranded DNA to form a double-stranded nicked circle and a linear single strand, although the mechanistic details of their actions differ somewhat (6-8).
We are especially interested in understanding the Rad51p-promoted
reaction in greater detail, particularly the
nucleotide-dependent binding of Rad51p to the two kinds of
DNA substrates. To this end, we examined the binding of Rad51p to a
modified single-stranded DNA, etheno-DNA (DNA), which has been used
previously to characterize RecA binding to ssDNA (13). Protein binding
to
DNA is readily monitored by an appreciable increase in the
fluorescence of the DNA. Our results show that Rad51p binding to
DNA
is strongly dependent on the presence of ATP and Mg2+. At
37 °C, ADP, ATP
S, and AMP-PNP cannot substitute for ATP in
promoting Rad51p binding to
DNA; however, at 30 °C, in the presence of high concentrations of Rad51p, ATP
S supports a low level
of binding. By contrast, RecA binds to ssDNA without ATP, but ATP
induces a transition to a high affinity binding form (13). The
nonhydrolyzable analogs ATP
S and AMP-PNP also increase the affinity
of RecA for DNA, but the RecA·DNA complex is more stable in the
presence of these nucleotides (13).
Under optimal conditions, the stoichiometry of Rad51p association with
DNA indicates a binding site size of 6.3 nucleotides/Rad51p and a
K (binding affinity) of 9 × 105
M
1 based on the value at half-saturated
binding. This binding site size is different from the previously
reported value of 3.6 nucleotides determined from studies of the strand
exchange reaction (7, 8). The difference in the binding stoichiometry
can be explained if the Rad51p monomer has two binding sites, each of
which can bind ~3 nucleotides/DNA strand. A similar difference in
binding stoichiometry exists when measuring the binding stoichiometry of RecA and
DNA and the binding site size at which the
DNA-dependent ATPase is maximal; these were 7.0 and 3.1 bases/RecA, respectively (17).
The greatest difference between RecA and Rad51p has been found in their
ability to interact with dsDNA. RecA binds very weakly to dsDNA at pH
7.5, and the efficiency is increased substantially at low pH or with
ATPS (14, 16, 24, 25). Rad51p, however, binds to dsDNA equally well
in the presence of ATP and Mg2+ over the pH range of
6.5-8.5. ATP
S and AMP-PNP are able to activate binding somewhat,
but only at higher concentrations of Rad51p, whereas ADP is only weakly
active. The binding site size estimated from the titration curve is
~3.3 base pairs/Rad51p, with a binding affinity (K) of
8 × 105 M
1; these values
are about the same as those for RecA binding to dsDNA (14).
Binding of Rad51p to DNA is maximal at ~5 µM ATP and
half-maximal at 1 µM; for binding to dsDNA, the values
are about two times higher, respectively. However, because binding to
ssDNA and dsDNA was measured in the presence of 1 and 2 µM Rad51p, respectively, the Kd value
for ATP binding could be considerably lower. The optimal amount of
Mg2+ is influenced by the amount of ATP; at 25-750
µM ATP, 2 mM Mg2+ is optimal, but
with excess ATP (3 mM), maximal binding requires 4-6
mM Mg2+. This suggests that the active species
for promoting Rad51p binding to ssDNA and dsDNA is a specific
ATP·Mg2+ complex, which requires for its formation a
large excess of Mg2+ over ATP. Quite possibly,
however, Mg2+ has an additional role in the binding
reaction, perhaps by its association with the DNA ligand or by action
on the protein itself.
At NaCl concentrations >0.1 M, the binding of Rad51p to
DNA is inhibited. The degree of inhibition increases with increasing salt concentration until complete inhibition occurs at 0.5 M. We have noted, but not investigated further, that other
anions, e.g. phosphate and sulfate, are far more inhibitory
than the chloride anion. Once formed at optimal levels of salt, the
Rad51p·
DNA complex is readily dissociated as the salt
concentration is increased. But at any particular salt concentration,
the complex is considerably more stable than would be expected from the
effect of that salt concentration on complex formation.
Once formed, the Rad51p·DNA complex is stable so long as ATP and
Mg2+ are maintained. Removal of either causes the complex
to dissociate. Addition of ADP or AMP-PNP at levels 10 times higher
than the ATP level does not affect the half-life of the complex.
However, the addition of ATP
S to a preformed Rad51p·
DNA complex
at a concentration 10 times that of the ATP concentration causes a relatively rapid dissociation of the complex. We surmise that ATP
S
competes with ATP for binding to Rad51p, but because the Rad51p·ATP
S complex is unable to bind to
DNA, dissociation of the Rad51p·
DNA complex ensues. The results of dissociation of the
Rad51p·
DNA complex formed in the presence of 2.5 µM
ATP show that ATP binding, not ATP hydrolysis, is required for Rad51p
binding to DNA. ATP hydrolysis serves to recycle Rad51p off the DNA.
The difference in the ability of Rad51p to bind DNA in the presence of
ATP and ATP
S suggests that only the interaction with ATP induces the
structural change needed to bind to DNA. An ATP-induced structural transition has also been proposed to activate RecA for DNA binding (13,
26).
Thus, as was the case for the strand exchange, there is only a partial similarity between Rad51p and RecA activities. Because Rad51p forms a helical filament with DNA very similar to the one formed with RecA, it is of interest to determine if ATP and Mg2+ are required to activate Rad51p for binding or to promote the formation of oligomers of Rad51p that then bind to DNA or whether ATP and Mg2+ influence the successive binding of monomers to already bound clusters of Rad51p. A relevant issue that needs to be investigated further is whether, like RecA, the association and dissociation of Rad51p with DNA occurs with a unique directionality.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank M. Campbell for providing the
DNA, I. R. Lehman for critical reading of the manuscript, and
E. Tolstova for excellent technical support.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant GM13235 from the National Institutes of Health.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.
On leave from the Petersburg Nuclear Physics Institute, Russian
Academy of Science, Gatchina, Russian Republic.
§ To whom correspondence should be addressed: Beckman Center, B062, Stanford University School of Medicine, Stanford, CA 94305-5425. Tel.: 650-723-6170; Fax: 650-725-4951; E-mail: pberg{at}cmgm.stanford.edu.
1
The abbreviations used are: ssDNA,
single-stranded DNA; dsDNA, double-stranded DNA; DNA,
1-N6-ethenoadenosine and
3-N4-ethenocytidine ssDNA; ATP
S,
adenosine 5'-O-(thiotriphosphate); AMP-PNP,
adenylyl-imidodiphosphate; Mes,
2-(N-morpholino)ethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|