From the Department of Biochemistry, The Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205
Received for publication, January 18, 2001
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ABSTRACT |
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We have prepared a mutant RecA protein in which
proline 67 and glutamic acid 68 in the NTP binding site were replaced
by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent
hydrolysis of ATP and is able to promote the standard
ATP-dependent three-strand exchange reaction between a
circular bacteriophage The RecA protein of Escherichia coli
(Mr 37,842; 352 amino acids) is essential for
homologous genetic recombination and for the postreplicative repair of
damaged DNA. The purified RecA protein promotes a variety of
ATP-dependent DNA pairing reactions that presumably reflect
in vivo recombination functions. The most extensively investigated DNA pairing reaction is the three-strand exchange reaction, in which a circular
ssDNA1 molecule and a
homologous linear dsDNA molecule are recombined to yield a nicked
circular dsDNA molecule and a linear ssDNA molecule. This reaction
proceeds in three phases. In the first phase, the circular ssDNA
molecule is coated with RecA protein, forming a helical nucleoprotein
filament known as the presynaptic complex. In the second phase, the
presynaptic complex interacts with the linear dsDNA molecule, the
homologous sequences are brought into register and pairing between the
circular ssDNA, and the complementary strand from the dsDNA is
initiated. In the third phase, the complementary linear strand is
completely transferred to the circular ssDNA by unidirectional
branch migration to yield the nicked circular dsDNA and displaced
linear ssDNA products (1, 2).
The x-ray crystal structure of the RecA protein indicates that the
phosphate groups of the nucleotide cofactor, ATP, are bound by a loop
consisting of amino acids 66-73 (3). The sequence of this loop
(66GPESSGKT73) corresponds to a variation of
the well known phosphate binding loop (P-loop) consensus sequence
(GXXXXGK(T/S)) found in many NTP-binding proteins (4). The
invariant lysine and threonine/serine residues in the P-loop motif are
generally found to interact directly with the As part of an investigation of the role of the P-loop in ATP
hydrolysis, we prepared a mutant RecA protein in which the proline and
glutamic acid residues at positions 66 and 67 were replaced by a
glycine and alanine, respectively. The biochemical properties of the
[P67G/E68A]RecA protein provide new insight into the role of ADP in
the DNA strand exchange reaction and are described in this report.
Materials--
Wild type RecA protein was prepared as described
previously (6). ATP was from Sigma; dATP, [ Preparation of the [P67G/E68A]RecA Protein--
The gene for
[P67G/E68A]RecA, in which the nucleotide sequence coding for proline
67 and glutamic acid 68 was replaced with a nucleotide sequence coding
for glycine and alanine, respectively, was produced using the
QuickChangeTM protocol (Stratagene). The initial mutagenesis template
consisted of a pET21a(+) vector (Novagen) containing the wild type
recA gene cloned into a NdeI/HindIII site. The mutagenesis primers that were used to introduce the E68A mutation were
5'-GAAATCTACGGACCGGCGTCTTCCGGTAAAACC-3' and
5'-GGTTTTACCGGAAGACGCCGGTCCGTAGATTTC-3' (the
codon for alanine 68 is underlined, and the nucleotide mismatch is in bold). The resulting plasmid, pETRecA(E68A), was then used as a
template for a second mutagenesis step. The mutagenesis primers that
were used to introduce the P67G mutation were
5'-CGTATCGTCGAAATCTACGGAGGGgcgTCTTCCGGTAAAACC-3' and
5'-GGTTTTACCGGAAGAcgcCCCTCCGTAGATTTCGACGATACG-3' (the codon for glycine 67 is underlined, the nucleotide mismatch is in
bold, and the codon for alanine 68 is in lowercase). The entire gene
for [P67G/E68A]RecA was sequenced to confirm that only the desired
changes had been introduced during the mutagenesis procedure. The
expression plasmid, pETRecA(P67G/E68A), was introduced into the
E. coli recA strain, BLR(DE3) (Novagen). The expression of
the [P67G/E68A]RecA protein was induced by addition of
isopropyl-D-thiogalactoside (1 mM final
concentration) at A600 = 0.6, followed by a 3-h
incubation at 37 °C. The [P67G/E68A]RecA protein was then purified
to greater than 95% homogeneity by methods that have been
described previously (6). The purified [P67G/E68A]RecA protein is
shown in Fig. 1.
ssDNA-dependent NTP Hydrolysis Activity of the
[P67G/E68A]RecA Protein--
The [P67G/E68A]RecA protein was
analyzed for ssDNA-dependent ATP and dATP hydrolysis
activity at pH 7.5 and 37 °C. The reaction solutions contained 1 µM [P67G/E68A]RecA protein and 30 µM
The [P67G/E68A]RecA protein catalyzed the hydrolysis of both ATP and
dATP (Fig. 2). The turnover number
(Vmax/[Etotal]) for hydrolysis was 6 min Three-strand Exchange Activity of the [P67G/E68A]RecA
Protein--
The DNA strand exchange activity of the [P67G/E68A]RecA
protein was evaluated using the three-strand exchange reaction. In this
reaction, a circular
The strand exchange reaction that was promoted by the wild type RecA
protein in the presence of ATP (3 mM) is shown in Fig. 3A. In this reaction,
partially exchanged DNA intermediates are visible within the first 3 min, and the fully exchanged circular dsDNA product can be detected
within 10 min. The strand exchange reaction appears to reach completion
within 60 min. Similar results were obtained when dATP was used in
place of ATP as the nucleotide cofactor (gels not shown). These results
are consistent with previous results (8).
The [P67G/E68A]RecA protein was also able to promote strand
exchange in the presence of ATP (3 mM) (Fig.
3A). Although partially exchanged DNA intermediates are
visible within the first 6 min of the reaction, the fully exchanged
circular dsDNA product is not detected until ~4 h after initiation of
the reaction. The strand exchange reaction appears to reach completion
after ~6 h. Similar results were obtained when dATP was used in place
of ATP as the nucleotide cofactor (gels not shown). These results indicate that the [P67G/E68A]RecA protein is able to form initial pairing intermediates with an efficiency similar to that of the wild
type protein, but that the formation of the fully exchanged circular
dsDNA product is delayed significantly relative to that with the wild
type protein.
The time course of the ATP hydrolysis reaction that occurred during the
[P67G/E68A]RecA protein-promoted strand exchange reaction is shown in
Fig. 3B. The hydrolysis of ATP followed a linear time course
for ~4 h, and then terminated abruptly after ~80-90% of the ATP
had been converted to ADP. A comparison of the time course of ATP
hydrolysis (Fig. 3B) with the time course of strand exchange (Fig. 3A) indicates that the fully exchanged circular dsDNA
product was just beginning to appear as the ATP hydrolysis reaction was ending. Under the same conditions, the ATP hydrolysis reaction of the
wild type RecA protein continued until ~50% of the ATP had been
converted to ADP (Fig. 3B), and the formation of the fully
exchanged product reached completion during the linear phase of ATP
hydrolysis (Fig. 3A).
Effect of ATP Regeneration on the Strand Exchange Activity of the
[P67G/E68A]RecA Protein--
The results in Fig. 3 indicated that
the pool of ATP in the reaction solution was being depleted by the ATP
hydrolysis activity of the [P67G/E68A]RecA protein before the strand
exchange reaction could be completed. Therefore, two additional sets of
strand exchange reactions were carried out. In the first set, the
strand exchange reactions were carried out in the presence of an ATP
regeneration system (creatine kinase/creatine phosphate), which
phosphorylates the ADP generated by the ATP hydrolysis reaction to
reform ATP. In the second set, the strand exchange reactions were
carried out in the absence of an ATP regeneration system, but with a
higher initial concentration of ATP.
As shown in Fig. 4, the formation of the
fully exchanged circular dsDNA product by the [P67G/E68A]RecA protein
was strongly inhibited when an ATP regeneration system was included in
the reaction solution. Similar results were obtained when dATP was used
in place of ATP as the nucleotide cofactor (gels not shown). By
comparison, the ATP regeneration system had little effect on the ATP or
dATP-dependent strand exchange activity of the wild type
RecA protein (Fig. 4, dATP results not shown).
The strand exchange activity of the [P67G/E68A]RecA protein was also
inhibited (in the absence of the ATP regeneration system) when the
initial concentration of ATP in the reaction solution was increased. As
shown in Fig. 5, when the initial
concentration of ATP was increased from 3 to 6 mM, the time
required for the appearance of the fully exchanged product increased
from 4 to 6 h. Conversely, when the starting concentration of ATP
was reduced from 3 to 1 mM, the time required for the
appearance of the fully exchanged product decreased from 4 to 2 h.
This same variation in ATP concentration had little effect on the
strand exchange activity of the wild type RecA protein (Fig. 5).
These results indicated that the strand exchange activity of the
[P67G/E68A]RecA protein was dependent not only on the presence of
ATP, but also on the presence of ADP. Under standard reaction conditions (in the absence of the ATP regeneration system), the requisite ADP would be generated from ATP by the ATP hydrolysis activity of the [P67G/E68A]RecA protein, and consequently, the fully
exchanged product does not form until a sufficiently high concentration
of ADP has accumulated in the reaction solution. The inclusion of the
ATP regeneration system prevents the accumulation of ADP and,
therefore, inhibits the strand exchange reaction. Furthermore, the
increase in the delay of the appearance of the fully exchanged product
that was observed when the ATP concentration was increased (in the
absence of the ATP regeneration system) suggests that the critical
parameter for the [P67G/E68A]RecA protein may not be the absolute
concentration of ADP (ADP is generated at approximately the same rate
with a starting concentration of either 3 or 6 mM ATP), but
rather the ratio of ADP to ATP that is present in the reaction solution.
Effect of ADP on the Strand Exchange Activity of the
[P67G/E68A]RecA Protein--
In order to explore the apparent
dependence of the [P67G/E68A]RecA protein-promoted strand exchange
reaction on ADP, a series of strand exchange reactions was carried out
in which the total concentration of nucleotide cofactor (ATP + ADP) was
kept constant (3 mM), but the percentage of ADP in the pool
was varied from 0 to 100%.
As shown in Fig. 6, when the starting
nucleotide pool contained 0% ADP, the fully exchanged circular dsDNA
products were detected ~4 h after the reaction was initiated,
consistent with the results described above (Fig. 3). As the percentage
of ADP in the initial nucleotide pool was increased, however, the time
required for the formation of the fully exchanged product decreased.
The most efficient reaction occurred when the starting nucleotide pool contained 90% ADP; under these conditions, the circular dsDNA product
could be detected within 1 h after the reaction was initiated. No
strand exchange was observed when the starting nucleotide pool contained 100% ADP, however, indicating that the [P67G/E68A]RecA protein-promoted strand exchange reaction is dependent on the presence
of both ATP and ADP. Similar results were obtained when dATP and dADP
were used in place of ATP and ADP as the nucleotide cofactors (gels not
shown). By comparison, the formation of the fully exchanged product by
the wild type RecA protein was inhibited as the percentage of ADP in
the starting nucleotide pool was increased from 0 to 30%, and was
completely eliminated when the starting ADP percentage was 40% or
higher (Fig. 6).
Strand Exchange Activity of the [P67G/E68A]RecA Protein Using a
Shorter Linear dsDNA Substrate--
Although the time required for the
appearance of the fully exchanged circular dsDNA product in the
[P67G/E68A]RecA protein-promoted strand exchange reaction was much
greater than that for the wild type RecA protein, the partially
exchanged DNA intermediates appeared to form at similar rates with the
two proteins (Fig. 3). Furthermore, the formation of the partially
exchanged intermediates by the [P67G/E68A]RecA protein did not appear
to be inhibited by the ATP regeneration system (Fig. 4). It is thought
that the partially exchanged intermediates are formed in an initial
pairing reaction that is dependent on ATP but does not require ATP
hydrolysis, and that the resulting nascent hybrid DNA intermediates
(generally about 1 kb in length) are then extended in an ATP
hydrolysis-dependent branch migration reaction to yield the
fully exchanged circular dsDNA product (9). Thus, the results in Figs.
3 and 4 suggest that the initial pairing reaction may occur normally
with the [P67G/E68A]RecA protein, and that it is the branch migration
phase of the reaction that is dependent on the presence of ADP. To test this idea, a series of strand exchange reactions was carried out with
the circular
As shown in Fig. 7, the [P67G/E68A]RecA
protein was able to recombine the circular Effect of Mg2+ Concentration on the Strand Exchange
Activity of the [P67G/E68A]RecA Protein--
The strand exchange
reactions described above were carried out with a standard
Mg2+ concentration of 10 mM; this concentration
of Mg2+ has been reported to be optimal for the strand
exchange activity of the wild type RecA protein (10). Recent studies,
however, indicate that the different phases of the RecA
protein-promoted strand exchange reaction may have different
dependences on Mg2+ concentration. It has been determined
(from studies of an ATP hydrolysis-deficient mutant RecA protein) that
although a Mg2+ concentration in excess of that needed to
form the Mg-ATP complex is required for the optimal formation of
initial pairing intermediates, the excess Mg2+ may inhibit
the formation of full-length strand exchange products. To account for
these observations, it has been proposed that excess Mg2+
favors the formation of discontinuous DNA pairing intermediates (containing both paired and looped regions) and that these
discontinuous intermediates are only slowly resolved into full-length
products during the branch migration phase of the reaction (9). Since the results in Fig. 3 were carried out under excess Mg2+
conditions (3 mM NTP and 10 mM
Mg2+), it was conceivable that the ADP dependence of the
[P67G/E68A]RecA protein was somehow related to a destabilization of
the initial pairing intermediates that was necessary in order for the
branch migration phase of the reaction to form the fully exchanged
products. To explore this idea, a series of strand exchange reactions
was carried out with the [P67G/E68A]RecA protein and the full-length 5.3-kb linear dsDNA substrate, in which the starting ATP concentration was fixed at 3 mM and the Mg2+ concentration
was varied from 4 to 10 mM.
As shown in Fig. 8, the [P67G/E68A]RecA
protein was able to recombine the circular
These results indicate that the [P67G/E68A]RecA protein-promoted
strand exchange reaction is stimulated (because the ADP dependence is
reduced) when the Mg2+ concentration is reduced to a level
just sufficient to complex all of the nucleotide cofactor present in
the reaction solution. By contrast, the wild type RecA
protein-promoted strand exchange reaction proceeded optimally at 6-10
mM Mg2+, and decreased slightly in efficiency
when the Mg2+ concentration was reduced to 4 mM
(Fig. 8).
Under our standard reaction conditions (10 mM
Mg2+/3 mM ATP), the [P67G/E68A]RecA protein
is able to recombine a circular Consistent with the apparent requirement for ADP, the strand exchange
activity of the [P67G/E68A]RecA protein is strongly stimulated when
ADP is included in the starting reaction solution. The reaction appears
to be most efficient when the starting nucleotide pool consist of
~90% ADP and 10% ATP. Under these conditions, the time required for
the [P67G/E68A]RecA protein to form the fully exchanged product is
reduced from 4 h to ~1 h (close to the rate expected, based on a
rate of NTP hydrolysis about 3-4-fold lower than for the wild type
protein). By contrast, the wild type RecA protein-promoted strand
exchange reaction is completely eliminated at the high ADP
concentrations (90% ADP) that provide the maximal level of stimulation
of the [P67G/E68A]RecA protein-promoted reaction. A modest
stimulation of heteroduplex DNA formation by the wild type RecA protein
in the presence of low concentrations of ADP has been noted previously
by Cox and co-workers (12). Although the mechanistic basis for this
observation was not determined, it is likely that it is related to the
pronounced ADP dependence of the [P67G/E68A]RecA protein-promoted
strand exchange reaction.
Cox and co-workers have shown that the wild type RecA protein-catalyzed
ATP hydrolysis reaction terminates when ~40-60% of the available
ATP has been hydrolyzed to ADP, regardless of the initial ATP
concentration. To account for this observation, they have proposed that
the termination of ATP hydrolysis is due to a dissociation of the RecA
protein from the DNA that occurs when the ADP/ATP ratio exceeds ~1.0;
this dissociation is apparently due to a structural incompatibility of
the ADP and ATP states of the recA-ssDNA filament (13, 14). The greater
extent of ATP hydrolysis that was observed with the [P67G/E68A]RecA
protein (90% ATP hydrolyzed) suggests that the [P67G/E68A]RecA-ssDNA
complex may be more resistant to ADP-mediated dissociation than is the wild type RecA-ssDNA complex. Furthermore, the finding that the appearance of fully exchanged products in the [P67G/E68A]RecA protein-promoted reaction occurs when the ADP concentration reaches ~80% (and rate of ATP hydrolysis has begun to decrease sharply) suggests that an ADP-mediated destabilization of the
[P67G/E68A]RecA-ssDNA filaments may be necessary in order for the
initial pairing intermediates to be resolved into the fully exchanged
product. Consistent with this idea, the ADP dependence of the
[P67G/E68A]RecA protein-promoted strand exchange reaction appears to
be reduced or eliminated when a shorter 1.1-kb linear We have recently prepared two additional mutant RecA proteins, one
containing only the P67G mutation and the other containing only the
E68A mutation. Although both of these mutations reduce the rate of
ssDNA-dependent ATP hydrolysis ~2-fold (relative to the
wild type RecA protein), neither the [P67G]RecA protein nor the
[E68A]RecA protein exhibits the ADP-dependent strand
exchange activity that is observed with the [P67G/E68A]RecA protein
(data not shown). Thus, the effects that are described in this paper appear to depend on the presence of both the P67G mutation and the E68A
mutation. The structural basis for the effect of the [P67G/E68A]
mutation on the properties of the RecA protein is not yet clear. It is
likely, however, that the [P67G/E68A]RecA mutation has not
fundamentally altered the mechanism of the RecA protein, but rather has
magnified a requirement for ADP during the branch migration phase of
the strand exchange reaction that also exists (but is not as readily
apparent) in the reaction of the wild type RecA protein.
X174 (
X) single-stranded DNA molecule and
a homologous linear
X double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the
wild type RecA protein, however, in that it is (i) completely inhibited
by an ATP regeneration system, and (ii) strongly stimulated by the
addition of high concentrations of ADP to the reaction solution. These
results indicate that the strand exchange activity of the
[P67G/E68A]RecA protein is dependent on the presence of both ATP and
ADP. The ADP dependence of the reaction is reduced or eliminated when
(i) a shorter linear
X dsDNA fragment (1.1 kilobase pairs) is
substituted for the full-length linear
X dsDNA substrate, or (ii)
the Mg2+ concentration is reduced to a level just
sufficient to complex the ATP present in the reaction solution. These
results indicate that it is the branch migration phase (and not the
initial pairing step) of the [P67G/E68A]RecA protein-promoted strand
exchange reaction that is dependent on ADP. It is likely that the
[P67G/E68A]RecA mutation has revealed a requirement for ADP that also
exists (but is not as readily apparent) in the strand exchange reaction
of the wild type RecA protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
phosphates of
ATP and have been shown for the RecA protein (as well as many other
proteins) to be directly involved in the catalysis of phosphoryl
transfer (5). Interestingly, although the four variable residues
(XXXX) in this sequence can differ widely in different
classes of proteins, the specific sequence, GPESSGKT, is
highly conserved in over 60 different bacterial RecA proteins (1).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP,
and [
-32P]dATP were from Amersham Pharmacia Biotech.
E. coli SSB was from Promega. Circular
X ssDNA ((+)
strand) and circular
X dsDNA were from New England Biolabs.
Full-length linear
X dsDNA was prepared from circular
X dsDNA as
described (7). The 1.1-kb
X dsDNA fragment was generated by
digesting circular
X dsDNA with DraI (New England
Biolabs) and was purified by agarose gel electrophoresis. Single- and
double-stranded DNA concentrations were determined by absorbance at 260 nm using the conversion factors 36 and 50 µg/ml/A260, respectively. All DNA
concentrations are expressed as total nucleotides.
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Fig. 1.
SDS-polyacrylamide gel electrophoresis of
purified [P67G/E68A]RecA protein. Lane 1,
[P67G/E68A]RecA protein; lane 2, wild type RecA
protein; lane 3, molecular mass standards (Life
Technologies, Inc.; molecular masses given in kilodaltons). The
acrylamide concentration was 5% in the stacking gel and 10% in the
separating gel. The gel was stained in 0.1% Coomassie Brilliant Blue
R-250.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
X ssDNA; these conditions ensured that there was sufficient ssDNA to bind
all of the [P67G/E68A]RecA protein present. The dependence of the
rate of ssDNA-dependent ATP and dATP hydrolysis on NTP concentration is shown in Fig. 2, and the
kinetic parameters for the hydrolysis of each NTP are summarized
below.
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Fig. 2.
Single-stranded DNA dependent NTP hydrolysis
by the [P67G/E68A]RecA protein. The reaction solutions contained
25 mM Tris acetate (pH 7.5),10 mM
Mg(acetate)2, 30 µM X ssDNA, 5% glycerol,
1 mM DTT, 1.0 µM [P67G/E68A]RecA protein,
and the indicated concentrations of [
-32P]ATP
(open circles) or [
-32P]dATP (open
triangles). The reactions were initiated by the addition of
[P67G/E68A]RecA protein and were carried out at 37 °C. ATP and
dATP hydrolysis was measured by a thin layer chromatography method as
described previously (15). The points represent the initial
rates of ATP and dATP hydrolysis that were measured at the indicated
concentrations of NTP. The solid line (ATP) and
broken line (dATP) represent fits of the data by
the Hill equation.
1 for both ATP and dATP,
and the S0.5 values were 45 µM and 25 µM for ATP and dATP,
respectively.2 Under the same
conditions, the wild type RecA protein catalyzes ATP and dATP
hydrolysis with turnover numbers of 20 min
1
for ATP and 24 min
1 for dATP, and with
S0.5 values of 45 µM and 20 µM
for ATP and dATP, respectively (8). Thus, the [P67G/E68A]RecA
mutation lowers the rate of ssDNA-dependent hydrolysis of
ATP and dATP by ~3-4-fold, but has little effect on the
S0.5 values for these NTPs.
X ssDNA molecule (5386 bases) and a homologous
linear
X dsDNA molecule (5386 base pairs) are recombined to form a
nicked circular dsDNA molecule and a linear ssDNA molecule; the
substrates and products of this reaction are readily monitored by
agarose gel electrophoresis (7).
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Fig. 3.
Wild type and [P67G/E68A]RecA
protein-promoted strand exchange reactions: full-length linear dsDNA
substrate. Panel A, strand exchange assay. The reaction
solutions contained 25 mM Tris acetate (pH 7.5), 5%
glycerol, 1 mM DTT, 10 mM
Mg(acetate)2, 5 µM circular X ssDNA, 15 µM linear
X dsDNA, 0.5 µM E. coli SSB, 3 mM ATP, and 2.5 µM wild type
RecA protein (wt, left) or [P67G/E68A]RecA
protein (mutant, right). The reactions were
initiated by the simultaneous addition of SSB and ATP after
preincubation of all other components for 10 min at 37 °C. Aliquots
(20 µl) were removed at the indicated times and quenched with 1.3 µl of SDS (20%) and 0.7 µl of EDTA (0.5 M). The
samples were analyzed by electrophoresis on a 0.8% agarose gel using a
Tris acetate-EDTA buffer system. The substrates and products were
visualized by ethidium bromide staining. S, linear dsDNA
substrate; I, partially exchanged reaction intermediates;
P, fully exchanged nicked circular dsDNA products;
ss, single-stranded DNA. Under these reaction conditions,
the ssDNA (5 µM total nucleotide) is limiting relative to
the linear dsDNA (15 µM total nucleotide = 7.5 µM base pairs); the maximum amount of the linear dsDNA
that can be converted to nicked circular dsDNA product is therefore
67%. Panel B, ATP hydrolysis assay. Aliquots were removed
from the strand exchange reactions described above and assayed for ATP
hydrolysis using a thin layer chromatography method described
previously (15). The points represent the amount of ATP
hydrolyzed by the wild type RecA protein (open circles) and
[P67G/E68A]RecA protein (closed circles) at the indicated
times.
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Fig. 4.
Effect of ATP regeneration on the wild type
and [P67G/E68A]RecA protein-promoted strand exchange reactions.
Strand exchange reactions were carried out as described in the legend
to Fig. 3. The reaction solutions contained 25 mM Tris
acetate (pH 7.5), 5% glycerol, 1 mM DTT, 10 mM
Mg(acetate)2, 5 µM circular X ssDNA, 15 µM linear
X dsDNA, 0.5 µM E. coli SSB, 3 mM ATP, and 2.5 µM wild type
RecA protein (wt, left) or [P67G/E68A]RecA
protein (mutant, right). S, linear
dsDNA substrate; I, partially exchanged reaction
intermediates; P, fully exchanged nicked circular dsDNA
products; ss, single-stranded DNA. The reaction solutions
also included an ATP regeneration system consisting of 10 units/ml
creatine kinase (Sigma) and 12 mM phosphocreatine, where
indicated.
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Fig. 5.
Effect of ATP concentration on the wild type
and [P67G/E68A]RecA protein-promoted strand exchange reactions.
Strand exchange reactions were carried out as described in the legend
to Fig. 3. The reaction solutions contained 25 mM Tris
acetate (pH 7.5), 5% glycerol, 1 mM DTT, 5 µM circular X ssDNA, 15 µM linear
X
dsDNA, 0.5 µM E. coli SSB, 10 mM
(with 1 or 3 mM ATP) or 13 mM (with 6 mM ATP) Mg(acetate)2, 2.5 µM wild
type RecA protein (wt, left) or [P67G/E68A]RecA
protein (mutant, right), and the indicated
concentrations of ATP. S, linear dsDNA substrate;
I, partially exchanged reaction intermediates; P,
fully exchanged nicked circular dsDNA products; ss,
single-stranded DNA.
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Fig. 6.
Effect of ADP on the wild type and
[P67G/E68A]RecA protein-promoted strand exchange reactions.
Strand exchange reactions were carried out as described in the legend
to Fig. 3. The reaction solutions contained 25 mM Tris
acetate (pH 7.5), 5% glycerol, 1 mM DTT, 10 mM
Mg(acetate)2, 5 µM circular X ssDNA, 15 µM linear
X dsDNA, 0.5 µM E. coli SSB, 2.5 µM wild type RecA protein
(wt, left) or [P67G/E68A]RecA protein
(mutant, right), and a mixture of ADP plus ATP (3 mM total nucleotide, with the percentage of ADP indicated).
S, linear dsDNA substrate; I, partially
exchanged reaction intermediates; P, fully exchanged nicked
circular dsDNA products; ss, single-stranded DNA.
X ssDNA and a 1.1-kb DraI fragment of the
linear
X dsDNA.
X ssDNA with the 1.1-kb
linear
X dsDNA in the presence of ATP. In this reaction, the fully
exchanged gapped circular dsDNA product is detected within 5 min, and
the reaction reaches completion after ~120 min. Moreover, in contrast to the results that were obtained with the full-length linear
X
dsDNA substrate (Fig. 4), the inclusion of an ATP regeneration system
in the reaction solution had no effect on the rate of the reaction with
the 1.1-kb linear
X dsDNA substrate (Fig. 7). These results indicate
that the ADP dependence of the [P67G/E68A]RecA protein-promoted
strand exchange reaction is reduced or eliminated when the 1.1-kb dsDNA
fragment is substituted for the full-length 5.3-kb dsDNA substrate. The
wild type RecA protein was also able to recombine the circular
X
ssDNA with the 1.1-kb linear
X dsDNA with similar efficiencies in
the presence or absence of the NTP regeneration system (Fig. 7).
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Fig. 7.
Wild type and [P67G/E68A]RecA
protein-promoted strand exchange reactions: 1.1-kb linear dsDNA
substrate. Strand exchange reactions were carried out as
described in the legend to Fig. 3. The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5% glycerol, 1 mM
DTT, 10 mM Mg(acetate)2, 5 µM
circular X ssDNA, 3 µM linear
X dsDNA fragment (1.1 kilobase pairs), 0.5 µM E. coli SSB, 3 mM ATP, and 2.5 µM wild type RecA protein
(wt, left) or [P67G/E68A]RecA protein
(mutant, right). S, linear dsDNA
substrate; P, fully exchanged nicked circular dsDNA
products; ss, single-stranded DNA. The reaction solutions
also contained an ATP regeneration system consisting of 10 units/ml
creatine kinase (Sigma) and 12 mM phosphocreatine, where
indicated.
X ssDNA with the
full-length linear
X dsDNA in the presence of ATP over the entire
range of Mg2+ concentrations examined. However, whereas the
fully exchanged product was not formed until 4 h at 10 mM Mg2+, the same product was formed within 90 min when the Mg2+ concentration was reduced to 4 mM (Fig. 8). Furthermore, in contrast to the results
obtained at 10 mM Mg2+ (Fig. 4), the inclusion
of the ATP regeneration system had no effect on the rate of the
reaction at 4 mM Mg2+ (gel not shown).
View larger version (75K):
[in a new window]
Fig. 8.
Effect of Mg2+ concentration on
the wild type and [P67G/E68A]RecA protein-promoted strand exchange
reactions. Strand exchange reactions were carried out as described
in the legend to Fig. 3. The reaction solutions contained 25 mM Tris acetate (pH 7.5), 5% glycerol, 1 mM
DTT, 5 µM circular X ssDNA, 15 µM linear
X dsDNA, 0.5 µM E. coli SSB, 3 mM ATP, 2.5 µM wild type RecA protein
(wt, left) or [P67G/E68A]RecA protein
(mutant, right), and the indicated concentration
of Mg(acetate)2. S, linear dsDNA substrate;
I, partially exchanged reaction intermediates; P,
fully exchanged nicked circular dsDNA products; ss,
single-stranded DNA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
X ssDNA molecule with a linear
X
dsDNA molecule to form a fully exchanged nicked circular dsDNA reaction
product. Although it appears to form the initial pairing intermediates
with an efficiency similar to that of the wild type RecA protein, the
time required for the appearance of the fully exchanged product is much
greater for the [P67G/E68A]RecA protein (4 h) than for the wild type
protein (10 min). Since the turnover number for the [P67G/E68A]RecA
protein-catalyzed ATP hydrolysis reaction (6 min
1) is ~3-4-fold lower than that of the
wild type RecA protein (20 min
1), it might be
expected that the rate of strand exchange would also be 3-4-fold
lower, since we have shown (with a different mutant RecA protein) that
the rate of strand exchange can be correlated with the rate of NTP
hydrolysis (Ref. 8; see also Ref. 11). The pronounced delay in the
appearance of the fully exchanged product that is observed with the
[P67G/E68A]RecA protein, however, is much greater than can be
accounted for by this effect. Instead, our results indicate that, under
standard reaction conditions, the strand exchange activity of the
[P67G/E68A]RecA protein is dependent on the presence of both ATP and
ADP, and that the fully exchanged reaction product does not form until
the ATP hydrolysis activity of the [P67G/E68A]RecA protein has
converted ~80% of the starting ATP into ADP. Because of this
requirement for ADP, the strand exchange reaction of the
[P67G/E68A]RecA protein is inhibited when the starting ATP
concentration is increased, since this increases the time required for
the ATP hydrolysis activity of the protein to convert 80% of the ATP
to ADP. Moreover, the formation of the fully exchanged product is
completely prevented when an ATP regeneration system is included in the
reaction solution because this prevents any ADP from accumulating in
the reaction solution. By contrast, the strand exchange activity of the
wild type RecA protein is not inhibited by high starting ATP
concentrations or by the presence of an ATP regeneration system.
X dsDNA
fragment is used in place of the full-length 5.3-kb dsDNA. The
[P67G/E68A]RecA protein may be able to fully exchange 1.1 kb of DNA
via the initial pairing step, without the need for an extensive branch
migration reaction. Additionally, the ADP dependence of the full-length
5.3-kb strand exchange reaction is decreased when the free
Mg2+ concentration is reduced to 1 mM or less.
The lower free Mg2+ condition presumably disfavors the
formation of discontinuous loop intermediates during the initial
pairing step (9) and may allow the [P67G/E69G]RecA protein to form
the fully exchanged reaction product more readily via the branch
migration reaction.
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ACKNOWLEDGEMENTS |
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We thank Tomoko Hamma, Edward Luk, and Marty Romeo for their assistance with some of the preliminary experimental work that led to the results described in this paper.
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FOOTNOTES |
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* This work was supported by Grant GM36516 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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Tel.: 410-955-3895; Fax: 410-472-3378; E-mail: fbryant@jhsph.edu.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100470200
2 S0.5 is the substrate concentration required for half-maximal velocity.
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ABBREVIATIONS |
---|
The abbreviations used are:
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA;
X, bacteriophage
X174;
SSB, E. coli single-stranded DNA binding protein;
kb, kilobase pair(s);
DTT, dithiothreitol.
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REFERENCES |
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