From the RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan, the ¶ Cellular
Signaling Laboratory, RIKEN Harima Institute at SPring-8, 1-1-1 Kohto,
Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, the § Department
of Biophysics and Biochemistry, Graduate School of Science, University
of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the
** Cellular & Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan, and the
Core
Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation, 1637 Yana, Kisarazu, Chiba 292-0812, Japan
Received for publication, October 24, 2002, and in revised form, November 15, 2002
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ABSTRACT |
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The human Rad51B protein is involved in the
recombinational repair of damaged DNA. Chromosomal
rearrangements of the Rad51B gene have been found in
uterine leiomyoma patients, suggesting that the Rad51B gene
suppresses tumorigenesis. In the present study, we found that the
purified Rad51B protein bound to single-stranded DNA and
double-stranded DNA in the presence of ATP and either Mg2+
or Mn2+ and hydrolyzed ATP in a DNA-dependent
manner. When the synthetic Holliday junction was present along with the
half-cruciform and double-stranded oligonucleotides, the Rad51B protein
only bound to the synthetic Holliday junction, which mimics a key
intermediate in homologous recombination. In contrast, the human
Rad51 protein bound to all three DNA substrates with no obvious
preference. Therefore, the Rad51B protein may have a specific function
in Holliday junction processing in the homologous recombinational repair pathway in humans.
Chromosomes are continuously subjected to attacks by exogenous and
endogenous mutagens, which damage the genomic DNA. Chromosomal double
strand breaks, which are potential inducers of chromosome aberrations and tumorigenesis, are caused by ionizing radiation, oxygen
free-radicals, DNA cross-linking reagents, and DNA replication failure
(1, 2). Homologous recombinational repair
(HRR)1 is an accurate pathway
for repair without base substitutions, deletions, and insertions, and
therefore, it is important to maintain chromosomal integrity (3-5). In
the HRR pathway, a single-stranded DNA (ssDNA) tail, which is produced
at the site, invades a homologous region of the intact sister
chromatid. Through this "homologous-pairing" step, an intermediate
structure, the Holliday junction, in which two double-stranded DNA
(dsDNA) molecules form a four-way junction (6), is generated between
the damaged and intact chromatids (7). The Holliday junction can move
along the DNA and has the ability to expand a heteroduplex region. This
step, termed "branch migration," is essential for the HRR pathway.
In humans, genes involved in the HRR pathway, including
Rad51, Rad52, Rad54,
Rad54B, Brca1, Brca2,
Xrcc2, Xrcc3,
Rad51B/Rad51L1/hRec2 (Rad51B), Rad51C/Rad51L2
(Rad51C), and Rad51D/Rad51L3
(Rad51D), have been identified (8, 9). Mutations in several
of these genes have been found in cancer patients but not in healthy
individuals (2). Notably, gross chromosomal rearrangements of the
Rad51B gene have been reported in uterine leiomyomas,
involving translocations with the HMG1C gene (10, 11), and
in a pulmonary chondroid hamartoma (12). In mice, disruption of the
Rad51B gene results in early embryonic lethality (13),
suggesting that the Rad51B gene product is essential for
development. The Rad51B knockout in chicken DT40 cells
impaired the HRR pathway and caused sensitivity to DNA-damaging agents
such as cisplatin, mitomycin C, and In the present study, we purified the human Rad51B protein as a
recombinant protein and biochemically characterized it. The purified
Rad51B protein bound to ssDNA and dsDNA in the presence of ATP and
Mg2+ or Mn2+ and hydrolyzed ATP in a
DNA-dependent manner. When the Holliday structure coexisted
with the replication-fork-like structure and B-form DNA, Rad51B only
bound to the synthetic Holliday junction, suggesting that Rad51B may
play a role in Holliday junction processing in the HRR pathway.
Overexpression and Purification of Rad51B--
The human
RAD51B gene was isolated from a brain cDNA library
(purchased from Clontech) by the polymerase chain
reaction and was cloned in the NdeI site of the pET15b
vector (Novagen). In this construct, the His6 tag sequence
was fused at the N terminus of the gene. The Rad51B protein was
expressed in the Escherichia coli JM109(DE3) strain, which
also carried an expression vector for the minor tRNAs (Codon(+)RIL,
Novagen). The cells producing the Rad51B protein were resuspended in a
buffer containing 50 mM sodium phosphate (pH 6.7), 0.3 M NaCl, 2 mM 2-mercaptoethanol, 5 mM imidazole, and 10% glycerol and were disrupted by
sonication. The cell debris was removed by centrifugation for 20 min at
30,000 × g, and the lysate was mixed gently by the
batch method with ProBond beads (Invitrogen) at 4 °C for one hour.
The Rad51B-bound ProBond beads were then packed into an Econo-column
(Bio-Rad) and were washed with 30-column volumes of a buffer containing 50 mM sodium phosphate (pH 6.7), 0.3 M NaCl, 2 mM 2-mercaptoethanol, 60 mM imidazole, and 10%
glycerol at a flow rate of about 0.3 ml/min. The
His6-tagged Rad51B was eluted in a 30-column volume linear
gradient from 60 to 400 mM imidazole in 50 mM
sodium phosphate (pH 6.7), 0.3 M NaCl, 2 mM
2-mercaptoethanol, and 10% glycerol. The His6 tag was
uncoupled from Rad51B by a digestion with 10 units of thrombin protease
(Amersham Biosciences) per mg of Rad51B, and the protein was
immediately dialyzed against a buffer containing 20 mM
sodium phosphate (pH 7.2), 100 mM NaCl, 5 mM
dithiothreitol, 0.1 mM EDTA, and 10% glycerol at 4 °C.
Then, the fractions containing Rad51B were mixed with 7 ml of
Heparin-Sepharose (Amersham Biosciences) at 4 °C for one hour and
were packed into an Econo-column (Bio-Rad). The Heparin-Sepharose beads
with Rad51B were washed with 10-column volumes of a buffer containing
20 mM sodium phosphate (pH 7.2), 100 mM NaCl, 5 mM dithiothreitol, 0.1 mM EDTA, and 10%
glycerol, and the Rad51B was eluted in a 10-column volume linear
gradient from 0.1 to 1.5 M NaCl in this buffer. The Rad51B
was dialyzed against a buffer containing 20 mM sodium
phosphate (pH 7.2), 100 mM NaCl, 5 mM
dithiothreitol, 0.1 mM EDTA, and 10% glycerol and was
subjected to Mono-Q (Amersham Biosciences) column chromatography. The
Rad51B was eluted in a 4-column volume linear gradient from 0.1 to 0.3 M NaCl in buffer containing 20 mM sodium
phosphate (pH 7.2), 5 mM dithiothreitol, 0.1 mM
EDTA, and 10% glycerol. The concentration of the purified Rad51B
protein was determined with a Bio-Rad protein assay kit, using bovine
serum albumin (BSA) as the standard.
Purification of the Human Rad51 Protein--
The Rad51
expression vector was constructed as described (18).
His6-tagged Rad51 was overexpressed in E. coli
strain JM109(DE3), as described above. Harvested cells were disrupted
by sonication in a buffer containing 50 mM Tris-HCl (pH
8.0), 0.5 M NaCl, 5 mM 2-mercaptoethanol, 10 mM imidazole, 10% glycerol, and protease inhibitors
(Complete EDTA-free, Roche Molecular Biochemicals). Lysates were mixed
gently by the batch method with nickel-nitrilotriacetic acid agarose
beads (Qiagen) at 4 °C for one hour. The Rad51-coupled nickel-nitrilotriacetic acid agarose beads were then packed into an
Econo-column (Bio-Rad) and were washed with 30-column volumes of a
buffer containing 50 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM 2-mercaptoethanol, 60 mM imidazole, and 10% glycerol at a flow rate of about 0.3 ml/min. The His6-tagged Rad51 was eluted in a 30-column
volume linear gradient from 60 to 400 mM imidazole in the
aforementioned buffer. The Rad51, which eluted in a broad peak, was
collected and treated with 1 unit of thrombin protease (Amersham
Biosciences) per mg of Rad51. The Rad51 protein was immediately
dialyzed against a buffer containing 50 mM Tris-HCl (pH
8.0), 0.2 M KCl, 0.5 mM EDTA, 2 mM
2-mercaptoethanol, and 10% glycerol at 4 °C. The Rad51 protein
precipitated overnight but re-dissolved after changing the dialysis
buffer the following day. After more than 24 h of dialysis, the
Rad51 protein was collected and filtered to remove the residual
precipitates. About 20 mg of HsRad51, which was more than 99% pure as
judged by SDS-PAGE and Coomassie Brilliant Blue staining, were then
applied to a 1-ml Mono Q column (Amersham Biosciences) for
concentration. The Mono Q column was washed with buffer (50 mM Tris-HCl (pH 8.0), 0.2 M KCl, 0.5 mM EDTA, 2 mM 2-mercaptoethanol, and 10%
glycerol), and the HsRad51 protein was eluted with a 20-ml linear
gradient from 0.2 to 0.6 M KCl in this buffer. The Rad51
protein eluted sharply between 0.3 and 0.4 M KCl, and the peak
fractions had concentrations of about 2 mg/ml. These fractions were
dialyzed against a buffer containing 20 mM Tris-HCl (pH
8.0), 0.1 M KCl, 0.5 mM EDTA, 2 mM
2-mercaptoethanol, and 10% glycerol and were used for subsequent
studies. Protein concentrations were determined using the Bio-Rad
protein assay kit with BSA as the standard.
Oligonucleotides--
An HPLC-purified 50-mer oligonucleotide
(5'-ATTTC ATGCT AGACA GAAGA ATTCT CAGTA ACTTC TTTGT GCTGT GTGTA-3') was
used for the D-loop formation assay. This sequence is
derived from a clone of human Preparation of Closed Circular Double-stranded DNA and Circular
Single-stranded DNA--
To avoid irreversible denaturation of the
dsDNA, we prepared plasmid DNA without any treatment that would
potentially cause denaturation, such as alkaline treatment, as
described previously (20). The plasmid DNA (pGsat4; 3216 base pairs)
containing the human Single-stranded DNA-binding Assay--
Double-stranded DNA-binding Assay--
Synthetic Holliday Junction, Half-cruciform, Single-stranded
Oligonucleotide, and Double-stranded Oligonucleotide-binding
Assays--
The indicated amounts of Rad51B were incubated with 2 µM single-stranded 49-mer oligonucleotide, 1 µM double-stranded 49-mer oligonucleotide, 1 µM half-cruciform, or 1 µM synthetic
Holliday junction in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP, 1 mM dithiothreitol, 100 µg/ml BSA, 1 mM
MgCl2, and 6% glycerol at 37 °C for 15 min. The samples
were analyzed by electrophoresis on a 2.75% Nusieve GTG agarose gel in
TBE buffer (90 mM Tris, 90 mM boric acid, and 2 mM EDTA (pH 8.3)). In the competitive DNA-binding assay,
the indicated amounts of Rad51B were incubated at 37 °C for 15 min in the presence of 33 nM of each DNA substrate, the
synthetic Holliday junction, the half-cruciform, and the dsDNA. The
products were analyzed by 10% polyacrylamide gel electrophoresis in
TBE buffer.
Assay for Homologous Pairing of ssDNA and dsDNA--
A
32P-labeled single-stranded 50-mer oligonucleotide (1 µM) was mixed with the indicated amounts of Rad51B or
RecA in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP, 1 mM dithiothreitol, 100 µg/ml BSA, 6% glycerol, and 1 mM MgCl2. The mixtures were incubated at
37 °C for 5 min, and the reaction was started by the addition of
pGsat4 form I DNA (20 µM). After an incubation at
37 °C for 15 min, the reaction was terminated by the addition of
0.5% SDS, and the proteins were removed from the DNA by
treatment with proteinase K (0.7 mg/ml) followed by an incubation at
37 °C for 15 min. The products of homologous pairing
(D-loops) were separated from the unreacted DNA substrates
by 0.8% agarose gel electrophoresis in 0.5 × TBE buffer.
ATPase Assay--
The ATPase activity of Rad51B was analyzed by
the release of 32Pi from
[ Purification of the Human Rad51B Protein--
To study the
function of Rad51B, we overexpressed Rad51B in the E. coli
recA The ATPase Activity of Rad51B--
The Rad51B protein contains the
Walker ATPase motifs (17). As shown in Fig.
2, the purified Rad51B protein hydrolyzed
ATP. The ATPase activity of Rad51B is enhanced in the presence of
ssDNA, whereas superhelical dsDNA slightly stimulated it (Fig. 2).
Therefore, Rad51B is a DNA-dependent ATPase like the
bacterial RecA and eukaryotic Rad51 proteins. It has been reported that
Rad51B directly interacts with Rad51C (21-28), and the
Rad51B·Rad51C complex hydrolyzes ATP. The ATPase activity of
the Rad51B·Rad51C complex is also significantly enhanced in the
presence of ssDNA and is moderately stimulated in the presence of dsDNA
(24). These characteristics of the Rad51B·Rad51C complex in the ATP
hydrolysis are similar to those of Rad51B alone.
The Homologous-pairing Activity of Rad51B--
We previously
reported that the other Rad51 paralogs, the Xrcc2·Rad51D and
Xrcc3·Rad51C complexes, catalyze homologous pairing, by which
homologous joint molecules between ssDNA and dsDNA are formed during
the HRR pathway (29, 30). A phylogenetic analysis has shown that Rad51B
is evolutionally close to the other Rad51 paralogs (17). Therefore, we
next examined whether Rad51B catalyzes the homologous pairing. To
examine the homologous pairing activity of Rad51B, we employed the
D-loop formation assay (Fig.
3A), which is a standard assay
to evaluate the homologous-pairing activity of the bacterial RecA,
RecT, and RecO proteins (31-33) and their eukaryotic homologs
(34-39). In contrast to the previous observations with Xrcc2-Rad51D
and Xrcc3-Rad51C, Rad51B did not catalyze homologous pairing by itself
(Fig. 3B). This result suggests that Rad51B may have a
different role from the other Rad51 paralogs in the HRR pathway.
The DNA-binding Activity of Rad51B--
We next examined the
DNA-binding ability of Rad51B. Rad51B Specifically Binds to the Synthetic Holliday
Junction--
Next, we tested the DNA binding of Rad51B to various DNA
structures, including a single-stranded oligonucleotide 49-mer (ssDNA 49-mer), a double-stranded oligonucleotide 49-mer (dsDNA 49-mer), a
replicational fork-like structure (half-cruciform), and a synthetic Holliday junction. The dsDNA, half-cruciform, and synthetic
Holliday-junction structures were made with 49-mer oligonucleotides.
The Holliday junction, a four-way junction of dsDNA molecules, is a key
intermediate in the HRR pathway (6). As shown in Fig.
5, Rad51B efficiently bound to the
synthetic Holliday junction (panel A), half-cruciform (panel B), and dsDNA (panel C) structures. Rad51B
also bound to the ssDNA 49-mer (Fig. 5D), but its ability
was weak, consistent with the experiment with the phage ssDNA (Fig.
4A).
Then, we next examined the DNA-structure specificity of Rad51B in DNA
binding. When the same amounts of the synthetic Holliday junction,
half-cruciform, and dsDNA structures were incubated together, Rad51B
only bound to the synthetic Holliday junction in the presence of these
three substrates (Fig. 6A). In
contrast, Rad51 bound to all three substrates without an obvious
preference (Fig. 6B). Therefore, we conclude that Rad51B has
specificity to the Holliday junction, and this specificity is not
conserved in Rad51.
As bacterial Holliday junction-binding proteins, the RuvA·RuvB
complex (RuvAB), which promotes branch migration of the Holliday junction, has been found (19, 40-42). Crystallographic and electron micrographic analyses have shown that RuvA forms a tetramer, whereas RuvB forms hexameric and heptameric ring structures (43-53). Both RuvA
and RuvB are ATP-binding proteins, and the branch migration promoted by
the RuvAB complex requires ATP (19, 40, 41, 54). In addition, the
E. coli RecG protein is a factor that creates a four-way
junction by reversing the stalled replication fork (55, 56). In the
present study we found that Rad51B specifically binds to the synthetic
Holliday junction, suggesting that Rad51B may have a role in processing
the Holliday-junction intermediate in HRR. However, we could not detect
branch migration activity with Rad51B (data not shown). Helicase
activity, which is required to promote efficient branch migration by
the RuvAB complex or RecG, also was not detected with Rad51B (data not
shown). Rad51B may require a subunit(s), like the bacterial RuvAB
complex, if it is involved in the branch migration process in the HRR pathway.
Thus far, a eukaryotic nuclease, Mus81, which resolves the Holliday
junction into two duplex DNAs, has been suggested to be a functional
homolog of the bacterial Holliday-junction resolvase, the RuvC protein
(57, 58). On the other hand, an ATP-dependent branch
migration activity, which co-fractionates with the Holliday junction
resolving activity, has been detected in mammalian cell-free extracts
(59, 60), suggesting that the Holliday junction migrase and resolvase
may form a complex in mammals, like the bacterial RuvA, RuvB, and RuvC
proteins (61). Rad51B, which can target the Holliday junction
structure, may be a subunit of the human Holliday junction-processing
complex. In humans, the TIP60 complex, which is composed of fourteen
subunits, was found to bind the Holliday junction structure and
exhibited helicase activity, but not branch-migration activity (62).
The TIP60 complex contains TIP49a (also known as RUVBL1, NMP238, or
Pontin52) and TIP49b, which have similarity to RuvB (63-67). Rad51B
may function in the Holliday junction processing with the TIP60
complex. Further studies are required to identify the factors that
process the Holliday junction during HRR in mammals.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-rays (14). Therefore, the
Rad51B gene product plays an essential role in the HRR
pathway in vertebrates. The human Rad51B protein is composed of 350 amino acid residues (15, 16) and has about 20% amino acid identity
with the human Rad51 protein, like other human proteins such as the
Xrcc2, Xrcc3, Rad51C, and Rad51D proteins (17). Therefore, these five
proteins, Xrcc2, Xrcc3, Rad51B, Rad51C, and Rad51D, which have
similarity with Rad51, have been classified as Rad51 paralogs.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-satellite DNA. The DNA substrates
used in the DNA-binding assays were identical to the synthetic Holliday
junction (I) designed by Iwasaki et al. (19). The synthetic
Holliday junction was composed of four 49-mer single-stranded
oligonucleotides, 1, 2, 3, and 4, with the sequences 5'-ATCGA TGTCT
CTAGA CAGCT GCTCA GGATT GATCT GTAAT GGCCT GGGA-3', 5'-GTCCC AGGCC ATTAC
AGATC AATCC TGAGC ATGTT TACCA AGCGC ATTG-3', 5'-TGATC ACTTG CTAGC GTCGC
AATCC TGAGC AGCTG TCTAG AGACA TCGA-3', and 5'-CCAAT GCGCT TGGTA AACAT GCTCA GGATT GCGAC GCTAG CAAGT GATC-3', respectively. The resulting junction contained a 12-bp homologous region at the center, and the
four 5' ends had one base overhangs. The half-cruciform, which was
produced by oligonucleotides 1 and 2, was composed of a 30-bp duplex
region, and two 18-bp and 19-bp heterologous single strands. The dsDNA
was produced by annealing oligonucleotide 2 with its complementary
oligonucleotide. All of the oligonucleotides were purified by HPLC, and
the DNA concentrations are expressed in moles of nucleotides.
-satellite sequence was introduced into the
E. coli DH5
strain, and the cells were cultured for
12-16 h at 37 °C. The cells were harvested, mildly disrupted with
0.5 mg/ml lysozyme and 0.1% sarkosyl, and centrifuged at 28,000 rpm
for one hour. The supernatant containing the pGsat4 plasmid DNA was
extracted with phenol/chloroform three times, and the pGsat4 DNA in the aqueous phase was precipitated by ethanol. The pellet was dissolved in
1 ml of TE buffer and was treated with 0.15 mg/ml RNaseA at 37 °C for 30 min. The pGsat4 DNA was purified by 5-20% sucrose gradient centrifugation at 24,000 rpm for 18 h. Phage circular ssDNAs (
X174 and M13mp19) were prepared as described previously (20). DNA concentrations are expressed in moles of nucleotides.
X174 ssDNA (40 µM) was mixed with the indicated amounts of Rad51B or
Rad51 in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM
ATP, 1 mM dithiothreitol, 100 µg/ml BSA, 1 mM
MgCl2, and 6% glycerol. The reaction mixtures were
incubated at 37 °C for 10 min and were analyzed by 0.8% agarose gel
electrophoresis in TAE buffer (40 mM Tris acetate, 1 mM EDTA). The bands were visualized by ethidium bromide staining.
X174 superhelical
dsDNA (10 µM) was mixed with the indicated amounts of
Rad51B or Rad51 in 10 µl of standard reaction buffer containing 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM ATP (or
dATP), 1 mM dithiothreitol, 100 µg/ml BSA, 1 mM MgCl2, and 6% glycerol. MgCl2
and ATP were omitted from the DNA-binding experiments testing for the
requirements of MgCl2 and ATP. In the experiments testing for the requirements of divalent cations in the DNA binding,
MgCl2 was substituted for the indicated amounts of
MnCl2 or ZnCl2 in the standard reaction buffer.
The reaction mixtures were incubated at 37 °C for 10 min, and the
samples were electrophoresed on a 0.8% agarose gel for 4 h at 3 V/cm in 0.5 × TBE buffer. The bands were visualized by
ethidium bromide staining.
-32P]ATP. The reaction mixtures contained 50 mM bis-Tris propane-HCl (pH 7.5), 1 mM
MgCl2, 10 µM ATP, 50 nCi
[
-32P]ATP, 60 mM NaCl, 5 mM
dithiothreitol, 6% glycerol, 100 µg/ml BSA, and 3 µM
Rad51B. After an incubation at 37 °C for 1 h, the reactions
were stopped by adding one-half volume of 0.5 M EDTA. The
samples were separated by thin layer chromatography on
polyethyleneimine-cellulose (Sigma) in a 0.5 M LiCl and 1.0 M formic acid solution and were quantified by a Fuji
BAS2500 image analyzer.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
JM109(DE3) strain as a fusion protein with an
N-terminal hexahistidine tag (His6 tag) containing a
cleavage site for thrombin protease. The His6-tagged Rad51B
protein was expressed by induction with isopropyl-1-thio-
-D-galactopyranoside (Fig.
1, lane 3) and was purified by
chromatography on a Ni2+-chelating column (ProBond,
Invitrogen) (Fig. 1, lane 4). The His6 tag was
uncoupled with thrombin protease (Amersham Biosciences) from the Rad51B
portion, which migrated slightly faster than the His6-tagged Rad51B protein upon SDS-PAGE (15-25%) (Fig.
1, lane 5). Then, Rad51B was further purified by
Heparin-Sepharose column chromatography (Amersham Biosciences) (Fig. 1,
lane 6) followed by Mono Q column chromatography (Amersham
Biosciences) (Fig. 1, lane 7).
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Fig. 1.
Purification of the human Rad51B
protein. Proteins (2 µg) from each purification step were
analyzed by 15-25% SDS-PAGE with Coomassie Brilliant Blue staining.
Lane 1 indicates the molecular mass markers. Lanes
2 and 3 indicate the whole cell lysates of the
E. coli JM109(DE3) cultures before and after induction
with isopropyl-1-thio- -D-galactopyranoside,
respectively. Lanes 4-7 indicate the samples
from the peak ProBond fraction, the fraction after the removal of the
hexahistidine tag, the peak Heparin-Sepharose fraction, and the peak
MonoQ fraction, respectively.
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Fig. 2.
The ATPase activity of Rad51B.
A, Rad51B (3 µM) was incubated for 1 h
with or without 75 µM of the M13mp19 circular ssDNA
(lane 3) or 50 µM of the X174 dsDNA
(lane 4) in the presence of [
-32P]ATP. The
samples were separated by thin layer chromatography and were visualized
with the BAS2500 image analyzer. Lanes 1 and 2 indicate the control experiments without protein and DNA, respectively.
B, graphic representation of the time course experiments for
the ATPase activities of Rad51B. The amounts of hydrolyzed
Pi at the indicated time points are presented. Open
circles and open triangles indicate the experiments
with ssDNA and dsDNA, respectively, and closed circles and
closed squares indicate the control experiments without
protein and DNA, respectively.
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Fig. 3.
The homologous-pairing activity of
Rad51B. A, schematic representation of the
D-loop formation assay. In the reaction, an oligonucleotide
50-mer, labeled by [ -32P]ATP and T4
polynucleotide kinase at the 5' end, invades the homologous region of
the superhelical dsDNA and forms joint molecules (D-loops).
B, Rad51B and RecA were preincubated with the
32P-labeled oligonucleotide 50-mer (1 µM) at
37 °C for 5 min, and the reactions were started by the addition of
the superhelical dsDNA (20 µM). The reactions were
incubated at 37 °C for 15 min and were terminated by the addition of
0.5% SDS. Then, the proteins were removed by a treatment with
proteinase K (0.7 mg/ml) at 37 °C for 15 min. The samples were
separated by 0.8% agarose gel electrophoresis in 0.5 × TBE
buffer and were analyzed with the BAS2500 image analyzer. Lane
1 indicates the negative control experiment without protein.
Lanes 2 and 3 indicate the experiments with
Rad51B, and lanes 4 and 5 indicate the
experiments with RecA. The concentrations of Rad51B and RecA were 5.2 µM (lanes 2 and 4) and 7.8 µM (lanes 3 and 5).
X174 phage circular ssDNA (5386 bases) and superhelical dsDNA, containing 30% nicked circular dsDNA,
were used for the ssDNA-binding and dsDNA-binding assays, respectively.
As shown in Fig. 4A, Rad51B bound to the phage ssDNA (lanes 2-5), but its binding
ability was extremely low as compared with that of Rad51 (lanes
7-9). In contrast, Rad51B clearly bound to both superhelical and
nicked circular dsDNAs (Fig. 4B, lanes 2-6). The
dsDNA binding by Rad51B was strongly dependent on the presence of both
ATP and Mg2+, in contrast to the Rad51-dsDNA binding, which
required neither ATP nor Mg2+ (Fig. 4C). Rad51B
bound to dsDNA in the presence of Mn2+ instead of
Mg2+; however, Zn2+ did not support its dsDNA
binding (Fig. 4D). Therefore, these results indicate that
DNA binding by Rad51B requires ATP together with either
Mg2+ or Mn2+.
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Fig. 4.
The DNA-binding activities of Rad51B.
A, circular ssDNA binding. X174 ssDNA (40 µM) was incubated with Rad51B or Rad51 at 37 °C for 15 min. The concentrations of Rad51B and Rad51 used in the ssDNA-binding
experiments were 0.5, 1, 2, 4, and 6 µM (lanes
2-6), and 0.5, 1, and 2 µM (lanes
7-9), respectively. The samples were separated by
0.8% agarose gel electrophoresis in TAE buffer and were visualized by
ethidium bromide staining. Lane 1 indicates the negative
control experiments without protein. B, superhelical and
nicked circular dsDNA binding.
X174 superhelical dsDNA containing
nicked circular dsDNA (10 µM) was incubated with Rad51B
or Rad51 at 37 °C for 15 min. The concentrations of Rad51B and Rad51
used in the dsDNA-binding experiments were 0.5, 1, 2, 4, and 6 µM (lanes 2-6), and 0.5, 1, and 2 µM (lanes 7-9), respectively. The
samples were separated by 0.8% agarose gel electrophoresis in 0.5 × TBE buffer and were visualized by ethidium bromide staining
(B-D). NC and SC indicate nicked
circular dsDNA and superhelical dsDNA, respectively. Lane 1 indicates the negative control experiment without protein.
C, ATP and Mg2+ requirements for dsDNA binding.
X174 dsDNA (10 µM) was incubated with Rad51B (4.4 µM) or Rad51 (4.4 µM) at 37 °C for 15 min in the presence of 1 mM MgCl2 and 1 mM ATP (lanes 4 and 7, respectively).
Lane 1 indicates the negative control experiment without
protein. Lanes 2-4 indicate the experiments with
Rad51B, and lanes 5-7 indicate those with Rad51.
Lanes 2 and 5 indicate the experiments without
Mg2+ and ATP, and lanes 3 and 6 indicate those without ATP. D, Mg2+,
Mn2+, and Zn2+ requirements for dsDNA binding.
X174 dsDNA (10 µM) was incubated with Rad51B (3.3 µM) at 37 °C for 15 min in the presence of 1 mM MgCl2 (lane 3), 10 mM
MgCl2 (lane 4), 1 mM
MnCl2 (lane 5), 10 mM
MnCl2 (lane 6), 1 mM
ZnCl2 (lane 7), or 10 mM
ZnCl2 (lane 8). Lane 1 indicates the
negative control experiment without protein. Lane 2 indicates a negative control experiment without divalent cation.
View larger version (37K):
[in a new window]
Fig. 5.
Structure-specific DNA binding of
Rad51B. The indicated amounts of Rad51B were incubated with 1 µM of the 32P-labeled synthetic Holliday
junction (A), half-cruciform (B), dsDNA 49-mer
(C), or 2 µM of ssDNA 49-mer (D).
Lane 1 indicates a negative control experiment without
protein. The samples were separated by 2.75% agarose gel
electrophoresis in TBE buffer and were analyzed by the BAS2500 image
analyzer.
View larger version (36K):
[in a new window]
Fig. 6.
Rad51B specifically binds to the synthetic
Holliday junction. Increasing amounts of Rad51B (A) or
Rad51 (B) were incubated with the DNA mixture containing the
three 32P-labeled DNA substrates, the synthetic Holliday
junction (33 nM), the half-cruciform (33 nM),
and dsDNA (33 nM). The samples were separated by
electrophoresis on a 10% non-denaturing polyacrylamide gel in TBE
buffer and were analyzed by the BAS2500 image analyzer. The
concentrations of Rad51B and Rad51 used in the DNA-binding experiments
were 0.44, 0.88, 1.3, 1.8, 2.2, 2.6, and 3.1 µM
(panel A, lanes 2-8), and 0.025, 0.05, 0.1, 0.25, 0.5, 1, and 2 µM (panel B,
lanes 2-8), respectively. Lane 1 indicates the negative control experiment without protein.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Hiroshi Iwasaki (Yokohama City University) for discussions. We also thank Takashi Kinebuchi, Wataru Kagawa, and Rima Enomoto (RIKEN Genomic Sciences Center) for technical assistance.
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FOOTNOTES |
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* This work was supported in part by the Bioarchitect Research Program (RIKEN), Core Research for Evolutional Science and Technology of Japan Science and Technology, and also by a grant-in-aid from the Ministry of Education, Sports, Culture, Science, and Technology, Japan.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 may be addressed. E-mail:
yokoyama@biochem.s.u-tokyo.ac.jp.
§§ To whom correspondence may be addressed. E-mail: tshibata@postman.riken.go.jp.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M210899200
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ABBREVIATIONS |
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The abbreviations used are: HRR, homologous recombinational repair; ss, single-stranded; ds, double-stranded; BSA, bovine serum albumin; HPLC, high pressure liquid chromatography; TAE, Tris-acetate/EDTA; TBE, Tris-borate/EDTA; SDS, sodium lauryl sulfate.
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