From the Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245-3207
Received for publication, November 3, 2000, and in revised form, December 12, 2000
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ABSTRACT |
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Human Rad51 (hRad51), a member of a conserved
family of general recombinases, is shown here to have an avid
capability to make DNA joints between homologous DNA molecules and
promote highly efficient DNA strand exchange of the paired molecules
over at least 5.4 kilobase pairs. Furthermore, maximal efficiency of
homologous DNA pairing and strand exchange is strongly dependent on the
heterotrimeric single-stranded DNA binding factor hRPA and
requires conditions that lessen interactions of the homologous duplex
with the hRad51-single-stranded DNA nucleoprotein filament. The
homologous DNA pairing and strand exchange system described should be
valuable for dissecting the action mechanism of hRad51 and for
deciphering its functional interactions with other recombination factors.
Genetic studies in various eukaryotic organisms have indicated
that homologous recombination processes are mediated by a group of
evolutionarily conserved genes known as the RAD52 epistasis group. As revealed in studies on meiotic recombination and mating type
switching in Saccharomyces cerevisiae, DNA double-strand breaks are formed and then processed exonucleolytically to yield long
single-stranded tails with a 3' extremity. Nucleation of various
RAD52 group proteins onto these
ssDNA1 tails renders them
recombinogenic, leading to the search for a homologous DNA target
(sister chromatid or homologous chromosome), formation of DNA joints
with the target, and an exchange of genetic information with it. The
repair by recombination of DNA double-strand breaks induced by ionizing
radiation and other DNA damaging agents very likely follows the same
mechanistic route, as it too is dependent on genes of the
RAD52 epistasis group (reviewed in Refs. 1 and 2).
Among members of the RAD52 group, the
RAD51-encoded product is of particular interest because of
its structural and functional similarities to the Escherichia
coli recombination protein RecA (2-5). RecA promotes the pairing
and strand exchange between homologous DNA molecules to form
heteroduplex DNA (4, 5), an enzymatic activity believed to be germane
for the central role of RecA in recombination and DNA repair processes.
Likewise, homologous DNA pairing and strand exchange activities have
been shown for S. cerevisiae Rad51 (yRad51) (6). Under
optimized conditions, the length of heteroduplex DNA joints formed by
yRad51 and RecA can extend over quite a few kilobase pairs (4, 5,
7).
In published studies, human Rad51 (hRad51) was found to have the
ability to make DNA joints but the maximal potential for forming only
about 1 kilobase pairs of heteroduplex DNA (8-11). Furthermore, while
yRad51 and RecA require their cognate single-strand DNA binding
factors, SSB and yRPA, for optimal recombinase activity, hRPA has been
suggested to stimulate the hRad51-mediated homologous pairing and
strand exchange reaction only when the hRad51 concentration is
suboptimal (9, 10).
Given the central role of hRad51 in recombination processes and the
fact that the activities of hRad51 are apparently subject to modulation
by tumor suppressor proteins such as BRCA2 (reviewed in Ref. 12),
establishing an efficient hRad51-mediated DNA strand exchange system
will be important for dissecting the functional interactions among
hRad51, other recombination factors, and tumor suppressors. In this
work, a variety of reaction parameters that could influence the
recombinase activity of hRad51 were explored. We demonstrate that under
certain conditions, hRad51 makes DNA joints avidly and promotes highly
efficient strand exchange over at least 5.4 kilobase pairs.
Importantly, under the new reaction conditions, the efficiency of the
hRad51-mediated DNA strand exchange reaction is strongly dependent on
hRPA over a wide range of Rad51 concentrations tested.
DNA Substrates--
Plasmids--
Plasmid pRh51.1 consists of human RAD51
K313 cDNA under the control of the T7 promoter in vector pET11
(Novagen). Plasmid pRh51.1 was then subject to in vitro
mutagenesis using the QuikChange kit (Stratagene), to change
Lys313 (AAA codon) to a glutamine
residue (CAA codon). The resulting plasmid, pRh51.2, was sequenced to
ensure that no other undesired change has occurred in the
RAD51 sequence. Plasmid p11d-tRPA (13), which coexpresses
all three subunits of hRPA, was used for purification of this factor.
Cell Growth--
Plasmids pRh51.1 and pRh51.2 were introduced
into the RecA-deficient E. coli strain BLR (DE3) with pLysS.
Following transformation, single clones were picked and grown for
15 h in 30 ml of Luria broth. The starter culture was diluted 200 times with fresh Luria broth and incubated at 37 °C. When the
A600 of the cultures reached 0.6 to 1, isopropyl-1-thio- Protein Purification--
All the following steps were carried
out at 4 °C. For the purification of hRad51 Lys313 and
hRad51 Gln313 proteins, E. coli cell paste,
30 g from 20 liters of culture, was suspended in 150 ml cell
breakage buffer (50 mM Tris-HCl, pH 7.5, 5 mM
EDTA, 200 mM KCl, 2 mM dithiothreitol, 10%
sucrose, and the following protease inhibitors: aprotinin, chymostatin, leupeptin, and pepstation A at 3 µg/ml each, and 1 mM
phenylmethylsulfonyl fluoride) and then passed through a French press
once at 20,000 p.s.i. The crude lysate was clarified by centrifugation
(100,000 × g, 120 min), and the supernatant (Fraction
I) was treated with ammonium sulfate at 0.23 g/ml to precipitate hRad51
and about 20% of the total extract protein. The ammonium sulfate
pellet was dissolved in 300 ml of T buffer (30 mM Tris-HCl,
pH 7.4, 10% glycerol, 0.5 mM EDTA, 0.5 mM
dithiothreitol) with the set of protease inhibitors used in extract
preparation, and then clarified by centrifugation (10,000 × g for 30 min). The cleared protein solution (Fraction II)
was then applied onto a column of Q-Sepharose (2.6 × 6 cm; total
30-ml matrix) equilibrated in T buffer with 100 mM KCl and
eluted with a 400 ml of gradient of 100 to 600 mM KCl in T
buffer. The peak of hRad51 (Fraction III), eluting at about 330 mM KCl (60 ml), was dialyzed against T buffer with 100 mM KCl and then fractionated in a column of Affi-Gel Blue (Bio-Rad; 1.6 × 5 cm; total 10-ml matrix) with a 100-ml gradient of 100 to 2000 mM KCl in T buffer. The hRad51 protein
eluted from Affi-Gel Blue at 800 to 1200 mM KCl, and the
pool of which (Fraction IV; 20 ml) was dialyzed against T buffer with
100 mM KCl and fractionated in a column of Macro
hydroxyapatite (Bio-Rad; 1 × 7.5 cm; total 6-ml matrix) with a
100-ml 30 to 320 mM KH2PO4 gradient
in buffer T. hRad51 eluted from 150 to 220 mM
KH2PO4, and the peak fractions were pooled
(Fraction V; 15 ml containing 7.5 mg of hRad51), dialyzed against T
buffer with 50 mM KCl, and applied onto a Mono S column (HR5/5), which was developed with a 30-ml 100 to 400 mM KCl
gradient in buffer T. The Mono S fractions containing the peak of
hRad51, eluting at about 250 mM KCl, were pooled (Fraction
VI; 4 ml containing 6 mg of hRad51), diluted with an equal volume of
10% glycerol and then fractionated in Mono Q (HR 5/5) with a 30-ml 100 to 600 mM KCl gradient. The final pool of hRad51 (Fraction
VII; 3 ml containing 5 mg of hRad51 in ~350 mM KCl) was
concentrated in Centricon-30 microconcentrators and stored at
For the purification of hRPA, extract was made from E. coli
BL21 (DE3) harboring the plasmid p11d-tRPA (13) and subjected to the
purification procedure we have used for yRPA (14). The concentration of
hRPA was determined by comparison of multiple loadings of hRPA against
known amounts of bovine serum albumin and ovalbumin in a Coomassie Blue
R-stained polyacrylamide gel.
DNA Strand Exchange System That Uses DNA Strand Exchange System That Employs
Oligonucleotides--
The reaction mixture had a final volume of 12.5 µl and the steps were carried out at 37 °C. hRad51 (7.5 µM) was incubated with oligonucleotide 2 (30 µM nucleotides) in 10 µl of buffer R. The reaction
mixture was completed by adding ammonium sulfate in 1 µl, 1 µl of
50 mM spermidine, and the radiolabeled duplex (30 µM nucleotides) in 0.5 µl. At all the times indicated,
a 3-µl portion of the reaction mixture was deproteinized as described above and then subjected to electrophoresis in 10% polyacrylamide gels
run in TAE buffer. The level of DNA strand exchange was determined by
PhosphorImager analysis of the dried gels.
Examination of Interaction between hRad51-ssDNA Filament and
Duplex DNA--
Oligonucleotides F1 and F1b (Midland) both have the
sequence
5'-TGGCTTGAACGCGTCATGGAAGCGATAAAACTCTGCAGGTTGGATACGCCAATCATTTTTATCGAAGCGCGCCGCCC-3', except that the latter also contains a biotin molecule positioned at the 5' terminus. In these oligonucleotides, nucleotide residues 11 to 72 are complementary to positions 5348 to 23 of the Recombination Factors--
The cDNA for hRad51 was amplified
from a human B-cell cDNA library. Sequencing of the
hRAD51 cDNA insert revealed that it was identical to one
of the published hRAD51 sequences (15) but differed from the
other sequence (16) at amino acid residue 313; the former has a lysine
(an AAA codon) while the latter has a glutamine (a CAA codon) at this
position. The cloned cDNA was subjected to targeted mutagenesis to
change lysine 313 to glutamine. Both hRad51 Lys313 and
hRad51 Gln313 variants were expressed in E. coli
and purified to near homogeneity (Fig.
1A). The two hRad51 isoforms
behaved identically during purification and gave indistinguishable
results in all the enzymatic assays described here. Only the results
with hRad51 Lys313 are shown. We presume that the two
hRad51 isoforms correspond to naturally occurring polymorphic variants.
For DNA strand exchange experiments, the human ssDNA binding factor
replication protein A (hRPA), a heterotrimer of 70-, 32-, and 14-kDa
subunits, was also purified to near homogeneity (Fig. 1B)
from E. coli cells harboring a plasmid which coexpresses all
three subunits of this factor (13).
System for ATP-dependent Homologous DNA Pairing and
Strand Exchange--
For characterizing the homologous DNA pairing and
strand exchange activity of hRad51, we used as substrates
We tested the effects of increasing concentrations of potassium
acetate, potassium chloride, potassium phosphate, potassium sulfate,
ammonium chloride, and ammonium sulfate, and found that while all of
these salts were stimulatory, ammonium sulfate produced the most
stimulation, followed by potassium sulfate. Panel I in Fig.
2B shows a time course experiment in which 7.5 µM hRad51 was used with 2 µM hRPA,
In published studies (9, 10), 80 mM potassium acetate was
employed. In agreement with the published work (9, 10), neither higher
(up to 200 mM in 20 mM increments) nor lower
concentrations of potassium acetate would improve homologous DNA
pairing and strand exchange efficiency beyond the level seen in
panel II of Fig. 2B. As expected from published
work (8), omission of ATP from the reaction abolished strand exchange,
either under our reaction conditions (Fig. 2B, panel I, lanes
10-12) or the published conditions (Fig. 2B, panel II,
lanes 11-13) (9, 10).
Thus, the inclusion of ammonium sulfate renders hRad51-mediated
ATP-dependent homologous DNA pairing and strand exchange
highly efficient. Under the new reaction conditions, the optimal levels of hRad51 for pairing and strand exchange were found to be between 2 and 4 nucleotides/protein monomer. Increasing hRad51 above 2 nucleotides/protein monomer resulted in gradual inhibition (data not
shown), which was likely due to binding of hRad51 to the duplex and its
sequestration from pairing with the hRad51-ssDNA complex (7, 9).
Dependence on hRPA--
In the yRad51-mediated DNA strand exchange
reaction that uses plasmid length DNA substrates, a strong dependence
of the reaction efficiency on yRPA has been observed (2, 4). However,
in the published work, when hRad51 was used at the optimal ratio of 3 nucleotides of ssDNA/hRad51 monomer, hRPA has no stimulatory effect on
the reaction efficiency, and relatively high levels of hRPA were in
fact strongly inhibitory (9, 10). We have examined whether under the
newly devised reaction conditions, hRPA is required for strand exchange
efficiency. Fig. 3 summarizes the results
obtained with 7.5 µM hRad51, 30 µM
nucleotides of ssDNA, 100 mM ammonium sulfate, and
increasing concentrations of hRPA, from 0.4 to 4.0 µM.
Whereas only negligible pairing and strand exchange was seen in the
absence of hRPA, increasing concentrations of hRPA gave progressively
higher levels of products (Fig. 3, panels I and II).
The optimal level of hRPA was ~2 µM, although addition
of as little as 0.4 µM hRPA gave highly notable
stimulation. Importantly, increasing the hRPA concentration to 4 µM did not lower the level of products, which is very
different from published studies (10) in which concentrations of hRPA
Additional experiments revealed that at levels of hRad51 higher (2 nucleotides/hRad51 monomer) or lower (6 nucleotides/hRad51 monomer)
than that (4 nucleotides/hRad51 monomer) used in Fig. 3, there is also
a similar dependence of homologous pairing and strand exchange on hRPA.
Likewise, at ammonium sulfate levels higher and lower than that used in
prior experiments, we have also observed a similar dependence of the
strand exchange reaction on hRPA. Control experiments confirmed that
hRPA by itself does not have homologous pairing and strand exchange
activity under the new conditions (data not shown). In summary, under
our reaction conditions, there is a uniform dependence of DNA strand
exchange on hRPA, regardless of the amount of hRad51 used.
Effect of Order of Addition of Salt and Heterologous DNA--
In
the experiments described thus far, ammonium sulfate was added to the
reaction mixture after hRad51 had already nucleated onto the ssDNA but
before the incorporation of hRPA. We have also examined whether the
addition of ammonium sulfate at other stages would affect the reaction
efficiency, as such an endeavor could yield important clues as to the
basis of stimulation. As shown in Fig. 4,
similar levels of homologous DNA pairing and strand exchange were
observed when ammonium sulfate was added at the beginning with hRad51,
after hRad51 but before the incorporation of hRPA (as in the standard
reaction), and after hRPA but before the incorporation of the duplex.
Interestingly, when ammonium sulfate was incorporated a few minutes
after the duplex, there was little product formed even at the reaction
end point of 60 min (Fig. 4A, lanes 11-13). Since dsDNA
coated with hRad51 or yRad51 has been found to be inactive in the DNA
strand exchange reaction (7, 9), we considered the possibility that
perhaps the suppression of DNA strand exchange seen with ammonium
sulfate being added after the duplex might have stemmed from free
hRad51 binding to the duplex (7). However, two lines of evidence
strongly suggest that this was not the main reason for the lack of
strand exchange stimulation. First, even at levels of hRad51 (6 nucleotides and 9 nucleotides of ssDNA/hRad51 monomer) lower than that
(4 nucleotides ssDNA/hRad51 monomer) used in Fig. 4 and with much longer preincubation of hRad51 with ssDNA to minimize the level of free
hRad51, addition of ammonium sulfate before the duplex molecule is
still necessary to see significant strand exchange (data not shown).
Second, an excess of a heterologous duplex (pBluescript) added together
with the homologous duplex or before the homologous duplex, in an
attempt to titrate out any free hRad51, also did not compensate for the
stimulatory effect of adding ammonium sulfate before the homologous
duplex (see below). Taken together, the results strongly suggested that
the lack of DNA strand exchange when ammonium sulfate was incorporated
after the homologous duplex was due to a reason other than free hRad51
coating the duplex molecule.
Interestingly, whereas the incorporation of increasing amounts of the
heterologous pBluescript duplex in the presence of ammonium sulfate
lowered the reaction efficiency only slightly (Fig.
5, A, panel I, and B,
panel I), the addition of pBluescript duplex before ammonium
sulfate resulted in much more pronounced inhibition (Fig. 5, A,
panel II, and B, panel II). These results, coupled with
those presented above, indicated that binding of duplex to the
hRad51-ssDNA nucleoprotein filament, regardless of whether the duplex
is homologous to the ssDNA situated in the hRad51 filament, has a
strong suppressive effect on pairing and strand exchange, unless salt
is already present.
In summary, the results have revealed a strict dependence of homologous
DNA pairing and strand exchange efficiency on ammonium sulfate being
incorporated into the reaction prior to the duplex, and they suggest
that ammonium sulfate exerts its stimulatory effect via modulation of
the interactions between the hRad51-ssDNA nucleoprotein complex and the
incoming duplex molecule. This premise is further tested and verified
in the experiments below.
Dependence of Strand Exchange Efficiency on Interactions between
Duplex and hRad51-ssDNA Complex--
Extensive biochemical studies
conducted with RecA have revealed that the incoming duplex molecule is
bound only transiently within the RecA-ssDNA nucleoprotein filament (4, 5, see "Discussion"). We reasoned that if the hRad51-ssDNA filament
has a relatively high affinity for the duplex, then the DNA homology
search process might occur efficiently only when the association of the
duplex molecule and the hRad51-ssDNA nucleoprotein filament is rendered transient, which could conceivably be realized by salt inclusion.
Intrinsic to this hypothesis are two predictions. First, it might be
expected that the salt dependence of the homologous DNA pairing and
strand exchange process would be lessened with reduction in the length
of the DNA substrates, such as when oligonucleotides are used (11) (see
Fig. 6A for schematic). This
is because the extent of interactions of a short duplex with the
limited length of hRad51-ssDNA nucleoprotein filament assembled on a
short single-strand would not be extensive. Furthermore, the search for
DNA homology with short DNA substrates would not be as rate-limiting as
when
Second, if salt indeed acted to weaken the interaction between the
duplex and the hRad51-ssDNA nucleoprotein complex, then we would expect
to see lessened binding of the duplex molecule to the hRad51-ssDNA
complex when ammonium sulfate was present. To test this premise
experimentally, we examined the interaction of
Once the utility of the assay system was verified, we proceeded to test
the effect of ammonium sulfate on the interactions of duplex DNA with
the immobilized hRad51-ssDNA complex. The results revealed gradual
weakening of the duplex/hRad51-ssDNA complex interactions by increasing
levels of ammonium sulfate. Specifically, whereas greater than 90%
retention of the duplex occurred in the absence of ammonium sulfate,
less than 10% of the duplex was bound at 100 mM of the
salt (Fig. 7, B, upper panel, and C). Analysis of
the amount of hRad51 in the various SDS eluates showed that even the
highest concentration of ammonium sulfate did not cause significant
turnover of hRad51 from the bound ssDNA (Fig. 7B, lower
panel). Taken together, we concluded that ammonium sulfate indeed
weakens the binding of duplex DNA to the hRad51-ssDNA complex. Other
experiments revealed that pBluescript duplex also binds to the
immobilized hRad51-
Interestingly, potassium acetate lessened the interaction between the
duplex and the immobilized hRad51-ssDNA complex only slightly (Fig.
7C), and as expected, the hRad51-ssDNA complex was stable to
potassium acetate (data not shown). Since potassium acetate is much
less effective in the homologous pairing and strand exchange reaction
(Fig. 2) (9, 10), the observation in Fig. 7C is again
consistent with the suggestion that ammonium sulfate stimulates
homologous pairing and strand exchange by attenuating the affinity of
the hRad51-ssDNA nucleoprotein filament for the incoming duplex.
In the DNA strand exchange experiments, we found that the order of
addition of ammonium sulfate relative to duplex DNA was important for
ensuring strand exchange efficiency, such that if duplex DNA was added
before ammonium sulfate, only negligible pairing and strand exchange
was observed (see Fig. 4). Given this observation, we wanted to test
whether the level of duplex retention by bead-immobilized hRad51-ssDNA
complex would change with the order of addition of ammonium sulfate. To
examine this, we used three different concentrations of ammonium
sulfate (25, 50, and 100 mM) and added the salt either
before the incorporation of duplex or after the duplex had already been
preincubated with the bead-immobilized hRad51-ssDNA complex. The
results from this experiment revealed that more of the duplex becomes
associated with the hRad51-ssDNA complex with preincubation of duplex
and Rad51-ssDNA complex prior to salt addition (Fig.
7D).
The experiments in Fig. 7 were conducted with hRad51-ssDNA
nucleoprotein complex assembled in the absence of hRPA. We have obtained similar results when hRPA was included in the binding reaction
(data not shown).
Homologous DNA Pairing and Strand Exchange by hRad51 and
hRPA--
Both hRad51 and yRad51 are related in amino acid sequence
and biological function to E. coli RecA. Like RecA, yRad51
forms nucleoprotein filaments on ssDNA and dsDNA in an
ATP-dependent manner. Biochemical studies have indicated
that the search for DNA homology in the incoming duplex DNA molecule
and formation of heteroduplex joints with the duplex occur within the
confines of the RecA-ssDNA and yRad51-ssDNA nucleoprotein filaments,
which are also referred to presynaptic filaments. The assembly of the recombinase-ssDNA nucleoprotein filaments and the efficiency of subsequent homologous pairing and strand exchange are stimulated by the
single-strand binding factor, SSB for RecA and yRPA for yRad51 (2, 4,
5). In the presence of ATP, hRad51 also forms a filament on ssDNA
similar in structure to the equivalent nucleoprotein filaments
assembled with RecA and yRad51 (3-5). However, published studies have
suggested that hRad51 has only a modest ability to make DNA joints and
an even lower capacity to promote DNA strand exchange. These published
observations have suggested that either hRad51 participates in making
DNA joints without catalyzing much DNA strand exchange, as discussed
before (3), or that other reaction conditions are in fact required to
reveal the strand exchange activity in hRad51. To entertain the latter
possibility, we have explored a variety of reaction parameters for
their effect on the hRad51 recombinase activity, and have shown that
when ammonium sulfate is included, an avid capability of hRad51 to
catalyze DNA joint formation and strand exchange is revealed. Moreover,
formation of nicked circular duplex, the product of full DNA strand
exchange, becomes completely dependent on hRPA. The dependence of
homologous DNA pairing and strand exchange on hRPA is seen over a wide
range of hRad51 concentrations, from below and above the optimal level.
The requirement for hRPA in homologous DNA pairing and strand exchange
is very likely due to its ability to minimize secondary structure in
DNA, thus facilitating the assembly of a contiguous hRad51-ssDNA
filament, as suggested in previous studies (2-5).
In summary, the results presented here demonstrate an intrinsic ability
of hRad51 to form DNA joints efficiently and to catalyze a substantial
amount of DNA strand exchange. These findings also reveal the
functional dependence of hRad51 recombinase on the ssDNA binding factor
hRPA. The ability of hRad51 and hRPA to promote DNA joint formation and
extension of nascent heteroduplex joints by strand exchange is likely
to be indispensable for various recombination reactions in
vivo.
Possible Basis for Salt Stimulation of Homologous DNA Pairing and
Strand Exchange--
Extensive biochemical studies have revealed the
presence of two distinct DNA-binding sites in the RecA protein
filament, with the initiating ssDNA substrate viewed as being situated
within the "primary" site, while the incoming duplex molecule is
bound within the "secondary" site of the presynaptic filament (4, 5). The search for DNA homology in the duplex DNA occurs by way of
reiterative binding and release of the duplex until homology is
located. For this random collision mode of DNA homology search to work
efficiently, the incoming duplex molecule must be retained only
transiently within the secondary site of the recombinase-ssDNA filament. Consistent with this deduction, evidence has been presented to suggest that the RecA-ssDNA filament has modest affinity for duplex
DNA (4).
Given the structural and functional similarities between hRad51 and
RecA, it seems reasonable to suggest that DNA homology conducted by the
hRad51-ssDNA presynaptic filament also occurs by means of random
association/dissociation of the duplex molecule with the former, with
efficient homology search to be dictated by transient, rather than
stable, association of the incoming duplex with the presynaptic
filament. We have presented two lines of evidence to support the notion
that salt exerts its remarkable stimulatory effect by weakening the
interactions of the duplex molecule with the hRad51-ssDNA filament,
thereby enhancing the rate of turnover and the efficiency at which the
duplex can be sampled for homology. First, we have shown that the
dependence of the homologous DNA pairing and strand exchange process on
salt is alleviated if the length of the DNA substrates is reduced, as
when oligonucleotides are used. This is likely due to the increased probability for productive collisions between the two oligonucleotide substrates that lead to homologous registry and also because the interactions between the duplex and the short presynaptic filament of
hRad51 assembled on the ss oligonucleotide are similarly minimized. Furthermore, we have provided direct evidence that binding of a duplex
molecule to hRad51 presynaptic filament is weakened by the inclusion of
ammonium sulfate.
In summary, we surmise from the biochemical results that the
hRad51-ssDNA filament binds duplex DNA in the available
secondary site with high affinity, thus limiting efficient
sampling of the duplex molecule for DNA homology. Accordingly,
stimulation of homologous DNA pairing is realized by salt addition,
with the degree of stimulation being dependent on the effectiveness of a particular salt to weaken the affinity of the hRad51-ssDNA
nucleoprotein filament for the duplex molecule. We further suggest that
the stimulation of strand exchange efficiency by ammonium sulfate is
also due to increased turnover of the duplex DNA from the secondary DNA-binding site, as the ease with which the initial DNA joint can be
extended by branch migration may be expected to be critically dependent
on the hRad51-ssDNA nucleoprotein filament being free of stably bound
duplex DNA molecules as well. These suggestions are summarized in our
working model in Fig. 8.
It is reasonable to ask whether the requirement for a high level of
salt for revealing the catalytic potential of the hRad51 recombinase is
physiological. Concerning this point, it is important to note that the
in vivo salt concentration is between 0.17 and 0.24 M (17). The specific stimulation of hRad51 strand exchange activity by salts could also be reflecting the requirement for a small
molecule (a polyanion, for instance), a certain post-translational modification of hRad51 (phosphorylation, for instance), or the involvement of other recombination factors (see discussion below) in
modulating the affinity of the hRad51-ssDNA filament for the incoming
duplex DNA molecule.
Significance of the in Vitro DNA Strand Exchange
System--
Studies on yRad51 using plasmid length DNA molecules as
substrates have been instrumental for formulating biochemical models for understanding the functions of various RAD52
group proteins. For instance, in addition to the well documented
stimulatory role of yRPA in yRad51-mediated DNA strand exchange,
experiments which varied the order of addition of reaction components
have revealed that yRPA, if added with or before yRad51 to the ssDNA
substrate, can also compete with yRad51 for binding sites on the ssDNA
and consequently suppress the assembly of yRad51-ssDNA nucleoprotein filament (2). The yeast RAD52 encoded product and the
heterodimeric molecule of yRad55 and yRad57 proteins, referred to as
recombination mediators, promote the assembly of the yRad51-ssDNA
filament and help overcome the suppression of DNA strand exchange
caused by coaddition of yRPA with yRad51 to the ssDNA substrate or by
preincubation of ssDNA with yRPA (2, 18).
We have verified that preincubation of ssDNA with hRad51 before the
incorporation of hRPA is in fact critical for homologous pairing and
strand exchange efficiency,2
providing evidence that hRPA competes with hRad51 for binding sites on
ssDNA. This observation suggests the existence of specific mediators in
the human recombination machinery for promoting hRad51-ssDNA filament
assembly when there is the need for hRad51 to compete with other
single-strand binding factors for sites on the initiating ssDNA
substrate. A possible mediator function may exist in various human
recombination factors including hRad52 and a number of Rad55/Rad57-like proteins, namely XRCC2, XRCC3, Rad51B, Rad51C, and Rad51D, which are
all known to be involved in recombination and either directly, or
through another recombination factor, physically interact with hRad51
(3, 19). Furthermore, it remains a distinct possibility that some of
these other recombination factors are in fact integral components of
the presynaptic filament, and as such, may modulate the dynamics of the
presynaptic filament to facilitate sampling of duplex DNA for homology
and to promote the formation of DNA joints once homology is located.
The in vitro DNA strand exchange system with the defined
biochemical parameters described herein should be well suited for
examining the function of various recombination factors and the role of
post-translational modifications of these recombination factors in the
DNA strand exchange reaction.
Recently, hRad51 was shown to interact with the breast tumor suppressor
BRCA2 (12). In the Capan-1 cell line defective in BRCA2 function, the
DNA damage-induced formation of hRad51 nuclear foci is defective,
suggesting the possibility that BRCA2 helps deliver hRad51 to the DNA
substrate (12). Whether or not BRCA2 functions as a mediator to promote
hRad51 nucleoprotein filament assembly can be tested with the in
vitro DNA strand exchange system described herein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
X174 viral (+)-strand was purchased from
New England Biolabs and
X174 replicative form I DNA was from Life
Technologies, Inc. The replicative form I DNA was linearized with
ApaLI. The pBluescript DNA was prepared from E. coli XL-1 Blue (Stratagene), purified by banding in cesium
chloride gradients, and linearized with BsaI. The
oligonucleotides (83-mer) used in strand exchange were: oligo 1, 5'-AAATGAACATAAGATAAATAAGTATAAGGATAATACAAAATAAGTAAATGAATAAACATAGAAAATAAAGTAAAGGATATAAA; oligo 2, the exact complement of oligo 1. Oligo 2 was labeled at
the 5' end with [
-32P]ATP and T4 polynucleotide kinase
and then annealed to oligo 1. The labeled duplex was purified from 10%
polyacrylamide gels by overnight diffusion at 4 °C into TAE buffer
(40 mM Tris acetate, pH 7.4, 0.5 mM EDTA). DNA
substrates were stored in TE (10 mM Tris-HCl, pH 7.5, with
0.5 mM EDTA).
-D-galactopyranoside was added to 0.4 mM and the induction of hRad51 continued at 37 °C for
4 h. Cells were harvested by centrifugation and stored frozen at
70 °C. Plasmid p11d-tRPA was introduced into E. coli
strain BL21 (DE3) and the induction of hRPA was carried out as
described previously (13).
70 °C. The hRad51 concentration was determined using the
calculated molar extinction coefficient of 12,800 M
1 cm
1 at 280 nM
(10).
X174 DNA--
All the
reaction steps were carried out at 37 °C. In Fig. 2, the reaction
(50 µl final volume) was assembled by mixing hRad51 (7.5 µM) added in 2 µl of storage buffer and
X174 viral
(+)-strand (30 µM nucleotides) added in 2 µl in 40 µl
of buffer R (40 mM Tris-HCl, pH 7.8, 2 mM ATP,
1 mM MgCl2, 1 mM dithiothreitol,
and an ATP regenerating system consisting of 8 mM creatine
phosphate and 28 µg/ml creatine kinase). After a 5-min incubation,
hRPA (2 µM) in 2 µl of storage buffer was added,
followed by a 5-min incubation, and then 5 µl of ammonium sulfate (1 M stock, final concentration of 100 mM),
followed by another 1-min incubation. To complete the reaction, linear
X174 replicative form I DNA (30 µM nucleotides) in 3 µl of TE and 4 µl of 50 mM spermidine (4 mM) were incorporated. At the indicated times, 4.5-µl
portions were withdrawn, mixed with 7 µl of 0.8% SDS and 800 µg/ml
proteinase K, incubated for 15 min before electrophoresis in 0.9%
agarose gels in TAE buffer. The gels were stained in ethidium bromide (2 µg/ml in H2O) for 1 h, destained for 12 to
18 h in a large volume of water, and then subjected to image
analysis in a NucleoTech gel documentation station equipped with a CCD
camera using Gel Expert for quantification of the data. Unless stated
otherwise, the reaction mixtures in other experiments were assembled in
the same manner with the indicated amounts and order of addition of reaction components, except that they were scaled down two and one-half times.
X (+)-strand DNA. These oligonucleotides were hybridized to
X (+)-strand by incubating a 3 M excess of the oligonucleotide with the
latter in buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 100 mM NaCl, and 1 mM dithiothreitol. The F1-
X (+)-strand and F1b-
X (+)-strand hybrids (30 µM nucleotides) were mixed with 10 µl of magnetic beads containing streptavidin (Roche Molecular
Biochemicals) in binding buffer containing 10 mM Tris-HCl,
pH 7.5, 100 mM KCl, and 1 mM EDTA for 10 min at
37 °C. About 70% of the F1b-(+)-strand hybrid was immobilized on
the beads, whereas, as expected, less than 5% of the F1-(+)-strand
hybrid was retained. To assemble hRad51 filament on the immobilized
X (+)-strand, magnetic beads preloaded with the F1b-
X (+)-strand
hybrid were incubated with 4 µM hRad51 in 20 µl of
buffer R. Reproducibly, ~85% of the hRad51 was bound to the magnetic
beads under the stated conditions, as determined by eluting the bound
hRad51 with 2% SDS followed by SDS-polyacrylamide gel electrophoresis
and staining with Coomassie Blue; this procedure gave an immobilized
hRad51-ssDNA nucleoprotein complex of ~3 nucleotides/hRad51 monomer.
The magnetic beads containing hRad51-ssDNA complex was then washed once
each with 20 µl of buffer R with 0.01% Nonidet P-40 and 20 µl
buffer R, before being incubated with linear
X duplex (8 µM nucleotides) for 3 min at 37 °C in 20 µl of
buffer R containing 4 mM spermidine and the indicated concentrations of ammonium sulfate. The beads were treated with 20 µl
of 2% SDS at 37 °C for 5 min to elute bound duplex and hRad51. The
supernatants and SDS eluates were analyzed in agarose gels followed by
staining with ethidium bromide to quantify DNA and in polyacrylamide
gels with Coomassie Blue staining to determine the amount of hRad51. As
controls, magnetic beads alone, magnetic beads preincubated with the
F1-
X (+)-strand hybrid, and magnetic beads preincubated with F1b but
without
X (+)-strand were similarly incubated with the linear
X
duplex and then processed for analyses.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Recombination factors. A,
purified hRad51 Lys313 (lane
2) and hRad51 Gln313 (lane 3), 3 µg each,
were analyzed in an 11% SDS-polyacrylamide gel and stained with
Coomassie Blue. B, purified hRPA, 3 µg in lane
2, was analyzed in a 12.5% SDS-polyacrylamide gel and stained
with Coomassie Blue. The three subunits of hRPA are denoted by the
arrows.
X 174 viral (+)-strand and linear duplex that are 5.4 kilobase pairs in
length (Ref. 6; schematic shown in Fig.
2A). In this system, hRad51 is
preincubated with the (+)-strand, followed by the addition of hRPA, and
the linear duplex is incorporated last. Pairing between the DNA
substrates yields a joint molecule, and branch migration, if
successful, over 5.4 kilobase pairs produces nicked circular duplex as
product (Fig. 2A). We have examined a variety of reaction
conditions including the levels of magnesium, pH, and various types of
salt on the hRad51 recombinase activity. As documented below, the most
dramatic effects were seen with the addition of salts.
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Fig. 2.
Homologous DNA pairing and strand exchange by
hRad51 and hRPA. A, schematic of the reaction. The
X174 viral (+)-strand (ss) is paired with the linear
homologous duplex (ds) to form joint molecules
(jm), and strand exchange over the length of the DNA (5.4 kilobase pairs) yields nicked circular duplex (nc) and
linear ssDNA as products. B, hRad51-mediated homologous DNA
pairing and strand exchange. Panel I shows reactions carried
out with ammonium sulfate as salt and with ATP (lanes 1-9)
or without ATP (lanes 10-12). The reactant concentrations
were: 7.5 µM hRad51, 2 µM hRPA, 30 µM (in nucleotides) each of ssDNA and dsDNA. Panel
II shows reactions carried out as described by Baumann and West
(9, 10), with ATP (lanes 1-10) or without it (lanes
11-13). The reactant concentrations were: 5 µM
hRad51, 1 µM hRPA, and 30 µM (in
nucleotides) each of ssDNA and dsDNA. C, in panel
I, the time courses of conversion of the input linear duplex into
nicked circular duplex in the experiments in panels I (
)
and II (
) of B are graphed. In panel
II, the time courses of conversion of the input linear duplex into
total products (joint molecules and nicked circular duplex) in the
experiments in panels I (
) and II (
) of
B are graphed.
X
(+)-strand (30 µM nucleotides), and
X linear duplex
(30 µM nucleotides) in pH 7.8 buffer and 100 mM ammonium sulfate. Following the published conditions of
Baumann and West (9, 10), another reaction was also carried out in which hRad51, at 5 µM, was used with 1 µM
hRPA and the same concentrations of DNA substrates and 80 mM potassium acetate at pH 7.5 (Fig. 2B, panel
II). The results showed a much higher level of DNA strand exchange
under the new conditions. Specifically, whereas no full strand exchange
product (nicked circular duplex) was detected under the published
conditions (Fig. 2, B, panel II, and C, panel I)
(9, 10), the inclusion of ammonium sulfate resulted in conversion of
~30 and ~60% of the linear duplex to nicked circular duplex after
30 and 60 min, respectively (Fig. 2, B, panel I, and
C, panel I). Overall, there was a 3-4-fold increase in
total products (joint molecules plus nicked circular duplex) in the reaction that employed ammonium sulfate (Fig. 2C, panel II).
Even though 100 mM ammonium sulfate was found to be
optimal, highly significant levels of homologous DNA pairing and
complete DNA strand exchange were seen at reduced concentrations (50 and 75 mM) of the salt (data not shown).
0.8 µM were found to be strongly inhibitory.
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Fig. 3.
Dependence of homologous DNA pairing and
strand exchange on hRPA. hRad51 at 7.5 µM was
incubated with 30 µM X ssDNA with or without
increasing concentrations of hRPA, and the resulting hRad51-ssDNA
nucleoprotein filaments were reacted with linear
X dsDNA for 20 (
), 40 (
), and 60 (
) min. In panel I, the level of
full DNA strand exchange, as measured by the percent conversion of the
input linear duplex to nicked circular duplex, was graphed. In
panel II, the level of total products, joint molecules plus
nicked circular duplex, was graphed.
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Fig. 4.
Order of addition of salt is critical for
reaction efficiency. A, ammonium sulfate (100 mM) was added at the same time as hRad51 to X ssDNA
(1o), after
X ssDNA had been preincubated with
hRad51 (2o), after
X ssDNA had been preincubated first
with hRad51 and then with hRPA (3o), or added 3 min after
the duplex had already been incorporated into the reaction
(4o). The complete reaction mixtures were incubated for 20, 40, and 60 min and processed for gel analysis. In lane 1,
DNA substrates were incubated in the absence of recombination proteins.
The reactant concentrations were: hRad51 at 7.5 µM,
X
ssDNA at 30 µM nucleotides, hRPA at 2 µM,
and
X dsDNA at 30 µM nucleotides. B, the
levels of total products, joint molecules plus nicked circular duplex,
were graphed.
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Fig. 5.
Effect of heterologous duplex.
A, increasing concentrations of pBluescript (1 to 3 times
the concentration of X dsDNA) was added in the presence of ammonium
sulfate (panel I) or 3 min before the incorporation of
ammonium sulfate (panel II) to presynaptic complex assembled
with hRad51 (7.5 µM), hRPA (2 µM), and
X
ssDNA (30 µM nucleotides). Following the incorporation of
X duplex (30 µM nucleotides), the reaction mixtures
were incubated for the indicated times. The concentration of ammonium
sulfate was 100 mM. In lane 1 of both panels,
DNA substrates were incubated in the absence of recombination proteins.
B, the levels of total products, joint molecules plus nicked
circular duplex, in A were graphed. Panel I shows
the levels of products (joint molecules and nicked circular duplex)
when pBluescript was added after ammonium sulfate and panel
II shows the levels of products when pBluescript was added before
ammonium sulfate.
X DNA substrates are used, because the probability of
productive collisions between two short substrates leading to their
homologous registry should be considerably higher. As predicted, when
the DNA substrates used were based on 83-mer oligonucleotides, the rate
of homologous pairing between the substrates was the same in the
absence of salt as when ammonium sulfate was present at 25 mM, and higher levels of ammonium sulfate were in fact
inhibitory (Fig. 6, B and C).
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Fig. 6.
Homologous pairing with oligonucleotide-based
substrates is independent of salt. A, pairing and
strand exchange between the unlabeled single-stranded oligonucleotide
with the 32P-labeled duplex results in displacement of the
radiolabeled single strand. B, hRad51 was incubated with the
83-mer oligonucleotide and the resulting nucleoprotein filament was
reacted with the homologous duplex in the absence of salt or with
increasing concentrations of ammonium sulfate, as indicated. In
lane 1, DNA substrates were incubated in the absence of
hRad51. C, the results from PhosphorImager analysis of the
gel in B were plotted.
X linear dsDNA with
hRad51-
X ssDNA complex immobilized on streptavidin magnetic beads
via a short biotinylated oligonucleotide, called F1b, which is
complementary to a portion of the
X (+)-strand (see schematic in
panel I of Fig. 7A
and "Experimental Procedures"). Analysis of the SDS eluate of the
magnetic beads allowed us to determine the amount of duplex DNA that
had bound to the immobilized hRad51-ssDNA complex (Fig. 7A, panel
I). As shown in Fig. 7A, lanes 1 and 2 in
panel II, incubation of the duplex with bead-immobilized hRad51-ssDNA complex in the absence of ammonium sulfate resulted in
>90% retention of the duplex on the beads. Binding of the duplex to
the magnetic beads was due to its interaction with the immobilized hRad51-ssDNA complex, because little retention of the duplex DNA occurred with magnetic beads pretreated with hRad51 and
X (+)-strand hybridized to an oligonucleotide, F1, that had identical sequence to
F1b but lacked the biotin tag of the latter (Fig. 7A, lanes 3 and 4 in panel II). As expected, only the
background level of duplex retention was seen with beads containing the
F1b-
X (+)-strand hybrid but without hRad51, with DNA-free beads
preincubated with hRad51, and with beads that contained only the F1b
oligonucleotide and preincubated with hRad51 (Fig. 7A, lanes
5-10 in panel II).
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Fig. 7.
Salt lessens interactions between duplex and
hRad51-ssDNA nucleoprotein filament. A, panel
I, the assay scheme is summarized. Briefly, X (+)-strand
hybridized to the biotinylated oligonucleotide F1b is immobilized on
streptavidin magnetic beads via the biotin tag. hRad51 filament is
assembled on the (+)-strand and then mixed with
X duplex DNA. The
bound duplex and hRad51 are eluted by SDS and analyzed. To verify the
utility of the assay (panel II), beads containing
X
(+)-strand-F1b hybrid (
X-F1b; lanes 1 and
2), beads preincubated with
X (+)-strand hybridized to
the nonbiotinylated oligonucleotide F1 (
X-F1; lanes
3 and 4), beads with F1b but no
X (+)-strand
(F1b only; lanes 5 and 6), and beads
that contained neither
X (+)-strand nor F1b (beads only; lanes
7 and 8) were incubated with hRad51 and then mixed with
X duplex. As an additional control,
X duplex DNA was incubated
with beads containing the
X (+)-strand-F1b hybrid without hRad51
(
X/F1b, No hRad51; lanes 9 and 10). The
supernatants (S) and the SDS eluates (E) from the
reactions were analyzed in an agarose gel for their content of duplex
DNA. B, salt weakens interaction of duplex with the
hRad51-ssDNA filament. Duplex
X DNA was incubated with the hRad51
filament assembled on the immobilized
X (+)-strand with increasing
concentrations of ammonium sulfate. The supernatants (S) and
SDS eluates (E) from the binding reactions were analyzed for
their contents of DNA duplex (upper panel) and hRad51
(lower panel). C, the results in B are
graphed (
), as are results from binding reactions in which potassium
acetate was used (
). D, effect of order of addition of
ammonium sulfate. Duplex DNA was added to binding reactions 3 min
before or immediately after the incorporation of increasing levels of
ammonium sulfate (panel I). The results with adding ammonium
sulfate before (shaded bar) and after (dark bar)
the incorporation of duplex are presented in the histogram in
panel II.
X ssDNA complex in a manner that is reduced by
ammonium sulfate (data not shown), indicating that the hRad51-ssDNA
complex can interact with both homologous and heterologous duplex molecules.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 8.
Model for hRad51-mediated DNA strand
exchange. The results suggest that duplex DNA molecules stably
bound to the hRad51-ssDNA nucleoprotein filament present a strong
impediment to the different reaction steps, including DNA homology
search, DNA pairing, and branch migration of the nascent DNA joint,
that lead to successful recombination between the DNA substrates.
Efficient DNA pairing and strand exchange is realized by lessening the
duplex/hRad51-ssDNA interactions, achieved by the inclusion of
salt.
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ACKNOWLEDGEMENTS |
---|
We are very grateful to Wen-Hwa Lee and Phang-Lang Chen for kindly providing the hRAD51 K313 cDNA and Marc Wold for the gift of plasmid p11d-tRPA.
![]() |
FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grants RO1 ES07061, RO1GM57814, and PO1 CA81020, Army Research Grant DAMD 17-98-1-8247, and Army Training Grant DAMD17-99-1-9402.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. Tel.: 210-567-7216;
Fax: 210-567-7277; E-mail: sung@uthscsa.edu.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M010011200
2 S. Sigurdsson and P. Sung, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: ssDNA, single-stranded DNA; dsDNA, double-stranded DNA.
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REFERENCES |
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