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INTRODUCTION |
The bacterial RecA
ATPase1 and its homologs
facilitate DNA recombination/repair, although the role of ATP
hydrolysis in these processes is not fully understood (1). All members
of the RecA family, including the human RAD51 protein (hRAD51), contain
classic Walker A/B motifs, which are fundamentally required for ATP
hydrolysis (1, 2). These motifs are generally conserved among proteins that bind and hydrolyze NTPs (3). RecA proteins mutated at the Walker
A/B motifs lack the ability to bind and/or hydrolyze ATP in
vitro (4). While cells expressing these RecA Walker A/B mutants
remain viable, they display dramatically reduced levels of
recombination and increased radiation sensitivity (5).
Multiple mitotic and meiotic RecA homologs contribute to eukaryotic
recombination/repair. Each of these homologs is likely to have distinct
requirements for ATP binding and/or hydrolysis in recombination/repair.
For example, four nonessential RecA homologs have been identified in
Saccharomyces cerevisiae: RAD51, RAD55, RAD57, and DMC1
(6-9). Mutation of the Walker A/B motifs of RAD51 and RAD55 results in
radiation sensitivity as well as meiotic recombination deficiency (9,
10). Mutation of the DMC1 Walker A motif results in a dominant meiotic
null mutant (11). However, similar mutations of RAD57 do not display
radiation sensitivity and are only modestly deficient for meiotic
recombination (9, 10).
The complexity of the recombination/repair system is further amplified
in higher eukaryotes, since eight vertebrate RecA homologs have been
identified (12). In contrast to RecA or the yeast homologs, the
vertebrate RAD51 gene appears to be required for cellular viability, since Rad51
/ mice
display early embryo lethality and embryo-derived cell lines could not
be established (13, 14). Similarly, chicken DT40 B-cells lacking
endogenous RAD51 were not viable (15). Taken together, these results
are consistent with the notion that the RecA homologs of higher
eukaryotes are not redundant. Interestingly, the chicken DT40
RAD51-deficient cells could be rescued by the overexpression of an
hRAD51 Walker A/B mutant protein that was able to bind but not
hydrolyze ATP (16). In addition, these rescued cells displayed no
increase in radiation sensitivity compared with wild type cells but
were less efficient at facilitating recombination-dependent gene targeting at several loci (16). This data suggests that the
contributions of hRAD51 ATP binding and hydrolysis to viability and
recombination/repair may be distinct.
Biochemical analysis has proven useful in elucidating the role of ATP
hydrolysis in the recombination functions of the RecA protein. It has
been suggested that cycles of ATP hydrolysis allow protomers within the
RecA nucleoprotein filament to alternate between distinct ATP- and
ADP-bound conformational states (1, 17). The cycling between
conformational states appears to drive directional strand exchange
during recombination. Historically, these alternating conformations
were suspected to facilitate strand exchange by redistributing
protomers within the nucleoprotein filament. This assumption was based
upon the differential affinities of RecA for DNA in the presence of
ATP
S (high affinity) versus in the presence of ADP (low
affinity) (1, 17). An alternative model suggested that strand exchange
is facilitated by ATP hydrolysis-dependent rotation of the
RecA nucleoprotein filament (18). Time lapse electron microscopy of
RecA nucleoprotein filaments formed in the presence of ATPS appeared
consistent with this latter proposal (19). These slowly hydrolyzing
nucleoprotein filaments remained in an extended yet dynamic state,
where the protomers appeared to rotate.
The coupling of ATP binding/hydrolysis to recombination by hRAD51 is
less certain. In comparison with electron microscopy images of RecA,
the hRAD51·ssDNA nucleoprotein filaments formed in the presence of
ATPS appeared less extended, suggesting a diminished response to ATP
(20). Yet, ATP was required for hRAD51 to extend the helical pitch
(DNA-unwinding) of dsDNA (20) as well as to promote strand exchange
between homologous DNA substrates (21-24).
Several recent studies suggest that hRAD51 can be induced to resemble
RecA, both structurally and functionally. hRAD51 forms an extended
nucleoprotein filament with the transition state mimetic ADP-AlF
that appears analogous to activated RecA (19). Two additional reports indicated that yeast and
human RAD51-mediated DNA strand exchange is greatly enhanced by
ammonium sulfate and/or spermidine (25, 26). The mechanistic basis for
ammonium sulfate and spermidine stimulation of RAD51 function is
unknown. It is important to note that the rate of RAD51 strand exchange
remains 3-5-fold lower than RecA (25, 26). Furthermore, RAD51 converts
a maximum of 30-60% of ssDNA to form II products in 60 min, whereas
RecA converts 100% ssDNA to form II in 15 min (25, 26).
Strand exchange by RecA has been shown to be largely independent of ATP
hydrolysis (27-30). However, RecA-dependent bypass of
heterologous DNA absolutely requires ATP hydrolysis (30-32). ATP
hydrolysis-dependent bypass of heterologous DNA has been
argued to require the generation of rotory torque and/or
recycling of the RecA protomers during the strand exchange reaction
(30, 33, 34). RAD51 displays a dramatically reduced capacity to bypass
heterologous DNA during strand exchange (26, 35-37). Taken together,
these observations suggest that altered ATP processing might account
for the disparities between hRAD51 and bacterial RecA activities.
To understand the ATP-dependent recombination functions of
hRAD51, we have performed the first quantitative examination of the
hRAD51 ATPase. Our results allow a detailed comparison with the
bacterial RecA protein, the only other homolog in this family in which
similar studies have been performed. Of particular importance is our
observation that hRAD51 appears to lack the magnitude of ATP-induced
cooperativity displayed by RecA. Our results suggest that other
regulatory factors or RecA homologs may be required to assemble and
control an hRAD51 nucleoprotein filament.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemicals of the highest grade were obtained from
Amresco (Solon, OH) or Sigma. Phosphoric/sulfuric acid-washed charcoal
was obtained from Sigma (catalog no. C-5510). ATP was purchased from Amersham Biosciences, dissolved in water, and adjusted for pH. ATP
concentration was determined by absorbance at 260 nm with 
= 1.54 × 104. [-32P]ATP was purchased
from PerkinElmer Life Sciences. Bacteriophage 
X174 DNA was purchased
from New England Biolabs (Beverly, MA). Linear, blunt-ended dsDNA
(RFIII) was prepared by treatment of bacteriophage X174 dsDNA (RFI)
with endonuclease StuI (New England Biolabs) followed by
phenol extraction and ethanol precipitation. The DNA was resuspended in
10 mM Tris-HCl (pH 8), 1 mM EDTA and analyzed
by agarose gel electrophoresis. The concentration of DNA is expressed
as mol of nt (ssDNA) or bp (dsDNA).
Purification of hRAD51--
hRAD51 was purified as previously
described (38) with several modifications. Briefly, hRAD51 cDNA was
subcloned into pET24d (Novagen), and induction was performed in the
E. coli strain BLR21pLysS. Cells were lysed by three
freeze/thaw cycles and centrifuged at ~160,000 × g
for 1 h. The supernatant was dialyzed overnight against 4 liters
of 100 mM Tris acetate (pH 7.5), 5% glycerol, and 7 mM spermidine HCl. The hRAD51 precipitate was collected by
centrifugation and resuspended (~10 mg/ml) in P buffer (100 mM potassium phosphate (pH 7.0), 10% glycerol, 150 mM NaCl, 1 mM EDTA, 1 mM
dithiothreitol) (fraction I). Fraction I was separated by
chromatography through Reactive Blue-4-agarose (Sigma). Protein was
eluted with a 750 mM step of NaCl and dialyzed overnight
against 4 liters of H buffer (20 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol) (fraction II). Fraction II was loaded
onto a heparin-Sepharose CL-6B column and eluted with a 750 mM NaCl step (fraction III). Fraction III was dialyzed
overnight against 4 liters of H buffer (fraction IV), loaded onto a
Mono-Q column (HR5/5 prepacked from Amersham Biosciences), and eluted
with a gradient of 150-750 mM NaCl over 20 ml (fraction
V). hRAD51 was dialyzed twice against 2 liters of modified H buffer
(containing 0.1 mM EDTA) and was stored on ice.
Occasionally, an additional purification step was necessary to remove
trace contaminants. In this case, the protein was dialyzed against
modified P buffer (P buffer containing 100 mM NaCl) after
Mono-Q, chromatographed on a Mono-S column equilibrated with modified P
buffer, and eluted with a 15-ml gradient of 100-750 mM
NaCl. This was followed by dialysis against modified H buffer (0.1 mM EDTA). Purified hRAD51 can be stored in modified H
buffer at 0 or 
80 °C for several months without appreciable loss
of ATPase activity. Protein concentration was determined by amino acid
analysis (Keck Facility, Yale University). Preparations were found to
be nuclease-free by incubation with both single- and double-stranded
phage DNA as well as small oligonucleotides.
ATP Hydrolysis--
Unless otherwise indicated, the 10-µl
reactions contained 1 µM hRAD51 and 6 µM
DNA (nt or bp). All reactions were performed in an A buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 2 mM magnesium acetate). In
addition to the indicated amounts of ATP, 1 µCi of
-[32P]ATP was included in each reaction. To achieve
consistency in such small reactions, mixes containing either DNA/ATP or
hRAD51 were added separately using a repeat pipettor (Eppendorf).
Reactions were initiated by adding the hRAD51 mix last and incubated at 37 °C for 30-60 min in an Ericomp thermal cycler with a heated lid
to prevent evaporation. 400 µl of ice cold 10% activated charcoal (Norit) in 10 mM EDTA was added to terminate the reactions.
Tubes were vortexed and placed on ice for a minimum of 3 h to
allow maximal binding of free ATP to the charcoal. After centrifugation for 30 min, two 50-µl aliquots were counted by the Cerenkov method. hRAD51 was omitted from two additional reactions, which contained either the lowest or highest amount of ATP. After processing with Norit, background hydrolysis determined for these reactions was averaged and subtracted from reactions containing hRAD51. In general, there was no significant difference in background between lowest and
highest ATP reactions. Total counts in each reaction were determined by
the Cerenkov method for duplicate reactions that had not been processed
with Norit. The average of at least three such duplicates was taken for
the total cpm/reaction. Specific activity of ATP (cpm/mol;
[-32P]ATP/ATP) in each was calculated by dividing the
total counts in each reaction by the amount of total ATP. The amount of
ATP hydrolyzed (mol) in each reaction was determined by dividing the adjusted hydrolysis value (cpm) by the specific activity of ATP in each
reaction. Molar ratios were varied by adjustment of the DNA or hRAD51
concentrations in otherwise identical reactions. In all reactions,
conditions were normalized for the DNA or hRAD51 storage buffers. The
pH was varied using similar reaction buffers except that MES was used
for the range between pH 6.2 and pH 7.2, and HEPES was varied between
pH 7.2 and 8.2. For ATPase reactions containing additional salts, 0.5 µl of 2 M (NH4)2SO4
(pH 8.0) and/or 0.5 µl of 80 mM spermidine HCl were added
to reactions with a final volume of 10 µl, yielding final
concentrations of 100 mM and/or 4 mM,
respectively. These reactions were processed as above.
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RESULTS |
ATP Dependence of Hydrolysis--
Using a modification of a
previously published method (38), we purified hRAD51 to near
homogeneity (Fig. 1). The ATPase activity
of hRAD51 was measured by the Norit method (see "Experimental Procedures"), and the unique conversion of ATP to ADP was confirmed by thin layer chromatography (TLC) (39). We found the Norit method to
be superior to TLC or PAGE analysis because of the ease of data
acquisition, reduced expense, and the large number of analyses that
could be performed in tandem. Past experience has suggested that the
separation efficiency of Norit exceeds 90% and can be increased by
prolonged incubation of the terminated reactions at 0 °C (39,
40).
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Fig. 1.
hRAD51 purified to near homogeneity.
hRAD51 (0.5 µg) was resolved by 10% SDS-PAGE and subsequently
silver-stained. Densitometric analysis performed with a Bio-Rad Gel Doc
System revealed better than 95% purity. The major contaminant, marked
by an asterisk, is ~30 kDa and is recognized by rabbit
polyclonal  -hRAD51 antibody.
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While a previous report indicated that ATP hydrolysis by hRAD51 was
very inefficient (kcat(ssDNA) = 0.2 min1, kcat(dsDNA) = 0.1 min1, kcat = 0.03 min1), these values were based on a single ATP
concentration (200 µM) (21). To confirm and extend these
studies and to examine the rate-limiting step(s) within the hydrolysis
cycle, we performed classic Michealis-Menten analysis to define the
Km and kcat
(Vmax/[hRAD51]o). We determined these
values for the hRAD51 ATPase activity in the presence of ssDNA,
supercoiled dsDNA (RFI), linear dsDNA (RFIII), and in the absence of
DNA. The kcat values
(kcat(ssDNA) = 0.21 min1;
kcat(dsRFI) = 0.1 min1;
kcat(dsRFIII) = 0.07 min1;
kcat = 0.07 min1; Fig.
2, A and B, and
Table I) agreed with values previously reported (21). These data indicate that the
kcat(ssDNA) for hRAD51 is ~150-fold lower than
bacterial RecA (kcat(ssDNA) = 28 min1), and the kcat(dsDNA) is
~220-fold lower than bacterial RecA
(kcat(dsDNA) = 22 min1 at pH 6.2)
(41-43). The kcat in the absence of DNA is
~4-fold higher than the bacterial RecA (kcat = 0.015 min1) (41-43). In addition, hRAD51 displays an
equal or higher apparent affinity for ATP
(Km(ssDNA) = 23 ± 3 µM; Km(dsDNA(RFI)) = 27 ± 4 µM;
Km(dsDNA(RFIII)) = 26 ± 5 µM; Km = 110 ± 22 µM) compared with bacterial RecA (S0.5(ssDNA) 
20-60 µM; S0.5(dsDNA(RFI)) 100 µM at pH 6.2; S0.5 100 µM)
(17, 41), the substrate concentration where half-maximal activity
occurs is S0.5 in a cooperative system and equals
Km in a noncooperative system (44). In general, DNA
appears to decrease the hRAD51 Km equivalently (Fig.
2, A and B, and Table I). Calculation of the
hRAD51 ATPase catalytic efficiency
(kcat/Km) suggests that ssDNA
(150 s1 M1) induces the ATPase
no more than 2-3-fold more than dsDNA (~68 s1
M1) and at least 5-fold more than in the
absence of DNA (11 s1 M1). The
catalytic efficiency of hRAD51 is ~50-fold less than bacterial RecA
(8300 s1 M1).
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Fig. 2.
hRAD51-mediated ATP hydrolysis as a function
of ATP. ATPase assays were performed by incubating 1.0 µM hRAD51 and, if present, 6 µM DNA (nt or
bp) with the indicated amount of ATP (which was supplemented with
[-32P]ATP). Each reaction was incubated at 37 °C
for 1 h and then terminated by the addition of 10% activated
charcoal (Norit) in 10 mM EDTA. The samples were
centrifuged, and the supernatant containing free phosphate was counted
by the Cerenkov method. Data points represent the average of at least
three replicate experiments. A, hRAD51 ATPase data were fit
to the Michaelis-Menten equation for reactions performed with ssDNA
( ) (Km = 23 ± 3;
Vmax 0.21), dsDNA ( ) (RFI)
(Km = 27 ± 4; Vmax 0.11), and dsDNA ( ) (RFIII) (Km = 26 ± 5;
Vmax 0.07) as well as in the absence of DNA
( ) (Km = 110 ± 22;
Vmax 0.07). Note that all
Km values are expressed in µM and that
Vmax represents µM ATP
hydrolyzed·min1. B, double-reciprocal plot
of the same data. C, Hill plot of the same data
(nH 1 for all cases).
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hRAD51 Lacks ATP-induced Cooperativity--
A commonly used method
for determining ATP-induced cooperativity is by calculating the Hill
coefficient (44). The Hill coefficient was originally developed for the
analysis of cooperative fractional saturation for a ligand binding to
multiple interdependent sites (45). If one assumes that the rate of an
enzymatic reaction is proportional to the fractional saturation of the
enzyme, then a slope (Hill coefficient) greater than one derived from a
plot of the fractional rate [v/(Vmax v)] versus the substrate concentration ([S]) indicates positive cooperativity (44). In general, the largest
value for cooperativity occurs at half-maximal fractional saturation.
As the fractional saturation approaches unity, the Hill coefficient
also approaches 1. This appears to be the case for the RecA ATPase (41,
46). The Hill coefficient (nH) of RecA varies
from nH = 3 to nH = 11 at
ATP concentrations below or slightly above the S0.5. At ATP
concentrations above the S0.5 (>100 µM),
ATP-induced cooperativity becomes less apparent, since the Hill
coefficient equals 1.
Three methods of analysis of hRAD51 ATPase data indicate that hRAD51
lacks ATP-induced cooperativity. First, in the absence or presence of
DNA, the rate of ATP hydrolysis was easily fit to a simple hyperbolic
curve (the Michaelis-Menten equation; Fig. 2A). Second, the
slope of the same ATPase data plotted by the double-reciprocal method
is linear (not concave upward; Fig. 2B). Third, the slope of
hRAD51 ATPase data plotted as a fractional rate versus ATP
concentration (Hill coefficient) was ~1 in all conditions (ssDNA
nH = 0.79; dsDNA (RFI) nH = 0.76; dsDNA (RFIII) nH = 0.74; absence of DNA
nH = 0.70; Fig. 2C and Table I).
hRAD51 also failed to display ATP-induced cooperativity in the range of
ATP concentrations below the Km (Fig.
3). These data contrast with the
cooperativity shown by RecA.
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Fig. 3.
The hRAD51 ATPase does not display
ATP-induced cooperativity at ATP concentrations below the
Km. Each reaction contained 0.4 µM hRAD51, 1.7 µM ssDNA (nt), or dsDNA
(RFI) (bp) and the indicated amount of ATP. Each reaction was incubated
at 37 °C for 30 min and processed with Norit as in Fig. 2. Data
points represent the average of at least three replicate experiments.
A, ATPase data for either ssDNA () or dsDNA (RFI) ()
were fit to the Michaelis-Menten equation. B, in the
presence of ssDNA (), the Hill coefficient
(nH) = 0.91, and in the presence of dsDNA
(RFI) (), nH = 0.74.
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The Effect of pH on hRAD51 ATPase--
RecA can efficiently
utilize dsDNA as a cofactor for hydrolysis at pH 6.2. However, at pH
8.0, the dsDNA-dependent ATPase can only be measured after
a lag of several hours (1, 17, 42). This lag at higher pH is absent
when the carboxyl-terminal domain of bacterial RecA is deleted or when
purified unwound dsDNA (form X) is used as a cofactor (43, 47, 48). We
examined the pH dependence of the hRAD51 ATPase between 6.2 and 8.2 and found no difference in ATPase activity for either ssDNA or dsDNA (RFIII) (Fig. 4, A and
B, respectively). It is interesting to note that a
comparison of the hRAD51 and RecA sequences suggests that the
carboxyl-terminal domain is missing in hRAD51 (1, 2, 49).
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Fig. 4.
The hRAD51 DNA-dependent ATPase
is not affected by pH. Each reaction contained 1.0 µM hRAD51, 200 µM ATP (supplemented with
[-32P]ATP), and 6 µM either ssDNA (nt)
(A) or dsDNA (bp; RFIII) (B). Each reaction was
incubated for 1 h at 37 °C and was processed by the Norit
method as described in Fig. 2. The pH was varied by using buffers made
with either MES, pH 6.2-7.2 (left side), or
HEPES, pH 7.2-8.2 (right side). Each
bar represents the average of at least three separate
experiments.
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High Salt Activation of the hRAD51 ATPase--
In the absence of
DNA, high salt concentrations increased the kcat
of bacterial RecA ATPase to a value that is equivalent to the
kcat(ssDNA) observed at low salt concentrations.
However, the high salt-dependent RecA ATPase reaction
displays a Km that approaches 1 mM and
fails to display ATP-induced cooperativity (50). ATPase activity under these conditions has been generally attributed to salt-induced structural transitions within the RecA nucleoprotein filament (51).
High salt (1.5 M NaCl) also increases the hRAD51 ATPase activity (kcat = 0.40 min1; Fig.
5A and Table I) such that it
approaches the ATPase activity observed with low salt in the presence
of ssDNA (150 mM NaCl; kcat(ssDNA) = 0.21 min1; Fig. 2A and Table I). In addition,
hRAD51 fails to display ATP-induced cooperativity in high salt
(nH = 0.8; Fig. 5B and Table I). In
contrast to RecA, the Km of the hRAD51 ATPase in
high salt (Km = 17 µM) resembles the
Km at low salt when DNA is present
(Km 20 µM). While the Km represents a number of variables (44), these
results are consistent with the notion that hRAD51 is incapable of
forming an actively hydrolyzing aggregate (see Ref. 50).
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Fig. 5.
hRAD51 promoted ATP hydrolysis in the
presence of high salt. hRAD51 (1.0 µM) was incubated
with the indicated amount of ATP (mM) for 1 h at
37 °C in reactions that contained 1.5 M NaCl. This high
salt condition stimulates hRAD51 ATPase activity to the same extent as
ssDNA (both Km and kcat
(Vmax/[E])). A, ATPase
data were fit to the Michaelis-Menten equation (Km = 16.9 ± 2 µM; Vmax = 0.2 µM·min1). B, a Hill plot of
the same data indicates that the high salt-dependent ATPase
of hRAD51 also lacks ATP-induced cooperativity
(nH = 0.81).
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DNA Dependence of the hRAD51 ATPase--
Michaelis-Menten analysis
of the hRAD51 ATPase suggested that DNA concentration affects both the
Km and Vmax (Fig. 6, A and B, and
Table II). The RecA protein displays a
reduction in S0.5 at elevated DNA concentrations similar to
that observed with hRAD51 (52). However, the
Vmax of RecA appeared unaffected by elevated DNA
concentration, whereas the Vmax of hRAD51
displayed a peak between 3 and 9 nt of ssDNA per hRAD51 monomer and
then decreased at high ssDNA concentrations (52). Calculation of the
catalytic efficiency
(kcat/Km) with ssDNA appears to indicate an optimum ratio of 6-8 nt of ssDNA/1 hRAD51 monomer. In
contrast, the catalytic efficiency of the hRAD51 ATPase in the presence
of dsDNA did not display an optimum. The Hill coefficient appeared to
remain constant, nH 1 at all ratios of
DNA/hRAD51 (data not shown). This observation contrasts similar studies
with RecA, where a 20 nt/1 RecA ratio resulted in a Hill coefficient greater than nH = 11 (46).
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Fig. 6.
The molar ratio of DNA/hRAD51 affects both
Vmax and Km of
the hRAD51 ATPase. For each ratio of hRAD51/DNA, both
Km () and Vmax ()
represent average values for a set of eight different ATP
concentrations performed in triplicate. These values were obtained by
fitting the initial rate of hydrolysis to the Michaelis-Menten
equation. Each reaction within a set contained 1.0 µM
hRAD51, the indicated amount of nt of ssDNA (A) or bp of
dsDNA (RFI) (B), and a variable amount of ATP (supplemented
with [-32P]ATP). Each was incubated at 37 °C for
1 h and subsequently processed with Norit as in Fig. 2. These data
are summarized in Table II.
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Table II
DNA dependence of the Km and Vmax for
hRAD51-mediated ATP hydrolysis
Km and Vmax were determined by
Michaelis-Menten analysis (see also Fig. 6).
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The Site Size of the hRAD51 ATPase--
The hRAD51 ATPase appeared
to increase linearly with increasing hRAD51 protein (Fig.
7A). This linear increase was
evident in the absence of DNA as well as in the presence of ssDNA and dsDNA. A clear saturation of the hRAD51 ATPase (v) was
observed at a molar ratio of 3-4 nt/1 hRAD51 (Fig. 7B; see
also Fig. 6A). Interestingly, there appeared to be an
initial saturation of ATPase activity that occurred at approximately a
1 nt/1 hRAD51 monomer molar ratio (Fig. 7B). These data
suggest that the hRAD51 ATPase may possess two modes of DNA
stimulation.
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Fig. 7.
hRAD51 and DNA dependence of ATP
hydrolysis. ATPase reactions were varied by mixing the indicated
amount of hRAD51 with 6 µM DNA (nt or bp) (A)
or the indicated amount of DNA with 3 µM hRAD51
(B). Each reaction contained 250 µM ATP and
was incubated for 30 min at 37 °C. Each reaction was processed by
the Norit method as in Fig. 2. Each point represents the average of
three replicate experiments. , reactions where ssDNA was added; ,
reactions where dsDNA (RFI) was added; , reactions performed in the
absence of DNA.
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The bacterial RecA site size (N) appeared to vary between 3 and 6 nucleotides per monomer and was dependent upon whether ATP hydrolysis or DNA binding was measured (17). This paradox was resolved
by dividing the vATPase/[DNA] (at constant
excess protein) by the vATPase/[protein] (at
constant excess DNA) and revealed that N was ~3 (53).
Using this methodology, we examined the site size of hRAD51 for both
ssDNA and dsDNA (Table III and Table IV). We found that the
vATPase/[DNA] for RecA remained constant with
increased DNA concentrations, while the
vATPase/[DNA] of hRAD51 decreased
significantly (Table III). These results produced a range of site sizes
that depended upon calculations using the average
vATPase/[protein] and individual
vATPase/[DNA] values (Table III) or the average
vATPase/[DNA] and individual
vATPase/[protein] values (Table IV). While
averages of these calculations yielded a site size of ~3 nt (bp),
these values appeared to be too variable to provide a precise
determination.
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Table III
ATPase data as a function of DNA concentration to determine site size
(N)
v was determined by incubating the indicated amount of DNA
with 3 µM hRAD51. Each 10-µl reaction contained 250 µM ATP and was incubated at 37 °C for 45 min. Site
size (N) was determined by dividing v/[DNA] by
average kcat values obtained from experiments where
[hRAD51] was varied in the presence of excess DNA (Table IV). An
average of the ssDNA site sizes (when hRAD51 was in excess) indicated
that N ~ 2.85 ± 0.76 nt. Excluding the 0.5 µM hRAD51 concentration (N = 9.14), the
same analysis for dsDNA indicated that N ~ 3.08 ± 1.11 bp. NA, not applicable.
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Table IV
ATPase data as a function of hRAD51 concentration to determine site
size (N)
v was determined by incubating the indicated amount of
hRAD51 with 6 µM DNA. Each 10-µl reaction contained 250 µM ATP and was incubated at 37 °C for 30 min.
kcat values were averaged for reactions performed in
the presence of either ssDNA (average kcat = 0.170)
or dsDNA (average kcat = 0.116). These average
kcat values were used to determine site sizes by
comparison with experiments when DNA was varied (Table III). The site
size (N) was determined by dividing the average
v/[DNA] value obtained for ssDNA (0.486) or dsDNA (0.358)
by each individual v/[hRAD51] obtained at various hRAD51
concentrations. Using this approach, the site size (N) = 2.66 ± 0.62 nt of ssDNA or N = 2.81 ± 0.89 bp of dsDNA (see also Fig. 7).
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The Effect of Ammonium Sulfate and Spermidine on the hRad51
ATPase--
Two recent reports demonstrate that extensive DNA strand
exchange promoted by the yeast and human RAD51 proteins could be induced with ammonium sulfate and/or spermidine (25, 26). Under these
modified conditions, hRAD51 displays a reduced affinity for dsDNA (25),
and the transition from intermediates to products during DNA strand
exchange was enhanced (26). Although ATPase activity was not examined,
these studies suggested that ammonium sulfate and/or spermidine
provided a condition for efficient DNA strand exchange in the absence
of significant ATP hydrolysis. To address the effects of ammonium
sulfate and/or spermidine, we performed a detailed analysis of the
hRAD51 ATPase under similar conditions. We found that ammonium sulfate
increased the Km ~3-4-fold in the presence of
ssDNA (Fig. 8A) or dsDNA (Fig.
8B) cofactors (Table V). In
contrast, spermidine did not significantly affect the hRAD51 ATPase
(Fig. 8; Table V). Ammonium sulfate also elicited a modest decrease in
the rate of hydrolysis (Vmax) in the presence of
dsDNA (Fig. 8B; Table V). However, the rate of hydrolysis
was modestly enhanced in the presence of ssDNA (Fig. 8A;
Table V). It is worth noting that under both conditions (ammonium sulfate and/or spermidine) the Hill coefficient remained ~1 (data not
shown), and the data were easily fit to the Michaelis-Menten equation
(Fig. 8).
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Fig. 8.
The effect of ammonium sulfate and spermidine
on the hRAD51 ATPase. ATPase assays were performed by incubating
0.5 µM hRAD51 and, if present, 6 µM ssDNA
(nt) (A) or dsDNA (bp) (B) with the indicated
amount of ATP (which was supplemented with [-32P]ATP).
Each reaction was incubated at 37 °C for 1 h and then
terminated by the addition of 10% activated charcoal (Norit) in 10 mM EDTA. The samples were centrifuged, and the supernatant
containing free phosphate was counted by the Cerenkov method. Data
points represent the average of at least three replicate experiments.
ATPase data were fit to the Michaelis-Menten equation. ATPase reactions
were performed under standard buffer conditions () in the presence
of 100 mM (NH4)2SO4
( ), in the presence of 4 mM spermidine
( ), or in the presence of 100 mM
(NH4)2SO4 and 4 mM
spermidine (). The insets clearly indicate the effect of
(NH4)2SO4 on Km
values. These data are summarized in Table V.
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DISCUSSION |
ATP binding and hydrolysis are coupled to the recombinational
strand exchange function(s) of bacterial RecA (1, 4, 17). This is
exemplified by the observation that the free energy of strand exchange
and ATP hydrolysis appear equivalent in temperature-dependent studies (54). In addition, coordinated ATP hydrolysis appears exceedingly efficient between protomers within the RecA nucleoprotein filament (Hill coefficient of >11). The ATP hydrolysis activity of
other RecA family members has not been well characterized. Initial
studies suggested that hRAD51 was inefficient at promoting extensive
recombinational strand exchange and displayed a weak ATPase activity
compared with RecA (21-23). More recent studies have demonstrated
enhanced RAD51 strand exchange activity in the presence of ammonium
sulfate and spermidine (25, 26). Our studies were initiated to detail
the mechanistic differences between RecA and hRAD51.
The hRAD51 ATPase appears fundamentally distinct from RecA. As has been
previously reported (21, 22), the kcat for
hRAD51 is ~150-200-fold lower than the kcat
for the bacterial RecA. Combined with the 2-3-fold lower
Km of hRAD51 reported here, these observations
translate to an ~50-fold difference in the catalytic efficiency
(kcat/Km) of hRAD51 compared
with the bacterial RecA. The catalytic efficiency of hRAD51 is ~7
orders of magnitude below a diffusion-limiting process (55).
The majority of sequence homology between hRAD51 and RecA is within a
central domain containing classic Walker A/B adenosine nucleotide
binding motifs (1, 2, 49). hRAD51 contains an N-terminal domain that is
absent in RecA and is missing a C-terminal domain that is present in
RecA (1, 2, 49). Based upon NMR and mutagenesis data, it has been
suggested that the N terminus of hRAD51 may functionally substitute for
the C terminus of RecA (56). However, the hRAD51 ATPase activity
reported here appears to largely resemble the ATPase activity of a
C-terminal truncated mutant of RecA (RecA5327). The
kcat of the RecA5327 mutant was stimulated by
both ssDNA and dsDNA equally and without a lag (48). Likewise, the
kcat of hRAD51 in the presence of ssDNA or dsDNA appeared only modestly different. Moreover, acidic pH allowed the RecA
to utilize dsDNA as a co-factor for ATP hydrolysis without a lag, an
effect that has been generally ascribed to charge neutralization of the
C-terminal domain (1). However, a range of pH had no effect on the
hRAD51 ATPase. These data suggest that the hRAD51 N terminus may not
fully substitute for the RecA C terminus.
We determined the DNA site size of the hRAD51 ATPase to be ~3 nt (bp)
for ssDNA or dsDNA. Similar methodologies were used to determine that
the bacterial RecA has a site size of 3 nt for ssDNA (53). This
correlative site size (3 nt or bp) for hRAD51 appears to indicate that
most of the protein in the purified fractions was active. While we have
estimated the minimal site size of hRAD51 to be 3 nt, 6-8 nt of ssDNA
per hRAD51 monomer provoked the optimal catalytic efficiency of the
hRAD51 ATPase. This observation may indicate that hRAD51 binds an
additional ssDNA molecule, similar to RecA. Saturation of a second RecA
ssDNA binding site reduced the S0.5 from 60 to ~20
µM. However, the Vmax and
ATP-induced cooperativity of RecA remained unaffected by excess ssDNA
(52, 53). In contrast, excess ssDNA lowered the
Vmax of the hRAD51 ATPase, and the
Km approached the KD for ATP binding (57). While the effect of excess ssDNA on the S0.5
of RecA appeared similar to hRAD51, the RecA ATPase retained an
~10-fold difference between the S0.5 and
KD for ATP binding. The persistent difference
between the S0.5 and KD of RecA is
thought to correlate with a threshold of nucleoprotein filament ATP
saturation that must precede efficient/cooperative ATP hydrolysis (17).
It may be that the hRAD51 ATPase is not subject to regulation by a
threshold of ATP saturation. The reduced Vmax
displayed by the hRAD51 ATPase in the presence of excess ssDNA also
suggests a change in the rate-limiting step (see Ref. 57).
It is notable that the hRAD51 ATPase fails to display the magnitude of
ATP-induced cooperativity found with the RecA ATPase. Whether the lack
of ATP-induced cooperativity is responsible for the reduced catalytic
efficiency of the hRAD51 ATPase and/or the distinct recombinational
strand exchange is uncertain. It is suspected that ATP-induced
cooperativity augments the catalytic efficiency of the RecA ATPase at
two potential rate-limiting junctures, 1) achieving the active/extended
nucleoprotein filament by saturation of a threshold number of RecA
protomers with ATP (1, 17, 58) and 2) maintaining the active/extended
nucleoprotein filament during ATP hydrolysis by provoking rapid ADP
release (thereby preventing reversibility) (59, 60). In the case of
RecA, the cooperative state of the nucleoprotein filament appears
critical for recombinational strand exchange as well as bypass of
heterologous DNA during strand exchange (1, 17, 30-32).
Specialized cellular or biochemical conditions may induce hRAD51 to
achieve and/or maintain an active nucleoprotein filament that is
necessary for efficient recombination functions. Such conditions could
enhance the rate and cooperativity of the hRAD51 ATPase. Alternatively,
such conditions may allow hRAD51 to more effectively utilize ATP but
not dramatically affect ATP hydrolysis. In the presence of ammonium
sulfate, we observed a modest decrease in the rate of hRAD51-mediated
ATP hydrolysis when dsDNA was used as the cofactor. These data appear
to be consistent with the interpretation of Sigurdsson et
al. (25), which suggested that ammonium sulfate enhanced the
ability of hRAD51 to discriminate between ssDNA and dsDNA. The most
significant effect of ammonium sulfate is to increase the
Km for ATP 3-4-fold. The molecular effect of
ammonium sulfate on the hRAD51 protein is unknown. However, recent
studies with the F1 ATPase suggest that
SO is capable of occupying an
intermediate position near the region of the -phosphate that, with
ADP, mimics an ATP hydrolysis intermediate (61). We have found that ADP
release appears rate-limiting for the hRAD51 ATPase (57). Perhaps
ammonium sulfate provokes transient ternary
hRAD51·ADP·SO complexes that maintain and mimic a more active ATP-bound nucleoprotein filament. Such
complexes would be distinct from fully ADP-bound inactive nucleoprotein
filaments and may promote efficient DNA strand exchange without
dramatically enhancing the rate of hydrolysis. It is important to note
that even in the presence of ammonium sulfate and spermidine, RAD51
does not appear to be capable of bypassing heterologous DNA during
strand exchange (25, 26). Our studies appear to suggest that it is the
continued inability of RAD51 to coordinate ATP hydrolysis between
protomers that makes bypassing heterologous DNA during strand exchange
largely refractory.
The present studies are consistent with at least two possibilities. One
possibility is that the role of ATP binding and hydrolysis by hRAD51 in
recombination/repair is different from RecA. Alternatively, other
factors may be required to mimic the enhancing effect(s) of ammonium
sulfate and/or improve the efficiency of hRAD51.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence and reprint requests should be addressed:
Kimmel Cancer Center, BLSB 933, 233 S. 10th St., Philadelphia, PA
19107. Tel.: 213-503-1346; Fax: 215-923-1098; E-mail: rfishel@lac. jci.tju.edu.
