From the Department of Genetics and the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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The eukaryotic homologs of RecA protein are
central enzymes of recombination and repair, and notwithstanding a high
degree of conservation they differ sufficiently from RecA to offer
insights into mechanisms and biological roles. The yield of DNA strand exchange reactions driven by both Escherichia coli RecA
protein and its human homolog HsRad51 protein was inversely related to the GC content of oligonucleotide substrates, but at any given GC
composition, HsRad51 promoted less exchange than RecA. When 40% of
bases were GC pairs, the rate constant for strand exchange by
HsRad51 was unmeasurable, whereas the rate constants for homologous pairing were unaltered relative to more AT-rich DNA. The ability of
HsRad51 to form joints in the absence of net strand exchange was
confirmed by experiments in which heterologous blocks at both ends of
linear duplex oligonucleotides produced joints that instantly dissociated upon deproteinization. These findings suggest that HsRad51
acting alone on human DNA in vivo is a pairing protein that
cannot form extensive heteroduplex DNA.
Human Rad51 protein (HsRad51) is a member of the universally
distributed class of RecA proteins that play important roles in
homologous recombination and recombinational repair (1). In
prokaryotes, the RecA proteins play roles in recombination, post-replication repair, and the repair of double-strand breaks (2). In
eukaryotes, members of this class play roles in meiotic recombination,
double-strand break repair (3, 4), and possibly immunoglobulin switch
recombination (5). In the mouse, the requirement of Rad51 for embryonic
survival (6, 7) reveals a vital role that has not been found in
prokaryotes and lower eukaryotes.
RecA protein from Escherichia coli promotes a search for
homology by a single strand of DNA, and initiates an exchange between that strand and homologous duplex DNA. To carry out those complicated interactions, RecA protein forms a helical nucleoprotein filament on
single-stranded DNA; and Rad51 from Saccharomyces cerevisiae and Homo sapiens form nucleoprotein filaments that resemble
the one formed by RecA (8, 9). Once the nucleoprotein filament has been
formed, RecA requires no cofactors other than ATP to promote a rapid
search for homology and an extensive strand exchange. Yeast, frog, and
human Rad51, as well as human Dmc1, a homolog that is specifically
expressed in meiosis, are DNA-dependent ATPases that carry
out homologous pairing and strand exchange reactions that resemble
those catalyzed by RecA protein (10-14). However, the eukaryotic
homologs differ from RecA in several notable respects. None of the
eukaryotic enzymes appears to manifest the kinetic barrier to binding
to duplex DNA (15), which in the case of RecA favors the loading of
protein on single-stranded DNA; all hydrolyze ATP at a rate that is at
least an order of magnitude lower than hydrolysis by RecA, and all
promote recombination reactions much more slowly than RecA (10, 11, 12,
14). Observations on human Rad51 showed that both phases of the
recombination reaction, homologous pairing and strand exchange, are
markedly slower than the corresponding phases of the RecA reactions
(13).
Auxiliary proteins play important roles in the reactions catalyzed by
the eukaryotic homologs of RecA. Replication protein A, the eukaryotic
heterotrimeric single-stranded DNA-binding protein appears to stimulate
the formation of nucleoprotein filaments by yeast and human Rad51 (10,
16, 17), and recent experiments have demonstrated a physical
interaction between human Rad51 and human replication protein
A.1 Rad52 protein also
appears to play an early role in stimulating reactions of yeast and
human Rad51 (10, 18-20). In yeast and man, Rad52 has been shown to
interact physically with Rad51 (3, 21-23), as well as with replication
protein A (24, 25). Rad54 protein, another product of the epsitatic
group of yeast genes that encodes Rad52 and Rad51 proteins, is a
DNA-dependent ATPase, whose catalytic rate constant is 3 to
4 orders of magnitude greater than that of Rad51. The yeast and human
homologs of Rad54 interact physically with the respective Rad51
proteins, and yeast Rad54 strongly stimulates homologous pairing by
Rad51 (26-28).
The low ATPase activity of the human Rad51, its slow rates of pairing
and strand exchange (13), and its limited ability to carry out
extensive strand exchange (12) led us to postulate that it has a
relative inability to "open" duplex DNA and to investigate the
operationally and kinetically separable phases of homologous pairing
and strand exchange (29). The experiments reported here show that human
Rad51 can catalyze homologous recognition and the formation of
homologous joints in the absence of net strand exchange and suggest
that the function of Rad51 itself is limited to recognition of homology
and initiation of strand invasion. These studies may provide insights
into the roles of other components of the recombination machinery in
eukaryotes, the underlying mechanism of homologous recognition, and the
regulation of recombination.
Enzymes and Reagents--
DNase I and dithiothreitol
(DTT)2 were purchased from
Boehringer-Mannheim; ATP and phenylmethylsulfonyl fluoride from Sigma; isopropyl- DNA Substrates--
The single-stranded oligonucleotides used in
this study were designated as either (
RG1(
GC10(
Addition of a six-carbon primary amine linker, subsequent labeling of
the linker with fluorescein and rhodamine, purification of labeled
oligonucleotides, and preparation of duplexes were done as described
earlier (29). The ( Purification of HsRad51--
The protein was purified
from E. coli DH10B (Life Technologies, Inc.) carrying
plasmid pEG932 (13). IPTG-induced cells (120 g) were lysed in 3 volumes
of lysis buffer (20 mM potassium phosphate buffer, pH 7.4, 0.5 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 10% sucrose, 1 mg of lysozyme/ml, 0.5 M KCl) followed by
mild sonication. The lysate obtained on centrifugation was fractionated by addition of ammonium sulfate at 40% saturation. The precipitate, redissolved in buffer R51 (20 mM potassium phosphate
buffer, pH 7.4, 0.5 mM DTT, 0.2 mM EDTA, 50 mM KCl, 1 mM phenylmethylsulfonyl fluoride,
10% glycerol), was passed through Q-Sepharose (100 ml) pre-equilibrated with buffer R51. The column was washed, and bound proteins were fractionated by gradient elution with 50-800
mM KCl in buffer R51. Fractions containing HsRad51 were
pooled, dialyzed, and applied to a hydroxyapatite column (Bio-Gel HT,
100 ml) in buffer R51. HsRad51 protein was eluted by a linear gradient
of 20-500 mM potassium phosphate in buffer R51. The
dialyzate of fractions containing HsRad51 was loaded on a Mono-Q
(HR10/10) column. A steep gradient of 50 mM to 1 M KCl in buffer R51 eluted HsRad51 at approximately 350 mM KCl. Fractions containing HsRad51 were pooled, dialyzed,
and passed through 10 ml of a native DNA cellulose column. HsRad51
protein eluted at approximately 300 mM KCl when a gradient
elution of 50 mM to 1.2 M was used. Pure HsRad51 fractions were pooled, concentrated, and stored at The Gel Assay for Pairing and Strand Exchange--
Recombination
reactions mediated by RecA or HsRad51 acting on oligonucleotide
substrates were assayed by gel electrophoresis as described before
(13). HsRad51 or RecA (1.2 µM) was incubated with 3 µM single-stranded DNA oligonucleotide in 15 µl of
reaction buffer R (25 mM Hepes, pH 7.4, 1 mM
DTT, 2 mM ATP, 100 µg of bovine serum albumin /ml, 1 mM MgCl2). After an incubation at 37 °C for 5 min, MgCl2 was increased to 30 mM, followed
by the addition of homologous duplex DNA (2.5 µM) labeled
at the 5' end of the ( Annealing Assay--
Annealing was assayed as described (13).
32P-Labeled ( DNase I Protection Assay--
HsRad51 or RecA (1.2 µM) were preincubated with 3 µM
5'-32P-labeled oligonucleotides in buffer R (total volume
15 µl), as mentioned above for 5 min at 37 °C. DNase I (final
concentration of 0.15 units/µl) was then added and incubation was
continued for 2 min. The reaction was ended by addition of stop
solution containing 25 µg of yeast tRNA. DNA was then precipitated by
addition of cold 10% trichloroacetic acid and acid soluble
radioactivity in the supernatants was measured.
ATPase Activity--
Reaction mixtures contained 25 mM Hepes, pH 7.4, 1 mM DTT, 100 µg of bovine
serum albumin /ml, 6 mM MgCl2, 1.0 µCi of
[3H]ATP, and 0.5 mM ATP. HsRad51 or RecA (1.3 µM) was incubated with 30 µM
single-stranded oligonucleotides at 37 °C and the reaction was
arrested at different time intervals by the addition of ATPase stop
solution (final concentration of 1 mM ATP, 1 mM
ADP, 25 mM EDTA). Three µl of the reaction mixture was
processed for detection of ATP hydrolysis by thin layer chromatography
as described (30). ATPase activity was expressed as moles of ATP
hydrolyzed per mole of protein/min.
Fluorometric Assays for Pairing or Strand Exchange--
Pairing
or strand exchange were detected by two separate fluorometric assays as
described before (13, 29). For pairing, 1.2 µM HsRad51 or
RecA was preincubated with 3 µM 3' fluorescein-labeled (
Quantitation of fluorescence resonance energy transfer (FRET) due to
homologous pairing was done as described (13, 29). To detect the FRET
signal, the spectrum was taken from 502 to 620 nm upon excitation at
493 nm, before the addition of duplex and after the reaction is completed.
The Effects of Base Composition on Recombination Reactions in
Vitro--
Five different substrates with varying GC content were made
to explore the effect of base composition on pairing and strand exchange by HsRad51 and RecA proteins: A16 (16% GC), C16 (25% GC),
R16 (50% GC), W16 (40% GC), and G16 (71% GC). In the design of these
oligonucleotides, we minimized secondary structure as described under
"Experimental Procedures."
Nucleoprotein filaments formed on single-stranded oligonucleotides by
HsRad51 or RecA were paired with homologous duplex oligonucleotides for
1 h prior to assaying the yields of the reactions by gel
electrophoresis (Fig. 2A). For
both RecA and HsRad51 the yields were inversely related to GC content
of the substrates (Fig. 2A), but at any given GC content the
yield of reactions mediated by Rad51 was lower than that by RecA. The
parallel functions that fit the respective data on HsRad51 and RecA
suggest that a common mechanism is responsible for the inverse effect
of GC content on strand exchange; but the existence of parallel
functions instead of a single function suggests that the effect of base
composition is not exclusively attributable to secondary structure in
the single-stranded DNA. In other words, there is a common effect of GC
content, but each of the two proteins contributes to a quantitatively
different outcome.
Either of two general explanations might account for the common effect
of GC content on the yields of reactions by HsRad51 and RecA.
(a) Nucleoprotein filaments might form differently as a
function of the GC content of single-stranded DNA. (b) By
its stabilizing effect on duplex DNA, high GC content might affect the
ability of HsRad51 and RecA to carry out any of the steps that are
potentially involved in the conversion of parental homoduplex substrate
into the product heteroduplex DNA, including, among others, the
exchange of base pairs and the final displacement of one parental strand.
Effect of GC Content on the Interactions of HsRad51 and RecA
Protein with Single-stranded DNA--
We examined the effect of GC
content on three different indices of interactions of HsRad51 and RecA
with single-stranded DNA: ATP hydrolysis, protection of single-stranded
DNA from digestion by DNase I, and annealing of complementary strands
(Table I).
In the case of HsRad51, we could detect no difference in catalytic rate
constants (turnover number) among a set of substrates with varying GC
content. In the case of RecA protein, there was a reproducible decrease
in kcat at the highest content of GC residues (Table I).
We determined the amount of DNase I that was sufficient to convert the
32P label present at the end of these 83-mer
oligonucleotides into a form that is soluble in acid. HsRad51 or RecA
were allowed to form filaments on these oligonucleotides (at a ratio of
protein to nucleotide residues of 1:2.5) in the presence of 1 mM MgCl2 at 37 °C for 5 min, followed by the
addition of DNase I, and incubation for 2 more minutes. As shown in
Table I, both HsRad51 and RecA protected all three oligonucleotide
substrates. There appeared to be some increased digestion of the
substrate with the highest GC content, but there was no discernible
trend that correlated with base composition. The assay for acid
solubility of label at the end of an oligonucleotide should be
sensitive because only a few cuts need to be made by DNase I to make
the label acid-soluble.
For annealing reactions, filaments formed on the three single-stranded
oligonucleotides were paired with their respective 32P-labeled complementary strands for 1 min, and annealed
products were monitored by gel electrophoresis after deproteinization. For both Rad51 and RecA, the yields of the annealing reactions were
similar at the varying GC compositions of the oligonucleotide substrates (Table I). In aggregate, these data do not reveal a
systematic effect of GC content that is sufficient to explain the
inhibitory effect of GC content shown in Fig. 2.
Stabilization of Duplex DNA and Its Effect on Pairing and Strand
Exchange Activity--
The pairing and exchange activity of HsRad51
was checked at different concentrations of MgCl2 in the
presence of 100 mM NaCl, with oligonucleotides A16 (16%
GC) and G16 (71% GC) as substrates. We observed, as expected, a
stimulatory effect of MgCl2 at low concentrations and an
inhibitory effect at high concentrations (31) (Fig.
3). There was, however, a clear
difference between the effect of Mg2+ concentration on
GC-rich DNA versus AT-rich DNA. High concentrations of
Mg2+ were more inhibitory to reactions with GC-rich DNA
than AT-rich DNA. This observation is consistent with the
interpretation that high GC content inhibits pairing and strand
exchange either because it stabilizes secondary structure in
single-stranded DNA or because it stabilizes the secondary structure of
parental duplex DNA, either of which would be augmented further by high
concentrations of Mg2+.
A Stimulatory Effect of Base Mismatches in Isoenergetic Strand
Exchanges--
One expects a thermodynamic effect of mismatched bases
on the yield of products of pairing and strand exchange, reflecting the
net gain or loss of Watson-Crick base pairs in the reaction. We
designed experiments in which strand exchange promoted by HsRad51 resulted in either a net loss, a net gain, or no change in the number
of base pairs. This was accomplished by varying the strand in which
base substitutions were located.
When substitutions were located in the single strand used to make
filaments and these were reacted with perfectly paired duplex DNA
(Table II, row 1), the product of strand
exchange contained mismatched base pairs, which results in a net
energetic loss. As expected, the yield of the reaction decreased as the
number of mismatches increased (Table II, row 1). By contrast, when
base substitutions were located in the (+)-strand of the duplex
substrate and the latter was reacted with filament containing a
(+)-strand that lacked substitutions, the reaction produced a net
increase in Watson-Crick base pairs and the yield increased as the
number of mismatches increased (Table II, row 2).
Our aim, however, was to examine the situation in which an isoenergetic
exchange might occur with a duplex 83-mer that had been destabilized by
up to 7 mismatched pairs of bases. This was accomplished by the
substitution of bases in the strand that was complementary to the
strand in the filament, in which case the duplex substrate and the
duplex product had precisely the same number and kinds of mismatched
bases. As the number of mismatches increased up to 7, the yield of this
isoenergetic reaction increased by 80%. Thus, the yield of products of
pairing and strand exchange promoted by human Rad51 was inhibited by
three forces that stabilize duplex DNA, namely, high GC content,
Mg2+, and perfectly matched base pairs.
Discrimination of the Respective Effects of GC Content on
Homologous Pairing and Strand Exchange--
Assays based on FRET can
distinguish the initial rapid second order pairing of a single strand
with duplex DNA from the subsequent, slower, first order strand
exchange (13, 29, 32). In the pairing assay, fluorescein is conjugated
to one end of the strand that is used to make a filament, and rhodamine
is conjugated to the complementary strand in parental duplex DNA. When
pairing takes place, the two dye-conjugated strands come into
apposition and photoexcitation of fluorescein leads to non-radiative
transfer of energy to rhodamine, which then fluoresces at its emission wavelengths (Fig. 4A).
Transfer of energy is also manifested as a quenching of light emitted
by fluorescein at its own emission wavelengths, and for measurements of
kinetics the quenching of fluorescein emission is much more readily
measured (see Ref. 29). In the pairing assay, the two dyes remain in
apposition even after strand exchange takes place. Thus the yield
measured at any moment represents the sum of synaptic intermediates
(Wcw) plus one of the products of strand exchange (Wc), which is equal
to the cumulative fraction of substrate molecules that have undergone
homologous pairing (Fig. 4A). In the version of the assay
that detects strand exchange, the two dyes are conjugated instead to
the complementary strands of the parental duplex oligonucleotide (Fig.
4B). At the starting point of this assay, energy transfer is
maximal and the fluorescence of fluorescein is quenched. As strand
exchange separates the strands, energy transfer diminishes and the
fluorescence of fluorescein is enhanced (Fig. 4B).
In a previous detailed analysis of RecA reactions by these assays, we
were able by computer simulation to fit the data to the two-step
kinetic scheme shown in Fig. 4 (29). For that analysis, we assumed that
the quenching of fluorescence from fluorescein was the same in the
intermediate (Wcw) and product (Wc). The analysis was based on three
assays, one that detected Wcw plus Wc, one that detected only Wcw, and
one that detected only Wc. The coherence of the analytical data did not
reveal any major flaws in the analytical procedures and assumptions. We
used the same methods and assumptions to analyze the reactions of
HsRad51 described below. Justification for use of the same methods and
assumptions is provided by the parallel behavior of RecA and HsRad51
(Figs. 2 and 5 below), and by the fit that we obtained of
computer-generated simulations to kinetic data on HsRad51 (data not shown).
As shown in Fig. 2, oligonucleotides with increasing GC content
progressively inhibited the yield of Rad51 reactions as measured by gel
electrophoresis. At 71% GC content, the yield was negligible. From the
oligonucleotides studied in Fig. 2, we chose those with 40 and 16% GC
to examine the effects of GC content on the phases of the reaction
promoted by both Rad51 and RecA (Fig. 5).
These two oligonucleotides showed no indication of effects of base
composition on the formation of filaments (Table II).
In the case of Rad51, 40% GC content roughly halved the yield of
homologous pairing relative to 16% GC content (Fig. 5A), consistent with the data in Fig. 2. By contrast, the fluorometric assay
that detects the physical separation of fluorescent dyes on the ends of
strands, showed that the same increase in GC content from 16 to 40%
reduced strand exchange to unmeasurable levels (Fig.
5B).
With the same substrates, we compared the effects of increasing GC
content on homologous pairing and strand exchange by RecA protein (Fig.
5, C and D). The results confirm the general
conclusion drawn from Fig. 2 that increasing GC content inhibits RecA
reactions as well as HsRad51 reactions, but to a lesser degree at any
particular GC content. The comparison also shows more specifically that
at 40% GC content, where strand exchange promoted by Rad51 was
virtually eliminated, as measured by the fluorescence assay, RecA
protein still promoted substantial exchange.
We analyzed the data in Fig. 5, A and B, by
computer simulation, using Kinsim software as described previously
(29). The derived rate constants are shown in Table
III. Consistent with visual inspection of
the data, the increase from 16% GC to 40% GC reduced
k2, the rate constant for strand exchange by 2 orders of magnitude. By contrast, k1 and
k-1, the forward and reverse rate constants for
homologous pairing were not significantly changed. This calculation
suggests that the apparent decrease in homologous pairing at 40% GC
(Fig. 5A) is attributable to the elimination of strand
exchange, which otherwise drives the reaction to the right. In other
words, at 40% GC content, which is approximately the average base
composition of human DNA, HsRad51 promotes homologous pairing, but no
net strand exchange.
Evaluation of the Fluorometric Assays Versus the Gel
Electrophoresis Assay--
As just described, with substrates that
contained 40% GC, strand exchange promoted by Rad51 was completely
inhibited according to the fluorometric assay (Fig. 5B).
However, at the same GC content, the assay by gel electrophoresis
registered 30% of product (Fig. 2A). This discrepancy
required further comparisons of the two assay systems.
The physical basis of the fluorometric assays has been described above.
These assays can be done in two ways. One can measure FRET by exciting
fluorescein at its absorption maximum and observing the resulting
"sensitized" emission of light from rhodamine, or one can monitor
the quenching of light emitted from fluorescein as a result of the
energy that it has lost by non-radiative transfer to rhodamine. In the
experiments described in this section we used both methods.
The fluorometric assays are carried out in solution without deliberate
disruption of protein-DNA complexes. The assay by gel electrophoresis
requires deproteinization of intermediates and products, and
consequently has different properties as shown in the following
experiments. The electrophoretic assay detects the homology-dependent incorporation of a labeled single strand
into a heteroduplex structure or the displacement of one labeled strand from parental duplex DNA. By its nature, this assay is incapable of
distinguishing homologous pairing from strand exchange. The required
deproteinization of the reaction mixture prior to electrophoretic analysis creates uncertainty about how much of the measured outcome is
attributable to strand exchange that occurred in solution and how much
to spontaneous branch migration that occurred after deproteinization.
Using the same oligonucleotide substrates with varying GC content as
studied in Fig. 2A, we measured the steady state level of
homologous pairing as measured by FRET for both Rad51 and RecA. A plot
of the FRET values versus the electrophoretic values for each substrate of a given GC concentration revealed a linear
correlation encompassing the data obtained with both Rad51 and RecA
(Fig. 2B). Thus, these two assays revealed a remarkable
concordance in relation to GC content.
We then compared strand exchange, as measured by enhanced emission from
fluorescein, with the measurement provided by the electrophoretic
assay. From concurrent observations designed to test the polarity of
strand exchange (17), we had identified substrates that showed
different kinetics according to the fluorometric assay. One substrate
was the 83-mer A16 containing 16% GC bases. Two other substrates were
chimeric molecules in which one end was GC-rich and the other was
AT-rich, with an average GC content of 50%. In oligonucleotide RG1,
the 3' end of the strand in the Rad51 filament was GC-rich, whereas in
oligonucleotide RG2 the 5' end was GC-rich. These were reacted with
their respective duplex counterparts.
The time course of strand exchange with each of these substrates was
compared directly by the fluorometric and the gel assays (Fig.
6). Relative to the AT-rich reference
oligonucleotide A16, the yield with oligonucleotide RG1 was reduced
3-fold and that of RG2 was reduced more than 5-fold as measured by the
fluorometric assay. The outcome was quite different when measured by
the gel assay. There was no difference observed between substrates RG1 and RG2, and their yields were reduced only about 20% relative to the
reference oligonucleotide A16.
The observations described above show that the data obtained by the
electrophoretic assay correlate with those obtained by the measurement
of homologous pairing by FRET, but do not correlate with those obtained
by the fluorometric assay for strand exchange. The simplest
interpretation is that with oligonucleotides as substrates of reactions
promoted by Rad51, the electrophoretic assay provides a better measure
of homologous pairing than it does of strand exchange. We suggest that
after deproteinization, intermediates that have not completed exchange
in the presence of Rad51 undergo spontaneous branch migration resulting
in either the regeneration of substrates or the production of
additional strand exchange products. Thus, as in the fluorometric assay
for homologous pairing (see previous section above), the
electrophoretic assay at any point in time measures the sum of some
fraction of existing synaptic intermediates (Wcw) plus products of
strand exchange (Wc + w), which is proportional to the cumulative
fraction of substrate molecules that have undergone homologous pairing
(see Fig. 4). If strand exchange is blocked, as it is in substrates
containing 40% GC base pairs (Fig. 5B), the electrophoretic
assay still measures some fraction of the synaptic intermediate because
of the spontaneous branch migration that can occur upon deproteinization.
Homologous Pairing of Oligonucleotides with Heterologous
Ends--
As a further test of the ability of human Rad51 to promote
homologous pairing without strand exchange, we examined reactions of
83-mer oligonucleotides with blocks of 10 heterologous nucleotides at
both ends. The single-stranded oligonucleotide in these experiments was
A16(
The terminal blocks of heterologous sequences were as effective in
blocking strand exchange as a completely heterologous sequence (Fig.
7C), whereas homologous pairing was still substantial (Fig. 7A). In the assay for pairing, the amplitude of the change
in fluorescence was about half of that observed for completely
homologous substrates (Fig. 7B). This relative decrease in
apparent yield of the pairing reaction was the same as seen when strand
exchange was eliminated in substrates containing 40% GC base pairs
(Fig. 5). Some major part of this decreased amplitude of the pairing reaction is probably an indirect consequence of blocking strand exchange, as described above.
After pairing reactions reached their limits we added SDS to
deproteinize the reactions and as quickly as possible we resumed measuring the fluorescence output. When the substrates had heterologous ends, the addition of SDS resulted in the immediate restoration of
fluorescence to the level seen prior to pairing (Fig. 7A). Thus the synaptic intermediates in this case were unstable in the
absence of protein and whatever bonds mediated homologous recognition
were immediately broken. The same treatment of a reaction of completely
homologous substrates (Fig. 7B) resulted in the restoration
of only about a fifth of the fluorescence emission, indicating that a
smaller fraction of molecules were in the synaptic state. In the
reaction of completely homologous molecules any that had gone on to
completion of strand exchange would not contribute upon
deproteinization to restoration of the initial fluorescence amplitude.
Base Composition Versus Secondary Structure in Single
Strands--
In a previous study of the effect of base composition on
strand exchange promoted by RecA protein, Gruss et al. (33)
reported several effects of high GC content on the overall process of
strand exchange promoted by RecA protein. One effect of high GC content appeared attributable to more stable secondary structure in
single-stranded DNA. Since single-stranded DNA-binding protein, which
removes secondary structure from single-stranded DNA, was only
partially able to overcome the inhibitory effect of high GC content,
factors other than secondary structure presumably contributed to the
effect of GC bases. Gruss et al. (33) identified another
effect, manifested by inhibition when a GC-rich region in
single-stranded DNA was adjacent to the site of initiation of strand
exchange. Those observations identified effects of GC-rich sequences on
the formation or structure of nucleoprotein filaments but did not
exclude effects on strand exchange itself.
In the present comparative studies of Rad51 and RecA, we designed
oligonucleotide substrates to minimize secondary structure in single
strands, and along with other assays, we used fluorometric assays that
distinguish homologous pairing from strand exchange. We did not find
evidence of systematic effects mediated by alterations in the
nucleoprotein filaments, but we cannot exclude such effects either.
However, we found effects on the strand exchange process itself.
A Direct Effect of GC Content on Strand Exchange--
Evidence of
a direct effect of GC-rich sequences on strand exchange was provided
principally by the kinetic analysis of reactions by Rad51 protein. When
the substrates contained 40% GC, the forward rate constant for strand
exchange was unmeasurable, whereas the forward and reverse rate
constants for homologous pairing were unchanged relative to DNA with a
much lower GC content. However, the inhibitory effect of GC content did
not end there. When the substrate was 71% GC, Rad51 was unable to
promote any significant homologous pairing, as reflected either by the
electrophoretic assay (Fig. 2) or by the specific fluorescence assay
for homologous pairing (17).
This order of effects in relation to increasing GC composition, the
complete elimination of strand exchange first, and then the elimination
of homologous pairing, is the opposite of what one might expect if GC
content were interfering only with the formation of presynaptic
filaments, in which case the inhibition of homologous pairing and
subsequent strand exchange should go hand in hand: without homologous
pairing there can be no strand exchange. On the other hand, in the case
of RecA protein, we know that pairing can occur without any net strand
exchange (34); and the present experiments show that the same is true
of Rad51. The observed order of effects of increasing GC content can be rationalized readily if, as has been suggested (35), the switching of
base pairs is a step that is common to both recognition of homology and
strand exchange. Hsieh et al. (36) estimated that homologous
recognition promoted by RecA protein requires only about a half dozen
base pairs. Thus, to form a homologously aligned synaptic complex with
83 nucleotide residues in each strand, perhaps only a small subset of
bases needs at any instant to have switched from parental to
heteroduplex pairs, whereas the completion of strand exchange by the
separation of one parental strand from its erstwhile complement
obviously requires that all parental base pairs be ruptured. If
increasing GC content progressively inhibits the switching of base
pairs, it follows that net strand exchange would be abolished before
homologous recognition were eliminated.
Formation of Paranemic Joints by HsRad51--
Acting on circular
single-stranded DNA and circular duplex DNA, RecA protein promotes the
recognition of homology and the formation of joints whose stability
depends on the continued binding of the protein (34, 37). Such joints
are true paranemic structures in which the linking number of parental
duplex and parental single strand is zero, and no net strand exchange
can occur. When a region of homology is flanked on both sides by
heterologous sequences, joints with similar properties are formed and
are also called paranemic joints (34). The experiments described here
show that human Rad51 protein also promotes the formation of this kind
of paranemic joint from substrates whose ends are heterologous. By chemical probing, Adzuma (35) observed that when RecA protein forms
paranemic joints in the presence of ATP Function of Rad51 in Vitro Versus in Vivo--
The reaction
carried out by Rad51 protein with substrates that contain 40% GC bases
leads to an intermediate that also resembles a paranemic joint, one in
which no net strand exchange takes place. A direct comparison showed
that whereas 40% GC content completely inhibited strand exchange by
Rad51 (Fig. 5B), RecA was still able to perform strand
exchange, which supports the view that HsRad51 has a relative
deficiency in the ability to promote strand exchange. On the basis of
reconstitution experiments in which the E. coli RuvAB
complex dissociated RecA from joint molecules and then accelerated branch migration, West and colleagues (40) suggested that RuvAB is
primarily responsible for the extension of heteroduplex DNA in
vivo. On the other hand, RecA acting alone in vitro
catalyzes extensive heteroduplex formation, and using the energy of ATP hydrolysis RecA has an impressive ability to push strand exchange through sizable heterologous insertions (41, 42, 43). On the basis of
the present results it would seem that the ability of human Rad51, as
well as human Dmc1 (14), to promote the formation of heteroduplex DNA
is much more limited. Without the action of other proteins, the
function of human Rad51 and human Dmc1 may be limited to homologous
pairing. In relation to the observed blockage in vitro of
strand exchange promoted by HsRad51 when the substrate had 40% GC base
pairs, it is interesting to note that the average GC content of human
DNA is 39% (44). The ability of RecA and its homologs to promote
strand exchange to greater or lesser degrees presumably reflects in
part an essential mechanistic coupling between the switching of base
pairs and either homologous recognition itself or the stabilization of
a homologous joint. RecA and its homologs are key players in finding
homology and initiating strand invasion; they probably extend and
stabilize D-loops as well, but the subsequent creation of extensive
heteroduplex DNA, particularly following the formation of Holliday
structures is probably the task of other enzymes.
Origins of replication tend to be AT-rich, as are promoter regions
which are hotspots for the initiation of meiotic recombination in yeast
(45). Thus AT-rich sequences in the genome are sites of initiation of
replication and recombination, presumably because of the lower
stability of AT base pairs. The striking dependence of human Rad51 and
Dmc1 on high AT content of DNA is consistent with their postulated
biological roles in initiating recombination.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
-D-thiogalactopyranoside (IPTG) from American
Bioanalytical; Q-Sepharose, native DNA cellulose, and Mono-Q HR 10/10
from Pharmacia Biotech; hydroxyapatite Bio-Gel HT from Bio-Rad; and
polynucleotide kinase from New England Biolabs. RecA was purified as
described (30).
)- or (+)-strand. For example,
A16(
) is a single-stranded oligonucleotide while A16(+) is its
complementary strand. In the experiments reported here the strand used
to form Rad51 or RecA filaments was designated as the
(
)-strand. Sequences of 83-mer oligonucleotides A16(
) (16% GC) and
W16(
) (40% GC) have been described earlier (13). The sequences of
C16(
) (25% GC), R16(
)(50% GC), and G16(
)(71% GC) are as
follows: C16(
) (25% GC):
5'-ATATCCTTTCTTTAAACTTCTATCATTGATTCTTACTAGTCTTTACCTTACTATACTTCTATCAGTTTATCGATTCTTCTTTA-3'; R16(
) (50% GC):
5'-GTTCGACGTGACTGGATTCTCTCTCTGGCGCAGTGTGGTAACTTCCGATGATGGGTACTTGAGATTTGATGCTTCTGGTCCAC-3'; G16(
) (71% GC):
5'-GCGGTGGACGGCTGGGTCGGGTGGTGAGTGGGTTGGCGATGGGAGGTGGTGGGCTAGGGGGCTTAGGGGGGAGTCGGTGGTGG.
) (50% GC) was an oligonucleotide with a 5'-AT-rich half
(71% AT) and 3'-GC-rich half (71% GC):
5'-TTTACTTGTACTTCATTCATTCACATTCCTATCATGTTTCTACGCCACCTCCCGACCCTCCCCACCACTCACCCAACCGCTACC; while RG2(
) has a GC-rich 5' end and AT-rich 3' end
5'-CCATCGCCAACCCACTCACCACCCCTCCCAGCCCTCCACCGCATCTTTGTACTATCCTTACACTTACTTACTTCATGTTCATTT.
) was derived from A16 and has a 10-base pair heterologous
substitution at the 5' end (GGGCGGGCGG) and a similar 10-base pair
heterologous substitution at the 3' end (GGCGGGCGGG). To minimize
secondary structure, we used GCG software and required that each 83-mer
have fewer than 10 successive intrastrand hydrogen bonds separated by
less than 4 unpaired bases.
)- and (+)-strands contained a linker at the 3'
and 5' end, respectively. All DNA concentrations refer to moles of
nucleotide residues, except for stoichiometric ratios for duplex DNA,
which are expressed as moles of protein per mole of base pairs.
80 °C.
A single band of approximately 38 kDa could be seen on
SDS-polyacrylamide gel electrophoresis analysis of concentrated HsRad51
(Fig. 1).
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Fig. 1.
Purity of HsRad51. HsRad51 was purified
as described under "Experimental Procedures." Five µg of HsRad51
was subjected to SDS-polyacrylamide gel electrophoresis analysis.
Molecular weight markers, labeled on the left, are in
kDa.
)-strand with [
-32P]ATP. After
an hour of incubation at 37 °C, the reaction was terminated by
addition of 3 µl of stop solution (final concentration of 0.5% SDS,
50 mM EDTA, 0.04% bromphenol blue, 0.04% xylene cyanol) and subjected to electrophoresis in a 12% nondenaturing polyacrylamide gel. The gel was run at 16 V/cm for 1.5 h in TBE buffer. The gel was dried, and reaction products were quantitated on a PhosphorImager (Molecular Dynamics) using ImageQuant software.
)-strand oligonucleotides at 3 µM were incubated with 1.2 µM RecA or HsRad51 in reaction buffer R containing 1 mM
MgCl2 for 5 min. Complementary (+)-strands were then added
at 3 µM to the respective tubes and incubated further for
1 min. The reaction was quenched by addition of stop solution
containing 30 µM unlabeled (
)-strand and
electrophoresed through 12% polyacrylamide. Products were quantitated
by use of a PhosphorImager.
)-strand in reaction buffer R (total volume 150 µl) at 37 °C for 5 min. MgCl2 was then increased to 30 mM
followed by the addition of duplex that contained rhodamine at the 5'
end of the complementary (+)-strand. In the strand exchange reaction,
the (
)-strand used to form filament was unlabeled, whereas the duplex
had fluorescein at the 3' end of the (
)-strand and rhodamine at the
5' end of the (+)-strand. Quenching in fluorescein intensity in the
pairing assay and enhancement in the strand exchange assay were
monitored every 2 s at 520 nm upon excitation at 493 nm.
RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References
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Fig. 2.
Pairing and strand exchange by HsRad51 and
RecA versus GC content. Reactions were carried out and
analyzed as described under "Experimental Procedures" with
substrates containing the indicated content of G and C nucleotide
residues. A, pairing and strand exchange measured by gel
electrophoresis versus GC content of the substrates.
B, homologous pairing measured by FRET versus
pairing and strand exchange as measured by gel electrophoresis. Steady
state FRET assays were performed independently for each of the
substrates and for both enzymes, and the values were plotted
versus those obtained by gel electrophoresis in
A. , HsRad51;
, RecA.
Interactions of HsRad51 and RecA with single-stranded DNA versus GC
content
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Fig. 3.
Effect of MgCl2 on
HsRad51-mediated pairing and strand exchange with AT-rich and GC-rich
DNA. For comparison of the distributions at high and low GC
content, the plots have been normalized to percent of the highest value
for each of the two substrates. The actual yields of the two reactions
were 54% for 16% GC and 7% for 71% GC. Reactions were carried out
and analyzed as described under "Experimental Procedures."
Effect of destabilizing mismatches on isoenergetic strand exchanges
catalyzed by HsRad51
) and W16(+) to produce the mismatched substrates. Substrates
containing two, six, and seven mismatches, respectively, had
transversions at positions 24 and 60; and 12, 24, 36, 48, 60, and 72;
and 6, 18, 30, 42, 54, 66, and 78 (see Ref. 32).
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Fig. 4.
Schematic representation of the fluorometric
assays for pairing and strand exchange. A, pairing
assay. As described previously (29), fluorescein (F) and
rhodamine (R) were located to detect the
homology-dependent conjunction of the dye-labeled
complementary strands by FRET. The fluorescence emission from rhodamine
is enhanced and that from fluorescein is quenched, as indicated by the
highlighted symbols. Since strand exchange does not separate
the dyes, the signal at any moment represents the sum of synaptic
intermediates (Wcw) plus products of strand exchange (Wc + w), which is
equal to the cumulative fraction of substrate molecules that have
undergone homologous pairing (A). B, strand
exchange assay. The separation of dyes upon strand exchange results in
diminished fluorescence from rhodamine and enhanced fluorescence from
fluorescein.
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Fig. 5.
Preferential inhibition of HsRad51 strand
exchange by 40% GC DNA. Homologous pairing and strand exchange
were separately measured by the fluorometric assays (Fig. 4) as
described under "Experimental Procedures": quenching of
fluorescence associated with the conjunction of a single strand with
homologous duplex DNA (A and C), and enhancement
of fluorescence associated with displacement of one of three strands
within the nucleoprotein filament (B and
D).
Kinetic analysis of homologous pairing and strand exchange catalyzed by
HsRad51 from substrates with 16 or 40% GC content
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Fig. 6.
Strand exchange mediated by HsRad51:
comparison of the electrophoretic and fluorometric assays. Data
were obtained with 3 substrates, A16 (16% GC) and two chimeric
substrates, with average GC content of 50%, but with GC-rich regions
located either at the 3' or 5' end of the strand used in the Rad51
filament. The latter were RG1, 5'-(29% GC)42(71%
GC)42-3', and RG2, 5'-(71% GC)42(29%
GC)42-3' (see "Experimental Procedures" and Ref. 17).
A, observations made by the fluorometric assay for strand
exchange (Fig. 4B). B, observations made by gel
electrophoresis.
) which has 16% GC base pairs. The duplex oligonucleotide, GC10,
was derived from A16 by substituting heterologous blocks of 10 GC base
pairs at each end. As a standard for comparison, reactions were also
carried out between A16(
) and the completely homologous duplex
oligonucleotide (Fig. 7B).
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Fig. 7.
The formation of paranemic joints by Rad51
protein. In each of the three frames A-C, the
fluorometric tracings of amplitude versus time are labeled
as follows: 1) completely homologous DNA substrates, oligonucleotide
A16 and its double-stranded counterpart; 2) completely heterologous
substrates, A16 plus W16 duplex oligonucleotide; and 3) substrates A16
single strand and GC10 duplex DNA with blocks of 10 heterologous base
pairs at both ends. A and B, assay for homologous
pairing. The abrupt discontinuities followed the addition of SDS at
0.5% final concentration. C, assay for strand exchange. The
lower tracing, labeled 2 and 3, was
formed by completely overlapping data from two separate reactions
containing, respectively, completely heterologous substrates, and
substrates with 10 heterologous nucleotide residues at each end.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
S, a non-hydrolyzable analog
of ATP, bases are found in a switched heteroduplex configuration. In
the present experiments on HsRad51, the instantaneous dissociation of
paranemic joints upon deproteinization means either that in the
presence of ATP, no bases had switched, or more likely, that switched
bases were in an unstable or dynamic state from which they could
readily revert to the parental state. This is reminiscent of the
properties of other joints formed by RecA protein that cannot progress
to net strand exchange but instead undergo continuous turnover
(38, 39).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Efim Golub and Oleg Kovalenko for helpful comments on the manuscript, Jan Zulkeski for data processing, and Zhufang Li for technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant R37-GM33504.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: Dept. of Genetics,
Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-5034; Fax: 203-785-7023; E-mail:
rgupta{at}biomed.med.yale.edu.
The abbreviations used are:
DTT, dithiothreitol; IPTG, isopropyl--D-thiogalactopyranoside; FRET, fluorescence resonance energy transfer; ATP
S, adenosine
5'-O-(thiotriphosphate).
1 Golub, E. I., Gupta, R. C., Haaf, T., Wold, M. S., and Radding, C. M. (1998) Nucleic Acids Res. 26, 5388-5393.
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