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INTRODUCTION |
Targeted modification of the genome by gene replacement is
of value as a research tool and has potential application to gene therapy. However, although facile methods exist to introduce new genes
into mammalian cells, the frequency of homologous integration is
limited (1), and isolation of cells with site-specific gene insertion
typically requires a selection procedure (2). Site-specific DNA damage
in the form of double-strand breaks produced by rare cutting
endonucleases can promote homologous recombination at chromosomal loci
in several cell systems (3-7), but this approach requires the prior
insertion of the recognition sequence into the locus. Because
intermolecular triple helices can provoke DNA repair (8),
oligonucleotide-mediated triple helix formation has been proposed as a
potentially more general approach to sensitizing a target site to
homologous recombination (9-12).
TFOs1 can bind in the major
groove of DNA to polypurine/polypyrimidine sequences, forming specific
Hoogsteen or reverse-Hoogsteen hydrogen bonds with the purine strand of
the duplex (13, 14). Triplex formation has been shown to inhibit
transcription in mammalian cells (15) and can be used to deliver a
DNA-reactive conjugate to a specific target site both in complex DNA
mixtures in vitro (16, 17) and within mammalian cells in
culture (18-22), in some cases leading to site-directed mutations (19,
20). Triplex formation, by itself, can be mutagenic, and evidence
suggests that the nucleotide excision repair (NER) and
transcription-coupled repair pathways may play a role in the
triplex-induced mutagenesis (8).
In previous work, we found that triple helix-directed psoralen
cross-links could stimulate recombination in a plasmid substrate containing two tandem copies of the supFG1 reporter gene
(9). Subsequent work established that triplex formation, even in the absence of covalent DNA damage, could stimulate recombination between
repeated sequences, an effect that was absent in cells deficient in the
NER factor, XPA (10). Recent work has extended these findings to the
demonstration of TFO-induced recombination at a chromosomal locus
containing two tandem copies of the herpes simplex virus thymidine
kinase gene, following direct intranuclear microinjection of the
oligonucleotides (11).
Based on the ability of TFOs to mediate specific molecular recognition
of a DNA target site within a cellular genome and on the observation
that triplex formation can stimulate recombination, we also tested a
series of bifunctional oligonucleotides consisting of a TFO designed to
bind to bp 167-196 of the supFG1 reporter gene coupled to a
short (40 nt) segment of DNA homologous to bp 121-160 of the gene.
Such a hybrid molecule, designated a tethered donor-TFO (TD-TFO), was
found to mediate recombination with the supFG1 gene present
in an SV40-based episomal vector in monkey cells at frequencies in the
range of 0.1-1% (Ref. 23 and data not shown), demonstrating that TFOs
can promote intermolecular as well as
intramolecular recombination in mammalian cells. This result
is consistent with studies demonstrating that bifunctional oligonucleotides can mediate both triplex formation and strand invasion
on plasmid substrates in vitro (24, 25).
In the present study, we have used a plasmid-based assay to investigate
triple helix-induced recombination in human cell-free extracts. We find
that triple helix formation can stimulate recombination between a
plasmid and short homologous fragments in vitro. Stimulation was observed whether or not the donor fragment was directly linked to
the TFO. Recombination was reduced in the absence of the TFO as well as
when the TFO was substituted with a non-triplex-forming, scrambled
sequence oligonucleotide. To probe the mechanism of the induced
recombination, the roles of the NER damage recognition factor, XPA
(26), and the human recombinase, HsRad51 (27), were directly tested by
experimental manipulation of the respective protein levels in the
extracts, either via immunodepletion with specific antibodies or
supplementation with purified proteins. We report here that both XPA
and HsRad51 are required for triple helix-induced recombination, and
that increased HsRad51 levels can boost the efficiency of the reaction.
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EXPERIMENTAL PROCEDURES |
Plasmid Vector--
The shuttle vector plasmid pSupFG1/G144C,
containing a supFG1 gene with an inactivating G:C to C:G
point mutation at position 144, was described previously (23).
Oligonucleotides--
Oligonucleotides were synthesized
by the Midland Certified Reagent Co. (Midland, TX) and purified by
either gel electrophoresis or high pressure liquid chromatography
(HPLC), followed by Centricon-3 filtration in distilled water (Amicon,
Beverly, MA). The oligonucleotides consisted primarily of
phosphodiester linkages but were modified at the 3' end to resist
exonuclease activity by the inclusion of phosphorothioate linkages at
the terminal three residues. In the TD-TFO molecule (designated
A
AG30), the linker segment between the donor fragment and the TFO
domain consisted of the sequence 9TT9TT9, in which 9 indicates a 9-atom
polyethylene glycol linker (Spacer 9, Glen Research, Sterling, VA). The
specific TFO, designated AG30, has the sequence
5'-AGGAAGGGGGGGGTGGTGGGGGAGGGGGAG-3' and is designed to bind as a third
strand to bp 167-176 of the supFG1 gene. The donor domain
(A) consists of a 40-nt synthetic single-stranded DNA fragment
homologous to positions 121-160 of the supFG1 gene (5'-AGGGAGCAGACTCTAAATCTGCCGTCATCGACTTCGAAGG-3'). The scrambled sequence oligonucleotide, SCR30, has the same base composition as AG30
but differs at 12 positions: 5'-GGAGGAGTGGAGGGGAGTGAGGGGGGGGGG-3'.
Cells--
Construction of E. coli SY302
lacZ125(Am) recA56
hsdR2::Tn10 trp-49 has been described
previously (28). HeLa cells were maintained and grown by the National
Cell Culture Center (Minneapolis, MN) and were obtained as cell pellets
for extract preparation.
Proteins and Antibodies--
HsRad51 protein was purified from
E. coli DH10B (Life Technologies, Inc.) carrying plasmid
pEG932. Purification consisted of chromatography through Q Sepharose,
Bio-Gel-http, Mono-Q, and native DNA-cellulose. Other purification
details have been reported previously (29). Purification was documented
by SDS-polyacrylamide gel electrophoresis analysis, yielding a single
visualized band of 37 kDa following the DNA-cellulose purification
step. Purified HsRad51 protein was injected into rabbits to produce
high affinity polyclonal antibodies specific to the HsRad51 protein
(29).
XPA protein was produced using an Escherichia coli
expression vector (obtained from R. Wood) containing the human XPA
cDNA sequence, along with an N-terminal 6-histidine tag, in the
pET-15b plasmid (Novagen, Madison, WI). The protein was expressed in an E. coli expression strain, BL21(DE3) pLysS, as described by
Jones and Wood (30). Following expression, XPA was purified using immobilized metal-affinity chromatography (Talon resin;
CLONTECH, Palo Alto, CA) under native conditions.
Fractions were eluted with a buffer containing 20 mM Tris,
pH 8.0, 100 mM NaCl, and 100 mM imidazole.
Western analysis was used to determine specificity using antibodies to
the 6-His tag. Coomassie staining was used to determine purity. To
further confirm that the expressed and purified protein was the correct
species and present as soluble monomers in solution, XPA protein was
subjected to both mass spectrometry and HPLC size exclusion
chromatography/laser light scattering analysis. The results revealed a
monodispersed peak at a molecular mass of 36.8 kDa, indicating a
monomer of the correct size. Rabbits were immunized with the purified
XPA protein (100 µg/injection) to produce high affinity antibodies
specific to XPA.
Preparation of Cell-free Extract--
HeLa whole cell extract
was prepared as described previously (28). Briefly, HeLa cells were
washed with phosphate-buffered saline and resuspended in 0.01 M Tris-HCl, pH 7.9, 1 mM EDTA, 5 mM
DTT, followed by lysis using a Dounce homogenizer. The lysate was
diluted in four packed cell volumes of 0.05 M Tris-HCl, pH 7.9, 0.01 M MgCl2, 2 mM DTT, 25%
sucrose, 50% glycerol, and a protease inhibitor mixture (Sigma catalog
no. P8340). One packed cell volume of saturated
(NH4)2SO4 (0.33 g/ml of solution)
was added and then neutralized by 1 N NaOH, followed by
centrifugation at 15,000 × g for 20 min at 4 °C.
The pellet was resuspended in 0.025 M HEPES, pH 7.9, 0.1 M KCl, 0.012 M MgCl2, 0.05 mM EDTA, 2 mM DTT, 17% glycerol and was
dialyzed in the same buffer for 8-12 h. The sample was quick frozen in
liquid N2 and stored at
80 °C. The preparation
typically contained 15-20 mg of protein/ml.
In Vitro Assay for Recombination--
Reactions consisted of 3 µg of pSupFG1/G144C plasmid DNA, 3 µg each of selected
oligonucleotides (TFO, donor fragment, or both), 60 mM
NaCl, 2 mM
-mercaptoethanol, 3 mM KCl, 12 mM Tris-HCl, pH 7.4, 2 mM ATP, 0.1 mM each dNTPs, 2.5 mM creatine phosphate, 1 µg of creatine phosphokinase, 12 mM MgCl2,
0.1 mM spermidine, 2% glycerol, 0.2 mM DTT,
and 15-20 µl of cell-free extract in a 50-µl total reaction
volume. After incubating 2 h at 30 °C, the reactions were
terminated by the addition of 25 µM EDTA, 0.5% SDS, and
20 µg of proteinase K. After incubation at 37 °C for 1 h, the
plasmid DNA was isolated by phenol extraction and ethanol precipitation
and dissolved in 10 µl of H2O. 1 µl of the resulting sample was used to transform E. coli SY302 by
electroporation, as described (28), followed by growth of the cells on
indicator plates for genetic analysis of supFG1 gene
function as described previously (19).
Depletion of HsRad51 and XPA from Cell
Extracts--
Anti-HsRad51 or anti-XPA sera were adjusted to 1×
Tris-buffered saline (10 mM Tris-HCl, pH-7.5, 100 mM NaCl) and then incubated with pre-swollen Protein
A-Sepharose beads for 1 h at 4 °C. The beads were washed three
times with Tris-buffered saline buffer and incubated with 50 µl of
HeLa cell extract for 2 h on ice with gentle rotation. The
supernatant (HsRad51- or XPA-depleted extract) was recovered by
centrifugation and subsequently examined by Western blot and used in
the in vitro recombination assay.
Solubilization of RAD51 Immunoprecipitates--
After incubating
the cell extract with HsRad51 antibody-Protein A-Sepharose beads, the
beads were centrifuged and washed twice with 10 mM
phosphate buffer, pH 7.2. Then one bead volume of 100 mM
glycine, pH 2.5, and 3.5 M MgCl2 was added and
kept at 4 °C. After 1 h, the sample was centrifuged and the
supernatant was immediately dialyzed against a buffer containing 20 mM potassium phosphate, pH 7.4, 0.5 mM DTT, 0.2 mM EDTA, 10% glycerol, and 100 mM KCl. The
supernatant was directly used to supplement the depleted extracts for
the recombination assay without freezing.
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RESULTS |
Experimental Design--
The substrate for triplex-targeted
recombination was the plasmid vector pSupFG1/G144C, containing a
mutated version of the supFG1 amber suppressor tRNA gene,
supFG1-144, which has an inactivating G:C to C:G mutation at
bp 144. The function of this gene can be readily assayed in indicator
bacteria carrying an amber stop codon in the lacZ gene, and
so supFG1-144 is a useful reporter of recombination events
that revert the gene to the functional sequence. The
supFG1-144 gene also contains a 30-bp, G-rich site at the 3'
end of the gene to which the G-rich 30-mer TFO (AG30) can bind to form
a triple helix in the anti-parallel motif (Fig.
1).

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Fig. 1.
Schematic diagram depicting the binding of
the AG30 TFO to the supFG1-144 gene in the vector
pSupFG1/G144C. AG30 in this example is linked to a 40-nt donor DNA
fragment homologous to bp 121-160 of the supFG1-144 gene.
The G at position 144 of the donor fragment is intended to correct the
inactivating G:C to C:G mutation at bp 144 in the target gene. The
linker segment consists of the sequence, 9TT9TT9, where "9"
indicates a 9-atom polyethylene glycol linker. In some experiments, the
donor fragment and the TFO were used separately or were co-mixed but
not linked.
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In a strategy to promote targeted recombination, we designed a TD-TFO
molecule (A
AG30) in which the AG30 TFO is tethered to a donor DNA
fragment homologous to a region of the supFG1-144 target
gene via a mixed sequence linker (Fig. 1) (23). This arrangement
facilitates target site recognition via triple helix formation while at
the same time positioning the donor fragment for possible recombination
and information transfer. This strategy also is intended to exploit the
ability of a triple helix, itself, to provoke DNA repair, potentially
increasing the probability of recombination with the homologous donor
DNA. In the bifunctional A
AG30 molecule, the donor fragment, A,
consists of a single-strand of length 40 synthesized to be homologous
to positions 121-160 of the supFG1-144 gene except at
position 144, where the sequence matches that of the functional
supFG1 gene.
Triplex-induced Recombination in HeLa Cell-free Extracts--
In
previous work, we demonstrated the occurrence of triplex-induced
recombination upon transfection of A
AG30 into monkey COS cells
already carrying the pSupFG1/G144C vector as an episomal, SV40-replicon-based target (23). To investigate the mechanism of
triplex-induced recombination, in the present work we have tested the
ability of triplex formation to promote recombination within human
cell-free extracts.
Selected oligonucleotides were incubated with the target pSupFG1/G144C
vector in HeLa whole cell extracts supplemented with nucleotides and
ATP. Following a 2-h incubation, the plasmid vector DNA was isolated
and used to transform recA, lacZ(Amber) indicator E. coli to score for supFG1 gene function (Fig.
2). The results show that the
bifunctional oligonucleotide, A
AG30, was active in the extracts and
produced supFG1-144 gene reversion at a frequency of 45 × 10
5. Note that this effect occurred in the
extract and was not mediated by recombination in the indicator bacteria
because, without incubation in the extract, no recombinant products
were observed upon transformation of the A
AG30 sample into bacteria.
The A donor fragment was also somewhat active, as co-mixture of A plus
the pSupFG1/G144C plasmid led to a low level of supFG1
reversion, consistent with the ability of short fragments of DNA to
mediate recombination and marker rescue (31-33). However, the effect
of A
AG30 was 4-fold higher than that of A alone, demonstrating the
influence of the TFO domain and providing direct evidence for
triplex-induced recombination in vitro. By itself, however,
the TFO domain produced minimal reversion over background, indicating
the need for the sequence information provided by the A donor
fragment.

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Fig. 2.
Triplex-induced recombination in human
cell-free extracts. The pSupFG1/G144C plasmid DNA was
incubated in vitro with the indicated oligonucleotides in
the presence or absence of HeLa whole cell extracts. After 2 h,
the plasmid DNA was isolated and used to transform indicator bacteria
for genetic analysis of the supFG1 gene. A schematic diagram
of each oligonucleotide or oligonucleotide combination is presented to
the left. Plus sign (+) indicates that the
different oligonucleotides are mixed together but unlinked.
Minus sign ( ) indicates that the various
oligonucleotides are connected. The bars indicate the
frequency of blue colonies (representing recombinants) out of the total
colonies, with the actual count given to the right of each
bar. The results are cumulative data from three
experiments.
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Interestingly, the sample in which AG30 and the A donor oligonucleotide
were not linked but were simply co-mixed as separate molecules together
with the plasmid substrate also produced an increased level of
recombination, at a frequency of 40 × 10
5, almost as high as that produced by the
linked A
AG30. This result provides further evidence that a TFO can
stimulate recombination between a donor fragment and a target locus. In
addition, because the donor fragment in this case is separate from the
TFO, the result specifically demonstrates a role for the TFO in
stimulating recombination that is distinct from its ability to deliver
a tethered donor fragment to the target site.
In another sample tested, the A donor was linked to an oligonucleotide
segment designated SCR30, consisting of the same base composition as
AG30 but a scrambled sequence creating 12 mismatches. SCR30 does not
bind to the supFG1 gene and so does not form a triplex. It
also has no homology to the target gene. Linkage of SCR30 to the donor
fragment was found to actually inhibit recombination relative to the
donor fragment alone.
Role of HsRad51 in Triplex-induced Recombination--
The results
above establish that triplex-induced recombination can be reconstituted
in HeLa cell extracts in vitro. By using this in
vitro system, we sought to determine the role(s) of selected recombination and repair proteins in the pathway of triplex-induced recombination. HsRAD51 is a human recA homolog that functions in
homologous recombination and has been shown to mediate DNA pairing and
strand exchange reactions (34). To test the role of HsRAD51 protein in
this homologous gene conversion, we used a polyclonal rabbit
anti-HsRad51 antibody to deplete HsRad51 protein from the cell extract.
Successful depletion of HsRad51 from the extract was confirmed by
Western blot (Fig. 3). The depleted
extract was tested for the ability to support triplex-induced
recombination (Table I), both in the case
of the linked A
AG30 bifunctional molecule and in the case of the
co-mixed separate A and AG30 sample. Immunodepletion of HsRad51 was
found to substantially reduce the frequency of recombinants in both
cases. Control samples demonstrated that Protein A-Sepharose, in the
absence of the HsRad51 polyclonal antibody, had no effect.

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Fig. 3.
Immunodepletion of HsRad51 from the HeLa cell
extracts. Extract samples were immunodepleted using a polyclonal
HsRad51 antibody pre-mixed with Protein A-Sepharose beads. The
immunoprecipitate was removed by centrifugation, and the remaining
supernatant was examined by Western blot analysis. Samples were treated
as indicated.
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Table I
Effect of HsRad51 depletion on triplex-induced recombination in
cell-free extracts
The results represent the combined data from three independent
experiments.
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Limited Complementation by Addition of Purified HsRad51 to the
HsRad51-depleted Cell Extract--
Next, we tested the extent to which
the triplex-induced recombination in the HsRad51-depleted extracts
could be restored by the addition of purified, recombinant HsRad51
protein. Increasing amounts of HsRad51 protein were added to the
depleted extracts, and the recombination assay was carried out (Table
II). Even after the addition of a large
amount of HsRAD51 (up to 5 µg), only a portion of the triplex-induced
recombination activity was recovered. We hypothesized that the lack of
complementation by purified HsRad51 might reflect the removal from the
immunodepleted extracts of other factors physically associated with
HsRad51. To test this, we supplemented the immunodepleted extracts with
re-solubilized HsRad51 immunoprecipitate (Table II). Addition of the
solubilized immunoprecipitate to the depleted extracts was found to
almost completely restore the recombination activity, indicating that HsRad51 immunodepletion removes more than HsRad51 alone and that HsRad51 supplementation, by itself, cannot compensate for the loss of
the other factors. This result is not surprising in light of emerging
evidence that the recombination complex in human cells consists of
multiple factors, including Rad52, Rad54, XRCC2, and XRCC3, as well as
members of the RAD51 family, including Rad51B/Rad51L1, Rad51C/Rad51L2,
and Rad51D/Rad51L3 (35, 36).
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Table II
Effect of HsRad51 supplementation on triplex-induced recombination in
HsRad51-depleted cell-free extracts
The results represent the combined data from three independent
experiments.
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Addition of HsRad51 to the Complete Extract Boosts
Activity--
Increasing amounts of HsRAD51 (from 250 ng to 2 µg)
were added to non-depleted whole cell extracts, and triplex-induced
recombination was measured (Table III).
Both in the case of the linked donor fragment and TFO (A
AG30)
and the unlinked donor plus TFO (A+AG30), additional HsRad51 was found
to increase the frequency of the triplex-induced recombinants. In the
samples supplemented with amounts of HsRad51 in the lower range, there
was a minimal effect. However, at higher levels of supplementation,
increased yields of recombinants were seen. Hence, even though HsRad51,
by itself, cannot fully complement the activity of the immunodepleted
extracts, it can provide increased activity to otherwise complete whole cell extracts.
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Table III
Effect of HsRad51 supplementation on triplex-induced recombination in
cell-free extracts
The results represent the combined data from three independent
experiments.
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The Role of the Nucleotide Excision Repair Factor, XPA--
In
previous work studying TFO-induced mutagenesis and recombination within
SV40 vectors in human cells, we had obtained genetic evidence that the
ability of triplex formation to stimulate DNA metabolism is dependent
on the activity of the NER pathway (8, 10). To obtain direct
biochemical evidence in support of this proposed mechanism, we tested
the requirement for the NER damage recognition factor, XPA (26), in the
triplex-induced recombination in the HeLa cell extracts.
A rabbit polyclonal antibody was raised against recombinant human XPA
protein produced in E. coli and was found to recognize a
single protein in human cells of the expected size (data not shown).
Using this antibody, XPA was removed from the extracts by
immunoprecipitation. Depletion of XPA was confirmed by Western blot
analysis of the residual samples (Fig.
4). Depletion of XPA from the extracts
was found to substantially reduce the frequency of TFO-induced
recombination, whether or not the TFO was covalently linked to the
donor fragment (Table IV). With both the
A
AG30 and the A+AG30 samples, the depletion of XPA reduced the
frequency of recombinants to that mediated by the donor fragment alone
(Table I). Hence, the ability of a triple helix to stimulate
recombination depends on the XPA protein. This result supports the
hypothesis that the NER pathway can recognize a triple helix as a
"lesion," thereby provoking DNA metabolism that can lead to
recombination or mutation.

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Fig. 4.
Immunodepletion of XPA protein from the HeLa
cell extracts. Extract samples were immunodepleted using a
polyclonal XPA antibody pre-mixed with Protein A-Sepharose beads. The
immunoprecipitate was removed by centrifugation, and the remaining
supernatant was examined by Western blot analysis. Samples were treated
as indicated.
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Table IV
Effect of XPA depletion on triplex-induced recombination in cell-free
extracts
The results represent the combined data from three independent
experiments.
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Following XPA immunodepletion, we tested the ability of XPA to restore
the triplex-induced recombination activity (Table IV). The results show
that increasing amounts of XPA protein provide functional
complementation in the depleted extracts. These results establish a
direct role for XPA in mediating the ability of a triple helix to
stimulate recombination.
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DISCUSSION |
The work reported here establishes that triplex-induced
recombination can be detected in human cell-free extracts. A 30-mer TFO
that binds with high affinity to a portion of the supFG1
reporter gene within the pSupFG1/G144C vector was found to stimulate
recombination between the vector and a 40-nt donor fragment.
Recombination was induced both when the donor fragment was linked to
the TFO and when it was present as a separate, unlinked molecule. The
stimulation was determined to occur in the extracts and not in the
indicator bacteria because no recombinants were observed unless the
samples were incubated in the extracts. The donor fragment, by itself, was able to participate in recombination with the plasmid in the extracts, consistent with previous studies that have detected intermolecular recombination in similar mammalian cell extracts (37).
However, the present work establishes that such intermolecular recombination can be stimulated by third strand binding to one of the molecules.
Establishment of TFO-induced recombination allowed testing of the role
of selected factors in the process. Immunodepletion of HsRad51, a human
recombinase homologous to recA (34), from the extract reduced the yield
of induced recombinants, but purified HsRad51 did not fully compensate
for the immunodepletion. When the immunoprecipitate was re-solubilized
and used to supplement the depleted extracts, the induced recombination
activity was restored. These results suggest that the immunoprecipitate
contains factors in addition to HsRad51 that are essential for the
reaction. Such factors could include HsRad51-associated proteins that
are proposed to play a role in homologous recombination, such as
HsRad51a, HsRad51b, HsRad52, HsRad54, XRCC2, and XRCC3 (35, 36). On the
other hand, the addition of extra HsRad51 to the non-depleted extracts
produced an increased frequency of recombinants, suggesting that
HsRad51, itself, plays a critical role in the process. This result is
consistent with the observation that overexpressed HsRad51 can provide
a modest increase in the frequency of recombination in reporter gene
substrates in mammalian cells (38).
The NER damage recognition factor, XPA, was also found to play an
essential role in the triplex-induced recombination, as no induced
recombinants were seen in the extracts after XPA immunodepletion. Supplementation of the induced extracts with recombinant XPA protein restored the induced recombination activity. This result not only is
consistent with previous work showing that triplex-induced mutagenesis
and recombination are substantially reduced in human mutant cell lines
deficient in XPA (8, 10), it also demonstrates directly that XPA is
required for the process. Taken together, the data support a model in
which the oligonucleotide-mediated triple helix is recognized by XPA,
thereby initiating repair activity that can create recombinogenic
intermediates. Such intermediates may either be correctly repaired,
repaired with incorporation of mutation in an error-prone manner, or,
if homologous DNA is present, serve as substrates for repair by a
HsRad51-dependent pathway of homologous recombination.
In the extracts, the TFO was found to sensitize the plasmid to
recombination either with a linked donor fragment or with an unlinked
fragment, at approximately the same frequency. This ability of the TFO
to stimulate recombination between the target site and an unlinked
fragment is in contrast to a previous study examining TFO-induced
recombination in COS cells, in which the linked TD-TFO molecule was
found to be 4-fold more active than the mixture of the unlinked
molecules (23). In that study, the pSupFG1/G144C vector was
pre-transfected into COS cells, and the cells were transfected the next
day with the oligonucleotides. Two days later, the vector DNA was
isolated for analysis in indicator bacteria. We interpret this
difference between the previous cell study and the present work to
suggest that, in the in vitro reactions, the TFO and donor
fragment are present in adequate local concentrations whether or not
they are linked together covalently. Hence, in the extracts, the
ability of the TFO to deliver a linked donor fragment to the target
site and place it in juxtaposition with the region of homology is not
as important in promoting recombination as is the ability of the
triplex to provoke DNA metabolism. In contrast, in the cell
experiments, both properties of the TFO appear to be needed, although
it remains to be determined whether the need for linkage of the donor
to the TFO in cells can be overcome by increasing the efficiency of
donor fragment transfection. If so, it would allow a gene correction
strategy in which a TFO could be used in combination with larger donor
fragments, greater than those that can be synthesized in continuity
with the TFO. This would be advantageous, since previous studies
examining recombination between episomal or chromosomal targets and
transfected DNAs in mammalian cells have consistently shown that
fragments in the range of 500 bp or larger produce higher levels of
recombination than do short fragments in the size range used here
(31-33, 39, 40).
Overall, the work reported here demonstrates that triplex-induced
recombination can be detected in human cell free extracts, and it
provides insight into the underlying mechanism by identifying critical
roles for HsRad51 and XPA. The results suggest a pathway of
triplex-induced recombination that depends on NER and on homologous recombinational repair of NER-generated intermediates. The ability to
reconstitute TFO-induced recombination in vitro should serve as a basis for further elucidation of the manner in which triplex formation can provoke DNA metabolism, and may thereby guide refinements in strategies to use TFOs to promote targeted genetic changes in human cells.