From the Life Sciences Division, Lawrence Berkeley
National Laboratory, Berkeley, California 94720, the
¶ Department of Biochemistry, Drexel University College of
Medicine, Philadelphia, Pennsylvania 19102-1192, and the
Division of Biological Sciences, Section of Microbiology and of
Molecular and Cellular Biology, Center for Genetics and
Development, University of California, Davis, California 95616-8665
Received for publication, October 29, 2002, and in revised form, November 7, 2002
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ABSTRACT |
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The human Rad51 protein is essential for DNA
repair by homologous recombination. In addition to Rad51 protein, five
paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2,
Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of
these proteins, recombinant Rad51,
Rad51B-(His)6, and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from
Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B·Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and
Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability
to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with
mixed Rad51B and Rad51C, compared with that of the individual protein.
In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase
activity. Rad51C displays an ATP-independent apparent DNA strand
exchange activity, whereas Rad51B shows no such activity; this apparent
strand exchange ability results actually from a duplex DNA
destabilization capability of Rad51C. By analogy to the yeast Rad55 and
Rad57, our results suggest that Rad51B and Rad51C function through
interactions with the human Rad51 recombinase and play a crucial role
in the homologous recombinational repair pathway.
Homologous recombinational repair
(HRR)1 is an important
pathway in repairing DNA double-strand breaks (DSBs) with high
accuracy, which is indispensable for the maintenance of genome
stability (for review see Refs. 1-4). The human Rad51 protein, a
structural and functional homolog of Escherichia coli
recombinase RecA (5, 6), plays a central role in HRR. In
vitro studies have shown that Rad51 binds single-stranded and
double-stranded DNA (ssDNA and dsDNA), exhibits
DNA-dependent ATPase activity, and functions to catalyze
homologous pairing and DNA strand exchange (6-8). As a prerequisite
step for the action of Rad51 in HRR, Rad51 binds DNA to form highly
ordered nucleoprotein filaments (9, 10). It has also been shown that
these filaments form preferentially on tailed duplex DNA substrates
that mimic the 3'-overhanging ssDNA tails at the sites of DSBs (11).
Thus far, Rad51 is the only mitotic protein that catalyzes the key
reactions of homologous pairing and strand transfer.
In yeast, the Rad51-related proteins include Rad55 and Rad57, which
exist together as a tight heterodimer that interacts weakly with Rad51
(12). These two proteins appear to be Rad51 paralogs, probably derived
by gene duplication followed by the evolution of new functions. The
Rad55·Rad57 complex acts as a cofactor for the assembly of Rad51 onto
ssDNA and functions to promote the DNA strand transfer activity of
Rad51 (12). In human cells, in addition to meiotic Dmc1 protein, five
Rad51 paralogs have been identified:
Rad51B/Rad51L12 (13-15),
Rad51C/Rad51L2 (16), Rad51D/Rad51L3 (15, 17, 18), XRCC2 (19-21), and
XRCC3 (19, 22, 23). These Rad51 paralogs share 20-30% sequence
identity with Rad51 and with each other. Interactions between these
proteins have been shown by a yeast two-hybrid assay and by baculovirus
co-expression experiments (24). Recent evidence from mutations in
hamster and chicken DT40 cell lines (25-27), showing increased
sensitivity to DNA damage and increased spontaneous chromosomal
instability, suggests an important role for these paralogs in HRR.
With the aim of determining the function of these paralogs, recent
efforts have been devoted to purifying the proteins and to revealing
their biochemical properties. The Rad51D protein was reported to
display ssDNA binding and DNA-stimulated ATPase activity, and to
interact with XRCC2 in vivo (28). The co-purified XRCC3·Rad51C and XRCC2·Rad51D complexes bind to ssDNA and form protein·DNA networks, as visualized by electron microscopy (29-31). These heterodimers were also reported to exhibit homologous pairing activity (30, 31).
Here we report several in vitro activities of the human
Rad51B and Rad51C proteins, including DNA binding, ATP hydrolysis, and
apparent DNA strand exchange. We also compared these activities to
those of Rad51. The activities of mixed proteins (Rad51 plus Rad51C,
Rad51 plus Rad51B, and Rad51B plus Rad51C) were determined as well. Our
results demonstrate the formation of multiprotein complexes composed of
Rad51B·Rad51C and Rad51·Rad51C·Rad51B, and show that Rad51C both
separates duplex DNA and facilitates the binding of Rad51 to DNA.
Protein Expression--
The human Rad51, Rad51B, and Rad51C open
reading frames were cloned into the baculoviral vector, and the
recombinant proteins were expressed in Sf9 insect cells as
described previously (24). The human Rad51 protein was purified by
selective spermidine precipitation (32) with a modified step, an
additional Q-Sepharose purification after spermidine precipitation to
eliminate DNA.
The N-terminal His6-tagged Rad51B was initially
constructed and expressed in insect cells using the baculovirus system.
An attempt to purify this protein was unsuccessful, because the protein did not bind to a Ni-NTA column. It is possible that the N terminus of
the expressed Rad51B protein is folded into the structure and thus
inaccessible to bind a Ni-NTA column. Therefore, we switched to a
C-terminal-tagged construct. To do so, the C-terminal-tagged RAD51B gene was PCR-amplified from a human testis cDNA
library with a hexahistidine-encoded sequence in the reverse primer and cloned into a pFastBac1 baculoviral vector. The sequence of
RAD51B was confirmed to be identical to those published
previously. The C-terminal His6-tagged Rad51B protein was
then expressed in Sf9 insect cells. To study the interaction
between Rad51B and Rad51C, we chose to make an untagged Rad51C
construct, because the Rad51B was tagged with His6. The
RAD51C gene was also PCR-amplified from a human testis
cDNA library and cloned into a pFastBac1 vector. The sequence of
RAD51C was confirmed and the recombinant protein was
expressed in Sf9 cells.
Purification of Recombinant His6-tagged
Rad51B--
All purification procedures were carried out at 4 °C.
Frozen cells (from 2 liters of insect cell culture) were thawed on ice and resuspended in 120 ml of ice-cold lysis buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10% glycerol, 10 mM imidazole, and
protease inhibitors. The mixture was incubated on ice for 30 min, and
phenylmethylsulfonyl fluoride was added to a final concentration of 0.5 mM prior to lysis by sonication. The lysate was clarified
by centrifugation at 14,000 rpm for 40 min. The resulting supernatant
(soluble fraction) was then applied onto a Ni-NTA-agarose column (5 ml,
Qiagen), which had been previously equilibrated with start buffer (50 mM NaH2PO4, pH 8.0, 300 mM NaCl, 10% glycerol, and 10 mM imidazole).
The column was then washed with 10 column volumes of start buffer,
followed by 10 column volumes of wash buffer containing 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10% glycerol, and 20 mM imidazole.
Finally, elution buffer (50 mM
NaH2PO4, pH 8.0, 300 mM NaCl, 10%
glycerol, and 250 mM imidazole) was added, and the eluate
was collected in 2-ml fractions. The fractions containing Rad51B were
dialyzed at least 4 h against T buffer consisting of 25 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5 mM DTT, and 10% glycerol, and
further loaded onto a Q-Sepharose column (10 ml, Amersham Biosciences) pre-equilibrated with the same buffer. The column was subsequently washed with 5 column volumes of T buffer and eluted with a linear gradient of 0-0.8 M KCl in T buffer. The fractions
containing the large proportion of Rad51B were pooled and applied to a
Mono Q column (5 ml, Amersham Biosciences) pre-equilibrated with T buffer. The column was then washed with 5 column volumes of T buffer
and eluted with a linear gradient of 0-0.8 M KCl in T
buffer. The Rad51B protein was eluted in a peak at ~0.3-0.4
M KCl. The pure Rad51B protein, as determined by
SDS-PAGE, was finally dialyzed for 12 h against storage buffer
consisting of 25 mM Tris-HCl, pH 7.5, 100 mM
NaCl, 0.1 mM EDTA, 1 mM DTT, and 10% glycerol, and was stored in aliquots at Purification of Recombinant Rad51C--
Frozen cells (from 2 liters of insect cell culture) were thawed on ice and resuspended in
120 ml of ice-cold lysis buffer containing 100 mM Tris-HCl
(pH 8.0), 300 mM NaCl, 0.1% Triton X-100, 0.5 mM DTT, 2 mM EDTA, 5% glycerol, and protease
inhibitors. The mixture was incubated on ice for 30 min and was
sonicated briefly to reduce viscosity. Cell debris was removed by
ultracentrifugation in a Beckman 70Ti rotor at 39,000 rpm for 1 h.
The clarified supernatant was dialyzed two times against 4 liters of
spermidine buffer (20 mM Tris acetate, pH 7.5, 7 mM spermidine-NaOH, and 0.1 mM DTT) for a total
of 10 h. The precipitate was recovered by centrifugation at 12,000 rpm for 30 min and resolubilized in 40 ml of P buffer (0.1 M KH2PO4, pH 6.8, 0.1 M
KCl, 0.5 mM DTT, and 10% glycerol) by stirring slowly
about 2 h until the pellet had largely dissolved. Insoluble
material was removed by centrifugation in a Beckman 70Ti rotor at
39,000 rpm for 30 min. The clarified supernatant was loaded directly
onto a hydroxyapatite column (20 ml, Bio-Rad), which had been
pre-equilibrated with P buffer. After loading, the column was washed
with 10 column volumes of P buffer, then eluted with a linear gradient
of 0.1-0.8 M KH2PO4 in P buffer. The eluate was collected in 2-ml fractions. An aliquot (10 µl) of
each fraction was analyzed by SDS-PAGE, and peak fractions containing
Rad51C, which eluted at ~0.2-0.3 M phosphate, were pooled and dialyzed against T buffer for at least 4 h. The sample was then passed over a Q-Sepharose column (10 ml, Amersham Biosciences) pre-equilibrated with T buffer and washed with 5 column volumes of T
buffer. The bound proteins were eluted with a linear gradient of 0-0.8
M KCl in T buffer and collected in 1.2-ml fractions. The
fractions containing the large proportion of Rad51C were pooled and
applied to a heparin affinity column (5 ml, Amersham Biosciences), which had been previously equilibrated with T buffer. The column was
subsequently washed with 5 column volumes of T buffer and eluted with a
linear gradient of 0-0.8 M KCl in T buffer. The major peak
was eluted at 0.3 M KCl. The corresponding fractions were
pooled and applied to a Mono Q HR 5/5 column (Amersham Biosciences) pre-equilibrated with T buffer. The column was then washed with 5 column volumes of T buffer, and the Rad51C protein was eluted in a
sharp peak at 0.2 M KCl. The purified Rad51C was finally dialyzed against storage buffer and stored in aliquots as mentioned earlier.
Antibodies--
Based on amino acid sequence analysis, a unique
region on Rad51B was chosen for antibody production (CDAQLQGNLKERNKF,
Zymed Laboratory). Polyclonal antiserum against human Rad51B was raised in rabbits using this specific synthetic peptide as the immunogen. The
His6-tagged bacterial recombinant protein of human Rad51C was purified and used for polyclonal antiserum production in rabbits. Both Rad51B and Rad51C antibodies were affinity-purified from the
antiserum. The human Rad51 antibody was kindly provided by Dr. Akira Shinohara.
Ni-NTA Magnetic Agarose Beads Pull-down Experiments--
The
pull-down experiments were carried out with Rad51, Rad51C, and
His6-tagged Rad51B using the procedures as previously
described (24).
DNA Substrates--
The oligonucleotides used in this study
were: #1, 63-mer,
ACAGCACCAGATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGA; #2, 63-mer,
TCCTTTTGATAAGAGGTCATTTTTGCGGATGGCTTAGAGCTTAATTGCTGAATCTGGTGCTGT; #5,
32-mer, CCATCCGCAAAAATGACCTCTTATCAAAAGGA; #6, 32-mer,
TCCTTTTGATAAGAGGTCATTTTTGCGGATGG; #25, 48-mer,
GCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGA; #26, 48-mer,
TCCTTTTGATAAGAGGTCATTTTTGCGGATGGCTTAGAGCTTAATTGC; #70, 79-mer,
TACAACATGTTGACCTACAGCACCAGATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGA; #98, 59-mer,
ATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCG. The concentrations of the oligonucleotides were determined
spectrophotometrically using an extinction coefficient
( DNA Binding Assays--
63-mer (#2) was 5'-end-labeled with
[ ATPase Assays--
Reactions (25 µl) contained 1.25 µg of
ssDNA and 3 µM protein in 50 mM Tris-HCl (pH
7.5), 1 mM MgCl2, 40 mM KCl, 1 mM DTT, 100 µg/ml BSA, and 0.2 mM ATP (0.2 µCi of [ DNA Strand Exchange Assays--
To form nucleoprotein complexes,
Rad51C protein was incubated with ssDNA in the standard buffer
containing 33 mM HEPES (pH 7.0), 2 mM DTT, 100 µg/ml BSA, 2 mM ATP, and 3 mM magnesium
acetate at 37 °C for 15 min. DNA strand exchange was initiated by
the addition of dsDNA to the nucleoprotein complexes and incubated at
37 °C. Aliquots were withdrawn from the reaction mixture,
deproteinized by the addition of EDTA to 50 mM, SDS to 1%,
and proteinase K to 700 µg/ml, followed by incubation for 15 min at
37 °C, mixed with a 1/10 volume of loading buffer (30% glycerol,
0.1% bromphenol blue), and loaded onto a 10% polyacrylamide gel.
Products of DNA strand exchange were quantified using a Storm 840 PhosphorImager (Amersham Biosciences).
Nuclease Assays--
To control for nuclease activity, the
Rad51C protein preparation was assayed at the standard conditions for
DNA strand exchange. Rad51C nucleoprotein complexes were assembled on
63-mer ssDNA (#2). Duplex DNA (#1-2) was used as a substrate for DNA
strand exchange. The dsDNA was 32P-labeled at the 5'-end of
the identical DNA strand (#2) using T4 polynucleotide kinase and
[ Purification of Rad51B Protein--
After cells were lysed,
roughly 50% of the His6-tagged Rad51B protein was found in
the soluble fraction, and the protein bound to a Ni-NTA column. The
Ni-NTA column provides a powerful purification step yielding 90% pure
Rad51B. The eluate from the Ni-NTA column was then run through a
Q-Sepharose column to remove contaminating DNA from the protein
preparation. Finally, Rad51B was further purified to near homogeneity
by a Mono Q column (Fig. 1A).
About 400 µg of homogeneous Rad51B protein was obtained from two
liters of insect cell culture. The identity of the protein was
confirmed by Western blotting analysis with Purification of Rad51C Protein--
The solubility of Rad51C
protein was determined to be ~50%, and the soluble fraction was used
for purification. In light of the selective precipitation of the
E. coli RecA
(33)3 and human Rad51 protein
by spermidine (32, 35-38), the effect of spermidine on Rad51C was
examined. We found that Rad51C is indeed precipitated by spermidine in
a concentration-dependent manner (data not shown). Soon
after the soluble fraction of cell lysate was dialyzed against
spermidine buffer, a white precipitate was observed. The amount of
precipitate accumulated continuously up to 10 h with a substantial
increase following the second buffer change. Approximately 70% of the
Rad51C was precipitated by spermidine. The Rad51C protein was
subsequently recovered from the precipitate by slowly stirring the
precipitate ~2 h at 4 °C, with about 70% of the protein being
dissolved into the solution. The solution was then subjected to
sequential column chromatography using hydroxyapatite, Q-Sepharose,
heparin, and Mono Q columns. About 500 µg of homogeneous Rad51C
protein was obtained from 2 liters of insect cell culture (Fig.
2A). The identity of the
protein was confirmed using Complex Formation between Rad51, Rad51B, and Rad51C--
The
His6-tagged Rad51B and untagged Rad51 and Rad51C were
co-expressed in Sf9 cells. Using Ni-NTA beads, we found that
Rad51 could be pulled down from insect cell extracts by the
His6-tagged Rad51B through its interaction with Rad51C
(Fig. 3A). This result demonstrates a simultaneous interaction between Rad51, Rad51B, and
Rad51C, implying a heterotrimer formation between these three proteins.
However, the amount of Rad51 in the complex is much less than the
amount of Rad51C. This observation was found consistently in several
experiments, and may indicate that the association between Rad51C and
Rad51 is weak. In addition, to ensure that the observed protein
associations were not caused by DNA, the Ni-NTA pull-down experiments
were also carried out in the presence of an increasing amount of
ethidium bromide. We found that up to 700 µg/ml ethidium bromide did
not affect the pull-down (data not shown). In the presence of ethidium
bromide, a consistent amount of Rad51 was detected after pull-down,
suggesting that the presence of Rad51 in the complex is due to
protein-protein interactions rather than to DNA binding.
To further confirm this observation, individual purified proteins were
mixed and used in similar Ni-NTA pull-down experiments. Fixed amounts
of purified Rad51B and Rad51C (molar ratio = 1:1) were mixed with
increasing amounts of Rad51 in the absence and presence of 500 µg/ml
ethidium bromide. The results showed that an increasing amount of Rad51
was pulled down in both conditions (Fig. 3B), confirming
that the interaction is not due to DNA association. Importantly, no
detectable Rad51B and Rad51C were found in the supernatant (data not
shown), suggesting a strong interaction between Rad51B and Rad51C.
However, the majority of Rad51 remained in the supernatant, and only
~5-10% of Rad51 was pulled down by Rad51B (data not shown),
indicative of a weak interaction between Rad51B·Rad51C and Rad51.
Based on these results, we conclude that Rad51B and Rad51C form a
stable complex with a strong association constant and that Rad51,
Rad51C, and Rad51B form a heterotrimer involving a weak interaction
between Rad51 and Rad51C. To our knowledge, this is the first in
vitro evidence showing the simultaneous interaction between
Rad51B·Rad51C and Rad51.
Rad51B and Rad51C Preferentially Bind ssDNA-tailed
dsDNA--
Based on sequence alignments, we have found that four of
the human Rad51 paralogs, including Rad51B, Rad51C, Rad51D, and XRCC3, contain a conserved helix-hairpin-helix motif (39, 40) in their N
terminus.4 The
helix-hairpin-helix motif is a domain of around 20 amino acids widely
present in prokaryotic and eukaryotic proteins involved in
non-sequence-specific DNA binding, e.g. E. coli
RadC, eukaryotic ERCC1/Rad10 family, yeast SW10, and human Rad51 (41).
Therefore, we examined the DNA-binding ability of Rad51B and Rad51C
using an electrophoretic mobility-shift assay. Both Rad51B and Rad51C were found to bind
We also compared the DNA binding capabilities of Rad51B and Rad51C with
Rad51 using the same three DNA substrates: 63-mer, 63/63-mer, and
63/32-mer. We found that Rad51C possesses a higher affinity for all of
three DNAs than does Rad51 (Fig. 4A and data not shown). The
relative affinity of these three proteins for DNA was found to be:
Rad51C > Rad51 > Rad51B (Fig. 4A and data not shown).
In addition, we investigated the DNA binding capacity of various mixed
proteins. For the 63-mer ssDNA substrate, we observed enhanced DNA
binding with mixed Rad51 and Rad51C (Fig. 4B), as well as
with mixed Rad51B and Rad51C (Fig. 4C). As is particularly evident from substrate use, comparison of the DNA binding ability of an
individual protein (Rad51 or Rad51C) with the mixed proteins (Rad51
plus Rad51C) shows that the DNA binding activity of protein mixture is
elevated. Using 3'-tailed dsDNA (63/32-mer) as the substrate, DNA
binding by the Rad51B·Rad51C complex was also enhanced over the
individual proteins (data not shown). However, no stimulatory effect
was found with mixed Rad51 and Rad51B compared with individual Rad51 or
Rad51B (data not shown). It is likely that the direct interaction
between Rad51 and Rad51C, as well as between Rad51B and Rad51C, is
responsible for their increased ability to bind DNA.
Both Rad51B and Rad51C Exhibit DNA-stimulated ATPase
Activity--
Amino acid sequence analysis indicates that the five
human Rad51 paralogs contain the conserved ATP-binding domains (3), the
Walker A and B motifs, suggesting ATP binding and hydrolysis activities
for these proteins. Like human Rad51 and Rad51D (7, 28), both Rad51B
and Rad51C were found to display ATPase activity, and this activity was
stimulated by DNA (Fig. 5A).
Single-stranded DNA was found to be a better stimulator of the ATP
hydrolysis activity than dsDNA for both proteins (Fig. 5A).
The effect of various ssDNA species on the activity of each protein was
also determined, including
The ATP hydrolysis activity of Rad51B and Rad51C in the presence of
ssDNA was found to be lower than that of Rad51 (data not shown). When
the ATPase activity of mixed proteins was examined (Rad51 plus Rad51B,
Rad51 plus Rad51C, and Rad51B plus Rad51C), an additive effect on the
ATP hydrolysis activity was observed, rather than a stimulatory effect
(data not shown).
Rad51C Promotes an Apparent DNA Strand Exchange
Reaction--
Using oligonucleotide DNA substrates, we tested the
ability of Rad51B and Rad51C proteins to promote DNA strand exchange
in vitro. For this purpose, Rad51C nucleoprotein complexes
were formed with ssDNA 63-mer (#2), and these complexes were mixed with
dsDNA of three different lengths: 32, 48, and 63 bp. To detect the
products of DNA strand exchange, the strand in dsDNA that was identical in sequence to the ssDNA (#2) was labeled (i.e. the strand
that was expected to be displaced from the dsDNA). After incubation, we
observed the appearance of the displaced ssDNA, the product of DNA
strand exchange (Fig. 6A).
Rad51C generated ~20-30% displaced ssDNA product after 30 min of
incubation. As shown in Fig. 6B, the reactions work equally
well with the 32-, 48-, and 63-bp dsDNA substrates. The activity of
Rad51C protein only slightly depended on DNA length in the tested range
of dsDNA substrate, with the activity increasing slightly with
increasing dsDNA length. Consistent DNA strand exchange products were
observed using three independent Rad51C protein preparations. In
contrast to Rad51C, Rad51B protein did not show any activity under the
conditions examined (data not shown).
For comparison, the DNA strand exchange activity of Rad51 was examined
using the same DNA concentrations and assay conditions as used for
Rad51C, except that the optimal concentration of magnesium acetate (20 mM) was used and the concentration of Rad51 (0.6 µM) was the optimal 1:3 stoichiometric ratio relative to
ssDNA. Under these conditions, Rad51 catalyzed the formation of
~15-35% of ssDNA product after a 30-min reaction (Fig.
6C). However, Rad51 shows a preference for the shorter dsDNA
substrates. Thus, we conclude that Rad51C displays a significant
apparent DNA strand exchange activity, which is comparable to that of Rad51.
The Apparent DNA Strand Exchange Promoted by Rad51C Is Protein
Concentration and Magnesium Ion-dependent but Not
ATP-dependent--
To determine the reaction stoichiometry
between Rad51C and ssDNA, the DNA strand exchange reaction was carried
out with a fixed amount of ssDNA (1.5 µM) and various
amounts of Rad51C. Using 63-mer ssDNA and dsDNA substrates, the
reaction yield was found to increase with increasing concentrations of
Rad51C and saturated at ~5.5 µM (Fig.
7A). This amount greatly
exceeds the classic 1:3 stoichiometry for Rad51 and DNA, and it
indicates that the binding stoichiometry of Rad51C and DNA is different from that of the Rad51 protein.
Because both RecA and Rad51 proteins (7) require a
nucleotide cofactor for DNA strand exchange, we tested whether this requirement applied to the Rad51C-mediated reactions. We found that,
contrary to expectations based on the RecA/Rad51 family members, the
reaction promoted by Rad51C protein is independent of a nucleotide
cofactor (Fig. 7B). We also found that AMP-PNP had no
stimulatory effect on the reactions (Fig. 7B).
Because the DNA strand exchange activity of Rad51 shows a strong
dependence on magnesium ion concentration, we examined the effect of
magnesium acetate concentration on the activity of Rad51C. A titration
experiment (Fig. 7C) shows that the Rad51C-promoted reaction
exhibits a dependence on magnesium ion concentration, with maximal
activity at 3 mM magnesium that remains approximately constant to 20 mM. Because ammonium sulfate was found to
stimulate the DNA strand exchange activity of Rad51 (42), we also
tested its effect on Rad51C. However, under our conditions, 100 mM ammonium sulfate instead inhibited the reaction promoted
by Rad51C (Fig. 7C).
The Rad51C-promoted DNA Strand Exchange Reaction Results From Its
dsDNA-melting Ability--
An observed exchange of DNA strands can
occur by several different biochemical processes. The canonical
RecA-promoted reaction occurs in the absence of any nucleolytic
resection of the DNA duplex and in the absence of any detectable dsDNA
strand separation (i.e. generation of free ssDNA). To
investigate whether the observed Rad51C-mediated DNA strand exchange
occurs without the generation of free ssDNA from the dsDNA as an
intermediate, three control experiments were performed. The first was
to eliminate the possibility that DNA strand exchange was the result of
partial nucleolytic resection of the DNA strand in duplex substrate
that is identical to the ssDNA region, followed by annealing of this
recessed dsDNA to the homologous ssDNA; thermal branch migration would
lead to DNA strand exchange. To exclude this possibility, we examined the integrity of the identical strand that is displaced by DNA strand
exchange. For this purpose, the 32P-labeled displaced DNA
strand was analyzed by electrophoresis in a 15% polyacrylamide gel
containing 8 M urea. As shown in Fig. 8A, the length of ssDNA
displaced in the Rad51C-promoted reaction (lane 3) was
indistinguishable from the ssDNA displaced by heating (lane
2), and from the original 32P-labeled 63-mer
(lane 1). This result indicates that the observed DNA strand
exchange was not caused by the nucleolytic resection of dsDNA followed
by spontaneous branch migration of the annealed DNA molecules. A second
possibility is that Rad51C is simply destabilizing and melting the
dsDNA, resulting in the production of a free ssDNA strand that in a
reaction does not require the homologous ssDNA as a strand exchange
partner. Therefore, the homology dependence of the reaction was
examined. We found that no ssDNA was produced when the DNA substrates
were heterologous; in comparison, product formation was ~30% when
the partner DNA was homologous (Fig. 8B). This result
demonstrates that Rad51C protein-promoted DNA strand transfer is
homology-dependent. Finally, we were concerned that Rad51C
might still be melting the dsDNA but that the free ssDNA strands would
quickly re-anneal after deproteinization. Hence, the reaction would
appear homology-dependent, but only because the homologous
ssDNA was acting to trap the strand-separated DNA duplex. To
investigate this possibility, the standard
homology-dependent experiment was carried out, but in
addition, an excess amount of homologous ssDNA was added to the stop
buffer. This added homologous ssDNA, although complementary to the
displaced 32P-labeled ssDNA product (#2), was of a
different length (79-mer) than the original ssDNA (63-mer) present in
DNA strand exchange reaction. Thus, pairing that might occur during the
deproteinization step would be distinguished from DNA strand exchange
with the intended partner as diagramed in Fig. 8C,
panel I. We found that, in the absence of 79-mer ssDNA in
the stop/quench mix, a displaced 63-mer ssDNA product was generated as
observed before (Fig. 8C, panel II, lane
1). When the 79-mer ssDNA was present, the displaced 63-mer ssDNA
quickly annealed with 79-mer ssDNA to form a 79/63-mer duplex (Fig.
8C, lane 2); this result was fully expected and
showed that the 79-mer ssDNA was an effective quench for any free ssDNA produced. However, when the homologous ssDNA was replaced by
heterologous ssDNA (lane 3), the 79/63-mer duplex was still
formed, even though DNA strand exchange could not occur. The only way
that the 79/63-mer duplex could form in the presence of heterologous
ssDNA was if Rad51C was melting the 63 base pair duplex into separate
DNA strands that were then annealing with the 79-mer ssDNA when the
sample was being deproteinized. In agreement with this interpretation, omitting Rad51C results in only a background level of 79/63-mer duplex
(lane 4). To test whether a Rad51C·ssDNA complex was
required for this melting activity, the 63-mer ssDNA was omitted; the
79/63-mer duplex was nevertheless formed (lane 5). This
reaction was also Rad51C-dependent (lane 6),
showing that Rad51C indeed possesses the capability of melting duplex
DNA, which quickly anneals with the 79-mer ssDNA after
deproteinization. This interesting result indicates that the apparent
DNA strand exchange activity of Rad51C results from its ability to melt
and separate duplex DNA. The 79-mer quench experiment was also
performed with Rad51. No DNA melting or strand separation by Rad51 was
observed (data not shown), suggesting that the DNA melting/strand
separation activity is unique to Rad51C.
Because Rad51C was found to associate with Rad51, we were concerned
that the observed DNA strand exchange activity might originate from
contaminating Rad51 protein. To exclude the possibility that the
purified Rad51C (and Rad51B) preparation contains Rad51 from insect
cells, the same amount of the purified Rad51B and Rad51C preparation
used in all assays was examined by probing with Rad51 antibody. The
To establish a purification strategy for the Rad51 paralogs, we
discovered that spermidine selectively precipitates overexpressed Rad51C from cell extracts, resulting in ~70% purity of the protein. This step greatly facilitated the purification of Rad51C protein. Based
on our unpublished observations, Rad51B also favorably precipitates with spermidine. Thus, in addition to purification of RecA and Rad51 as
described previously (32, 33, 35-38),3 spermidine
precipitation is demonstrated here as a powerful strategy for
purification of Rad51-related proteins. Our results suggest that this
strategy might work for purifying other untagged Rad51 paralogs.
However, because spermidine also precipitates DNA, it is important to
include a Q-Sepharose column to eliminate DNA in the purification process.
In previous studies using a yeast two-hybrid assay and baculovirus
co-expression with Ni-NTA pull-down analysis (24), we reported that
Rad51B directly interacts with Rad51C, and that Rad51C weakly interacts
with Rad51. Here we demonstrate a simultaneous interaction among these
three proteins. This is the first in vitro evidence for
Rad51·Rad51C·Rad51B complex formation. However, in the course of
our study, it was reported that overexpressed His6-tagged Rad51C did not pull down Rad51 in human cells (43, 44). Because we
observed that the interaction between Rad51C and Rad51 is weak, only
~5-10% of the overexpressed Rad51 was pulled down by the Rad51B·Rad51C complex in insect cell extracts as well as by purified complex of Rad51B and Rad51C proteins, the failure to detect the Rad51C-Rad51 interaction in human cells (43, 44) is likely due to the
weak interaction between the Rad51B·Rad51C complex and Rad51.
To elucidate the mechanism of homologous recombination involving Rad51
and Rad51 paralogs and to understand the functions of each protein, the
biochemical activities of these proteins were determined. Using
established in vitro assays for Rad51, including DNA binding
and ATPase and DNA strand exchange assays, the biochemical properties
of Rad51B and Rad51C were determined and compared with those of Rad51.
To catalyze DNA strand transfer, Rad51 must first bind to ssDNA to form
a nucleoprotein filament (9, 10). Because the Rad51 paralogs play a
role in HRR, a key question is whether the paralogs are involved in the
DNA binding step. We found that four of the five Rad51 paralogs,
including Rad51B, Rad51C, Rad51D, and XRCC3, contain a
non-sequence-specific DNA binding domain, a helix-hairpin-helix motif
(39, 40). The DNA binding ability of each paralog was assessed using an
electrophoretic mobility-shift assay. In addition to the previously
reported DNA binding activity of Rad51D (28), we demonstrated that both
Rad51B and Rad51C proteins exhibit DNA binding capacity. This finding
suggests a role for the Rad51 paralogs in the DNA binding step. Because
the break sites of DNA are nucleolytically processed after DSB
formation to produce ssDNA with a 3'-overhanging end for binding by
RPA, Rad51, and other associated proteins, we examined the substrate binding preferences of Rad51B and Rad51C. Our data, showing that both
Rad51B and Rad51C proteins bind the 3'-end-tailed dsDNA substrates in
preference to either ssDNA or dsDNA, support the notion that the Rad51
paralogs bind the damaged sites and play a role in the repair
processes. The observation that the DNA binding activity of Rad51C is
higher than that of Rad51 is somewhat interesting. It may suggest that
Rad51C plays a crucial role in the DNA binding processes of the
HRR-related proteins. Another significant finding is that the complex
formed by Rad51 and Rad51C, as well as by Rad51B and Rad51C, displays a
higher DNA binding activity than that of the individual proteins. This
result suggests that the direct protein-protein interaction serves
possibly to recruit Rad51, and perhaps other proteins as well, to the
processed ssDNA.
Alignments of the amino acid sequences of Rad51 and five Rad51 paralogs
revealed that all proteins contain the Walker A and B motifs (3),
suggesting ATP binding and hydrolysis capability. The ATPase activity
of each paralog was confirmed in vitro. We showed that
purified Rad51B and Rad51C proteins display ATPase activity that is
stimulated by DNA. Little activity was observed in the absence of DNA,
implying that DNA is the key cofactor in activating ATP
binding/hydrolysis by Rad51B and Rad51C. The role of ATP binding and
hydrolysis by the Rad51 paralogs in HRR is still unclear. It was
reported that the ATPase mutant of XRCC2 does not affect the formation
of Rad51 foci (45), indicating that XRCC2 does not use ATP binding or
hydrolysis to promote its function. Whether the ATPase mutants of
Rad51B and Rad51C are altered in their DNA binding activity remains to
be determined.
Thus far, Rad51 is the only protein found to catalyze the key reactions
of homologous pairing and DNA strand transfer that are needed by HRR to
repair DNA double-strand breaks in human mitotic cells. Current
evidence supports a mediating role for Rad51 paralogs in HRR, where
these proteins may function as accessory cofactors assisting the action
of Rad51 (4). During the course of our research, the Rad51B·Rad51C
complex was purified in the Sung laboratory (46). They reported that
the Rad51B·Rad51C complex acts to facilitate the replacement of RPA
from the nucleofilaments by Rad51 and to promote the DNA strand
exchange activity of Rad51 (46), and they suggest a mediator role for
the Rad51B·Rad51C complex in HRR. We found, using oligonucleotide DNA
substrates, that Rad51C, but not Rad51B, promotes a DNA strand exchange
reaction. After further investigation, we discovered that the observed
DNA strand exchange products were generated by a DNA melting and strand separation activity of Rad51C. This result implies a distinct role for
Rad51C in HRR and suggests that Rad51C may have a more direct function
in addition to its mediator role. It is very possible that Rad51C
recruits Rad51 to ssDNA and mediates the separation of duplex DNA to
promote DNA strand exchange. This proposed function is based on several
aspects of our work: (i) Rad51 and Rad51C (with and without Rad51B)
physically interact; (ii) Rad51 and Rad51C show synergy in their DNA
binding affinity; and (iii) Rad51C promotes dsDNA separation for 32-, 48-, and 63-bp substrates equally well, but Rad51 acts preferentially
on the shorter substrates, suggesting that Rad51C might assist in the
melting of longer duplex regions. Together, these results demonstrate
that Rad51C behaves very differently from Rad51, indicating the Rad51C
may serve a different role from Rad51 in the DNA strand exchange stage
of homologous recombination. It is most likely that Rad51C "helps" with the strand exchange by Rad51 in several ways: Rad51C may not only
directly bind DNA at DSB sites but also facilitate the DNA binding of
Rad51 by complex formation and, subsequently, catalyze the separation
of duplex DNA for strand exchange. It was recently reported that Rad54
promotes transient separation of the strands in duplex DNA via its ATP
hydrolysis-driven DNA supercoiling function and the ability is
stimulated by Rad51 (47). It will be interesting to see whether Rad51C
functions with Rad54 in mediating the separation of duplex DNA for
strand exchange.
Among the protein-protein interactions between the five Rad51 paralogs,
Rad51C has been shown to be a central player that interacts directly
with Rad51B, Rad51D, XRCC3, and weakly with Rad51 (24), and which is
present in various multiprotein complexes in human cells, including
Rad51B·Rad51C, Rad51C·XRCC3, and Rad51B·Rad51C·Rad51D·XRCC2 (43, 44, 46, 48, 49). The evidence of multiple protein interactions
supports the notion that Rad51C may play multiple roles in the
recombinational repair processes. In addition, two hamster cell lines
that are mutated in Rad51C, irs3, and CL-V4B, were recently identified,
and these cells show a reduction in sister chromatid exchange and
genomic stability (34, 50), emphasizing a key role for Rad51C in HRR.
Here we described several biochemical properties that help define the
functions of Rad51C in this process, including complex formation, DNA
binding, ATP hydrolysis, and DNA melting/separating activities. These
findings underpin the significant biological function of Rad51C protein in the DNA strand exchange events of homologous recombination.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
260) of 9833 M
1cm
1 (1 A260 = 33 µg/ml). All DNA
concentrations are expressed in moles of nucleotides. Oligonucleotides
were stored at
20 °C.
-32P]ATP using standard protocols. 63-mer (#1) was
annealed to 63-mer (#2) to create blunt-ended dsDNA (63/63-mer). 32-mer
(#5) was annealed to 63-mer (#2) to create 3'-end-tailed dsDNA
(63/32-mer). The protein (2 µl) was incubated with
32P-labeled DNA (3 µM nucleotides) in a
buffer consisting of 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 60 mM KCl, 2 mM ATP, 1 mM DTT, and 100 µg/ml BSA in a
total volume of 10 µl at 37 °C for 30 min. The reaction mixture
was then mixed with a 1/10 volume of loading buffer (30% glycerol,
0.1% bromphenol blue). Samples were loaded onto a 10% polyacrylamide
gel and analyzed by electrophoresis in TBE at 9 V/cm for 2 h. Gels
were dried on filter paper, and 32P-labeled DNA·protein
complex was detected by autoradiography.
-32P]ATP) and were incubated at 37 °C. At
the indicated time points, a 5-µl aliquot was removed, and the
reaction was quenched by the addition of 2.5 µl of 0.5 M
EDTA. 1 µl of the samples was spotted on a thin layer chromatography
plate, and the plate was developed in 0.75 M
KH2PO4. The percentage of
[
-32P]ATP hydrolysis was quantified with a Model 425E
PhosphorImager (Amersham Biosciences).
-32P]ATP. DNA strand exchange was carried out for 30 min at 37 °C. After deproteinization, the products of this reaction
were separated using a 10% polyacrylamide gel run in TBE. The DNA
strand that was displaced from dsDNA was eluted from the gel, and its
integrity was confirmed by electrophoresis in a 15% polyacrylamide gel
containing 8 M urea.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Rad51B antibody (Fig.
1B).
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Fig. 1.
Purification of Rad51B protein.
A, Coomassie Blue-stained 10% SDS-polyacrylamide gel
of extracts of Sf9 cells before (C) and after
(E) expression of Rad51B protein. Soluble fraction
(S) was purified by Ni-NTA-agarose (Ni),
Q-Sepharose (Q), and Mono Q (MQ) column
chromatography. Molecular weight markers were shown in lane
M, and their sizes in kilodaltons are shown on the
left. The Rad51B protein is indicated by an
arrow. B, Western blot of the purified Rad51B
protein eluted from the Mono Q column using -Rad51B antibody.
-Rad51C antibody (Fig.
2B).
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Fig. 2.
Purification of Rad51C protein.
A, Coomassie Blue-stained 10% SDS-polyacrylamide gel of
extracts of Sf9 cells before (C) and after
(E) expression of Rad51C protein. Soluble fraction
(S) was purified by spermidine precipitation (SP)
and column chromatography using hydroxyapatite (HA),
Q-Sepharose (Q), heparin affinity (Hp), and Mono
Q (MQ). Molecular weight markers were shown in lane
M, and their sizes in kilodaltons are shown on the
left. The Rad51C protein is indicated by an
arrow. B, Western blot of the purified Rad51C
protein eluted from the Mono Q column using -Rad51C antibody.
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Fig. 3.
Rad51, Rad51B, and Rad51C form a heterotrimer
in vitro. A, Ni-NTA pull-down
experiments using baculovirus co-expressed insect cells. Lane
1, uninfected Sf9 cell extract; lane 2,
Sf9 singly infected with His6-tagged Rad51B;
lane 3, Sf9 doubly co-infected; and lane
4, Sf9 triply co-infected. Each gel was run in triplicate,
and three Western blots were done, probing with three different
antibodies. B, Ni-NTA pull-down experiments using purified
Rad51, Rad51B, and Rad51C proteins. Rad51B and Rad51C were mixed with
increasing amounts of Rad51 in the presence of 500 µg/ml ethidium
bromide. The pull-down samples were subject to Western blot analysis
with -Rad51,
-Rad51B, and
-Rad51C.
X174 single-stranded DNA (data not shown). To
further determine the substrate preference of these two proteins, oligonucleotide substrates were employed: ssDNA (63-mer), dsDNA (63/63-mer), and tailed dsDNA with 3'-tailed ssDNA (63/32-mer). Both
Rad51B and Rad51C preferentially bind 3'-tailed dsDNA over ssDNA, and
they have the lowest affinity for dsDNA (Fig.
4A). Two forms of Rad51C·DNA
complexes were observed: an aggregate that was trapped in the loading
well, and a protein·DNA complex that entered the gel (as indicated in
Fig. 4A). However, for Rad51 (data not shown) and Rad51B,
only the aggregates were observed. It is possible that Rad51C exists as
multimers of limited size, some of which form a large aggregate of
protein·DNA complexes. Our gel filtration results demonstrating
tetramer formation by Rad51C (data not shown) indeed support this
speculation. Because the repair of DNA double-strand breaks (DSBs) by
homologous recombination requires processing of the break to produce a
3'-overhanging ssDNA tail, our observations indicate that Rad51B and
Rad51C proteins preferentially bind the 3'-overhanging ssDNA tail
created during the repair process.
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Fig. 4.
DNA binding activity of Rad51B and Rad51C
proteins. A, gel shift assays, using oligonucleotide
substrates, were carried out in the presence of various amounts of
Rad51B or Rad51C. B, Rad51C promotes the DNA binding
activity of Rad51. Equal molar amounts of individual proteins (Rad51
and Rad51C) and the mixed proteins (Rad51 plus Rad51C) were added to
separate reaction mixtures. To do so, a half-molar amount of each
protein was mixed to make the final molar amount of total protein equal
in the three different samples (Rad51, Rad51C, and Rad51 plus Rad51C).
A gel shift assay was performed to determine their DNA binding
activity. C, Rad51B promotes the binding activity of Rad51C.
The molar amounts of each individual protein (Rad51B and Rad51C) and of
the total amount of mixed proteins (Rad51B plus Rad51C) are
indicated.
X174 ssDNA, M13 ssDNA, poly(dT), and a 63-mer oligonucleotide (Fig. 5B). Each ssDNA stimulated the
activity of Rad51B and Rad51C, indicating that the secondary structure of ssDNA has no significant effect on the stimulation. The ATPase activity of Rad51B was shown to be dependent on the presence of a
divalent cation, Mg2+, and the activity is Mg2+
concentration-dependent; the ATPase activity of Rad51B
increased with higher concentration of Mg2+. In addition,
Mn2+ was able to partially substitute for Mg2+
(Fig. 5C). Similar effects by a divalent cation were also
found for Rad51C (data not shown) and were reported for Rad51D
(28).
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Fig. 5.
ATP hydrolysis activity of Rad51B and Rad51C
proteins. A, time course for ATPase activity of Rad51B
(panel I) and Rad51C (panel II). Rad51B or Rad51C
was incubated in the absence ( ) or presence of single-stranded (
)
or double-stranded (
)
X174 DNA as described under "Experimental
Procedures." At the indicated time points, an aliquot was removed and
the reaction was quenched by EDTA. B, the ATPase activity of
Rad51B and Rad51C was stimulated by various single-stranded DNA. ATPase
activity was quantified for Rad51B (panel I) and Rad51C
(panel II) after 2-h incubation in the absence of DNA and in
the presence of circular
X174 ssDNA, circular M13 ssDNA, poly[dT]
DNA, and a 63-mer oligonucleotide (76 µM nucleotides).
Each value represents an average from two independent determinations.
C, the ATPase activity of Rad51B is dependent on a divalent
cation. Rad51B was incubated with
X174 ssDNA and three different
amounts of Mg2+ (
) or 2.5 mM of
Mn2+ (
) at 37 °C for 2 h, and the ATP hydrolysis
activity was determined.
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Fig. 6.
Rad51C protein displays an apparent DNA
strand exchange activity. A, DNA strand exchange
between labeled dsDNA and homologous ssDNA, as a function of dsDNA
length. Nucleoprotein complexes were formed by incubating 1.5 µM of ssDNA (63-mer, #2) with 6 µM of Rad51C protein. To initiate DNA strand exchange,
these complexes were mixed with 32P-labeled dsDNA and
incubated for various time points at 37 °C as indicated. The
nucleotide concentration of dsDNA was 1.5 µM for 32-bp
(#5-6), 2.25 µM for 48-bp
(#25-26), and 3.0 µM for 63-bp
(#1-2) duplexes. The concentration of dsDNA was equimolar
to ssDNA in terms of molecule concentrations. Control reactions without
protein were carried out under identical conditions. The products of
DNA strand exchange were determined as described under "Experimental
Procedures." The products of DNA strand exchange reaction were
quantified and plotted in panel B. The open
symbols represent the protein-free controls. C, DNA
strand exchange activity of Rad51. The DNA concentrations and buffer
conditions were the same as used for Rad51C except that 20 mM magnesium acetate and 0.6 µM of Rad51 was
used for the reactions. The products of DNA strand exchange were
quantified and plotted.
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Fig. 7.
Effects of Rad51C, ATP, and magnesium acetate
concentration on DNA strand exchange. A, the reaction
catalyzed by Rad51C is protein-concentration dependent. Nucleoprotein
complexes were formed by incubating 1.5 µM ssDNA (63-mer,
#2) with Rad51C protein at concentrations of 0, 1, 3, 4, 6, or 9 µM. To initiate DNA strand exchange, these complexes
were mixed with 3 µM of 32P-labeled 63-bp
dsDNA (#1-2) and incubated for 30 min at 37 °C. The
products of DNA strand exchange were determined as described under
"Experimental Procedures" and plotted. B, the reaction
catalyzed by Rad51C protein is ATP-independent. The reaction conditions
of DNA strand exchange were identical to those described in panel
A, except that 6 µM Rad51C was used and the
reactions were assayed in the presence ( ) or absence (
) of ATP,
or ATP was replaced with AMP-PNP (
), when indicated. The control
reaction without protein (
) was carried out under identical
conditions. The aliquots were withdrawn from the reaction mixtures at
the indicated time points, deproteinized, and analyzed by
electrophoresis in a 10% polyacrylamide gel run with TBE buffer.
C, the reaction catalyzed by Rad51C protein is dependent on
the magnesium ion concentration. The reaction conditions for DNA strand
exchange were identical to those described in panel A,
except that the Rad51C protein concentration was 6 µM and
assayed in the presence of 100 mM ammonium sulfate or
increasing concentrations of magnesium acetate.
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Fig. 8.
Rad51C-promoted DNA strand exchange is not
due to nuclease activity but is mediated by a DNA melting
activity. A, the ssDNA strand displaced during strand
exchange promoted by Rad51C is intact. The reaction conditions for DNA
strand exchange were identical to those described above. Rad51C was
mixed with 1.5 µM ssDNA (63-mer, #2). DNA
strand exchange was initiated by addition of homologous 3 µM of 63-bp dsDNA (#1-2), in which the strand
that would be displaced during DNA strand exchange (#2) was
32P-labeled. The ssDNA displaced was isolated, and its
integrity was analyzed by electrophoresis in a 15% polyacrylamide gel
containing 8 M urea (lane 3) along with
ssDNA-displaced strand produced in a spontaneous DNA strand exchange
(lane 2) and with the original 32P-labeled ssDNA
63-mer (lane 1). B, Rad51C-promoted DNA strand
exchange requires a homologous DNA. Either 1.5 µM
heterologous 59-mer (#98) or homologous 63-mer
(#2) ssDNA was separately incubated with 6 µM
Rad51C protein. To initiate DNA strand exchange, these nucleoprotein
complexes were mixed with 3 µM 32P-labeled
63-bp dsDNA (#1-2) and incubated for 10 min at 37 °C.
The products of DNA strand exchange were determined as described under
"Experimental Procedures." C, Rad51C displays DNA
melting activity. The reactions are carried out in the same conditions
as used in panel B except that 13.1 µM of a
79-mer ssDNA (#70) was included in the reaction stop
buffer.
-Rad51 recognized the insect Rad51 in control samples from
Sf9 cells, but no Rad51 was detected in either preparation (data
not shown), indicating that the DNA binding, ATPase, and apparent DNA
strand exchange activities are intrinsic to Rad51B and Rad51C proteins,
rather than due to Rad51 contamination.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Saira Mian for helping with amino acid alignments. We also thank Larry Thompson and David Schild for critical reading of the manuscript and Kevin Peet for his editorial contribution.
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FOOTNOTES |
---|
* This work was supported by the U.S. Department of Energy under contract DE-AC03-76SF00098, National Institutes of Health Grant CA 74046, U.S. Department of Defense Grants DAMD 17-99-9170 and DAMD 17-01-1-0542, and California Breast Cancer Research Program Grant 7KB-0019 (to Y.-C. L. and D. J. C.); National Institutes of Health Grants GM-62653 and CA-092584 (to S. C. K.); and Drexel University College of Medicine Startup funds and Pennsylvania Health Research Formula Funds from the Tobacco Settlement Act (to A. V. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: MS74-157, Life Sciences Division, 1 Cyclotron Rd., Lawrence Berkeley National Laboratory, Berkeley, CA 94720. Tel.: 510-486-5861; Fax: 510-486-6816; E-mail: YLio@lbl.gov.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M211038200
2 Rad51L is the recommended symbol for the Rad51-like genes/proteins (Human Gene Nomenclature Committee).
3 J. Mirshad and S. C. Kowalczykowski, submitted for publication.
4 Y.-C. Lio, S. Mian, and D. J. Chen, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
HRR, homologous
recombinational repair;
DSB, double-strand break;
ssDNA, single-stranded DNA;
dsDNA, double-stranded DNA;
Ni-NTA, nickel-nitrilotriacetic acid;
DTT, dithiothreitol;
BSA, bovine serum
albumin;
RPA, replication protein A;
AMP-PNP, 5'-adenylyl-,
-imidodiphosphate.
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REFERENCES |
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