From the Laboratory of Molecular Genetics, NIA,
National Institutes of Health, Baltimore, Maryland 21224 and
§ Imperial Cancer Research Fund Laboratories, Institute of
Molecular Medicine, University of Oxford, John Radcliffe Hospital,
Oxford OX3 9DS, United Kingdom
Received for publication, July 28, 2000, and in revised form, November 2, 2000
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ABSTRACT |
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Bloom syndrome and Werner syndrome are genome
instability disorders, which result from mutations in two different
genes encoding helicases. Both enzymes are members of the RecQ family
of helicases, have a 3' Despite the obvious importance of genetic stability, the mammalian
genome has an abundance of sequences that are potentially destabilizing. Elements that can form non-duplex structures, such as
DNA triple helices (1, 2), G quartets (3-5), hairpins (6, 7), and
cruciforms (8), all have the capacity to interfere with transcription
and replication. Moreover, many studies have described the role of
these elements in DNA rearrangements such as deletions, sister
chromatid exchanges, homologous and illegitimate recombination events,
etc. (reviewed in Refs. 9 and 10). With such an array of provocative
sequence elements, it seems likely that cells would have developed the
capacity for controlling the potential of these sequences for genome destabilization.
Some insight into the nature of the enzymology involved in the
maintenance of genetic integrity has come from the study of two
"genome instability" disorders, Bloom and Werner syndrome. Patients
with Werner syndrome (WS)1
are characterized by premature aging (11) and a high incidence of
certain cancers. They have elevated frequencies of spontaneous deletion
mutations in the HPRT gene (12) and show a variety of
karyotypic abnormalities including inversions, translocations, and
chromosomal losses (13, 14). The mutant gene in WS has been identified
as a member of the RecQ helicase family, and the protein is a
3'-5'-helicase (15, 16) and a 3'-5'-exonuclease (17-19). A role in
replication is implied by the demonstration that the WRN helicase
interacts with and is stimulated by the human replication protein A
(RPA) (20, 21). The protein has been recovered from cells in a
replication complex (22), and the Xenopus homologue, FFA-1,
is required for the formation of replication foci (23). In addition,
recent work (24-26) suggests a role for the WRN helicase in telomere
maintenance. Finally, the chromosomal instability of WS cells suggests
that the enzyme plays an anti-recombinogenic role in wild type cells.
Bloom syndrome (BS) patients display a variety of symptoms, among them
growth retardation, immunodeficiency, sun sensitivity, and a marked
propensity to develop cancers of all varieties (27-29). At the
cellular level there is an increased frequency of spontaneous mutation
(30) and somatic recombination (31). Elevated levels of sister
chromatid exchanges have long been associated with the disorder (32).
As with Werner syndrome there is an enhanced frequency of large
deletion mutations in the HPRT gene (33). The mutant
gene in Bloom syndrome patients also encodes a RecQ family
helicase with 3'-5' polarity although it lacks an exonuclease activity
(34-36). It is found associated with other proteins involved in DNA
metabolism (37), including RPA (38). The marked chromosomal instability
of BS cells and recent biochemical evidence (39) argue for an
anti-recombinogenic activity for the wild type BLM protein as well.
Whereas the defining function of these and all other helicases is the
unwinding of duplex nucleic acid, recent work indicates that the BLM
and WRN enzymes are active on alternate DNA structures. In
particular, they can unwind tetrahelical structures that can form in
stretches of G-rich DNA (G DNA) (40, 41). Such sequences are widely
distributed in the genome (5) and are found, among other places, at
telomeres (42-44) and in the triplet repeats such as the CGG
associated with Fragile X syndrome (45). They can form under
physiological conditions and inhibit DNA synthesis in vitro
(46). It has been suggested by many authors that these complexes,
appearing as the result of single strand exposure during replication,
recombination, or transcription, could play a causal role in
genomic rearrangements, including strand exchange, strand slippage, and
the size changes seen in the triplet expansion disorders. The
possibility that the genomic instability of the WS and BS disorders
might be due to a failure to resolve alternate DNA structures has
generated considerable interest (47).
The triple helix is another alternate DNA structure that can be formed
by sequences that are widely distributed throughout the human genome
(1). Triplexes have been known for many years (Ref. 48 and reviewed in
Refs. 49 and 50) and are generated when a third strand lies in the
major groove of duplex DNA. They occur most readily on
polypurine:polypyrimidine sequences, and the third strand may be
composed of either pyrimidines or purines, with the most stable
resultant structure dictated by the specific sequence (51). They can
form when an appropriate sequence partially melts with one of the
single strands folding back to complex with the adjacent duplex (52)
and have been demonstrated in chromosomes and nuclei (53, 54). These
complexes can also be formed by oligonucleotides directed against
specific target sequences. This has prompted their consideration as
gene-targeting reagents (55-58).
Although there is a 4-decade literature on triple helices and a
considerable interest in the role of helicases in DNA transactions, there have been remarkably few studies on the activity of these enzymes
on triplexes. The DDA protein of phage T4 can unwind a triplex
(59), as can the SV40 T antigen (60). However, there has been no report
of the activity of human helicases with these substrates. In this
report we describe the activity of the BLM and WRN helicases on a
pyrimidine-purine:pyrimidine triple helix and corresponding duplex structures.
Proteins--
Recombinant hexahistidine-tagged human WRN protein
was overexpressed in Sf9 insect cells and purified as described
elsewhere (20). Recombinant hexahistidine-tagged human BLM protein was overexpressed in Saccharomyces cerevisiae and purified as
previously described (61). UvrD (DNA helicase II) was kindly provided
by Dr. Steven Matson (University of North Carolina, Chapel Hill). T4
polynucleotide kinase was obtained from New England Biolabs.
Oligonucleotides, Nucleotides, and DNA--
The following
oligonucleotides were prepared: TC30,
5'-TCTTTTTCTTTCTTTTCTTCTTTTTTCTTT; TC30 5' tail,
5'-TGACGCTCCGTACGA-TC30; TC30 3' tail, 5'TC30-TCACGCTCCGTACGA. The
cytosines in the TC30 portion were 5 methylcytosines. This modification
stabilizes the triplexes at physiological pH (62). The 28-mer
oligonucleotide 5'-TCCCAGTCACGACGTTGTAAAACGACGG-3' was from Life
Technologies, Inc. M13mp18 ssDNA was from New England Biolabs.
Ribonucleotides, deoxyribonucleotides, and ATP Triplex Substrates--
The plasmid psupF5 contains a duplex
sequence that serves as a target for TC30 and related oligonucleotides.
Cleavage of the plasmid with NdeI released fragments of 4 and 0.6 kilobase pairs. The triplex site lies 1800 bases from one end
of the large fragment. Triplexes were prepared by incubation of 3 pmol
of 5'-32P-labeled oligonucleotide (based on the molecular
weights of the reaction components) overnight at room temperature with
6 pmol of NdeI-cleaved plasmid in a buffer containing 33 mM Tris acetate, pH 5.5, 66 mM KOAc, 100 mM NaCl, 10 mM MgCl2, and 0.1 mM spermine. The complexes were then separated from unbound
oligonucleotide by gel filtration chromatography using Bio-Gel A-5M
resin. The 6-base pair duplex extension substrate was prepared as above
by incubation of the 3'-tailed third strand with plasmid that was cleaved with BglII and filled in by incubation with Klenow
polymerase. This construction placed the triplex at the extreme end of
the duplex DNA with a 6-base pair duplex extension. The 3'-tailed "blunt triplex" was prepared by incubation of a 30-mer duplex oligonucleotide with the 3'-tailed third strand. This duplex contained the triplex region without extensions.
Tm Determination--
A triplex was prepared by
incubation of the 3'-tailed third strand with a duplex 30-base pair
oligonucleotide consisting of the triplex target sequence. The triplex
was diluted into the buffer used for the helicase assays (see below),
and the thermal stability was determined in a PerkinElmer Life Sciences
spectrophotometer with a Peltier controlled thermal unit attached.
Restriction Protection--
The triplex target site overlaps an
XbaI site, which is blocked by triplex formation. 2 µg of
psupF5 were incubated overnight with 2 µM of the 5', or
3', or blunt TC30 oligonucleotides in the buffer described above. The
complexes were then separated from free oligonucleotide by ethanol
precipitation from 2.5 M ammonium acetate. They were
suspended in 50 mM NaCl, 10 mM Tris-HCl, pH
7.0, 10 mM MgCl2, 1 mM
dithiothreitol, 0.1 mM spermine, 100 µg/ml bovine serum
albumin and digested with XbaI and PstI at 37 °C. Although in these experiments the triplexes were formed on
circular plasmids prior to restriction digestion, triplex formation was
equally efficient on linear DNA.
Duplex Helicase Substrates--
To prepare the TC30 duplex DNA
substrate, the TC30 3' tail oligonucleotide was 5'-end-labeled and
annealed to a 30-mer oligonucleotide complementary to TC30 (TC30'). The
annealing mixture contained 50 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10 pmol TC30', and 4 pmol of
32P-labeled TC30. The annealing mixture was incubated at
90 °C for 5 min and allowed to cool to room temperature over the
course of 3 h. The forked duplex substrate was prepared by
annealing a 50-mer that contained the 30-base complement of TC30 and a
20-base noncomplementary 5' tail. The labeled annealed DNA duplexes
were electrophoresed on a 12% polyacrylamide (19:1,
acrylamide:bisacrylamide) 1× Tris borate, EDTA gel, and the duplex
fragment was excised. The DNA fragment was purified using the QIAEX II
polyacrylamide gel extraction kit (Qiagen) and eluted with 10 mM Tris-HCl, pH 8.0. The 28-base pair M13mp18 partial
duplex substrate was constructed with a 28-mer complementary to
positions 6296-6323 in M13mp18. The 28-base pair M13mp18 partial
duplex substrates were constructed as described previously (63) with
the following modifications. The 28-mer oligonucleotides were labeled
at their 5' ends using T4 polynucleotide kinase and
[ Helicase Assays--
Helicase assay reaction mixtures (20 µl)
contained 40 mM Tris borate, pH 7.4, 5 mM
MgCl2, 5 mM dithiothreitol, 2 mM
ATP (or the indicated nucleoside 5' triphosphate), and the indicated
amounts of WRN or BLM helicase. The amount of the TC30 triplex or
3'-tailed duplex molecules in the reaction mixtures was ~15 fmol. The
amount of the M13 partial duplex molecules was 4 fmol. Reactions were initiated by the addition of WRN or BLM protein and incubated at
24 °C for 30 min. At the end of the incubation, a 10-µl aliquot of
loading buffer (40% glycerol, 0.9% SDS, 0.1% bromphenol blue, 0.1%
xylene cyanol) was added to the mixture, and the products of helicase
reactions were resolved on nondenaturing polyacrylamide gels (12%
acrylamide, 40 mM Tris acetate, pH 5.5, 5 mM
MgCl2, 25% glycerol) at 4 °C. The 30-mer 3'-tailed
"blunt triplex" substrate was resolved on a 10% polyacrylamide gel
containing 5% glycerol. An excess of unlabeled third strand was added
to the reaction mixtures at the end of the reaction to preclude
reassociation of the unwound labeled third strand with the duplex.
Radiolabeled DNA species in polyacrylamide gels were visualized using a
PhosphorImager or film autoradiography and quantitated using the
ImageQuant software (Molecular Dynamics). The percent helicase
substrate unwound was calculated by the following formula: % Displacement = 100 × P/(S + P). P is the product and S is the
substrate. The values for P and S have been
corrected after subtracting background values in the no enzyme and
heat-denatured controls, respectively. Helicase data shown are
representative of at least three independent experiments.
Enzyme Preparations--
The recombinant WRN and BLM proteins were
characterized by standard helicase and ATPase assays (not shown). The
activity of the WRN enzyme with a duplex substrate, formed by the
hybridization of a 28-base oligonucleotide to single strand circular
M13, was measured. This substrate was almost completely unwound at
higher concentrations of enzyme (30-60 nM) (data not
shown). Unwinding of the M13 partial duplex by the BLM enzyme was
virtually complete at enzyme concentrations Triplex Substrates--
We prepared triple helices with a
pyrimidine motif third strand with 15 nucleotide 3' or 5' single strand
tails or with no tail. The sequences of the target duplex and the third
strands are shown in Fig. 1A.
These triplexes were stable at physiological pH. In the buffer used for
the helicase assays the Tm of the TC30 triple helix was
57.9 °C, and the Tm of the underlying duplex was 62.9 °C.
Duplex unwinding by both helicases is dependent on a single strand 3'
tail. All the triplexes used in these experiments were based on blunt
duplexes, eliminating the possibility that triplex unwinding was due to
the disruption of the underlying duplex.
Restriction Site Protection by Triplex Formation--
Triplex
formation on the target sequence in psupF5 partially occludes an
XbaI site. As shown in Fig. 1B protection of the site from XbaI cleavage could be demonstrated following
triplex formation with the oligonucleotides, whereas cleavage by
PstI, whose sites are not near the triplex target, was
unaffected. Another plasmid (psupF12), which does not have the TC30
triplex target sequence, was also incubated with the TC30
oligonucleotides. However, as expected, there was no protection from
XbaI digestion.
Activity of WRN and BLM Helicase on Triplexes with Different
Third Strands--
WRN and BLM helicases are most active on duplex DNA
with 3' single strand tails (16,
40).2 We asked if the
helicases would be active with DNA triplexes with a 3', 5', or no
single strand tail as depicted in Fig. 1A. Each substrate
was incubated with increasing amounts of the individual enzymes. As
shown in Fig. 2A, the WRN was
active against the 3' tail triplex with increasing amounts of triplex
unwinding as a function of enzyme concentration. The extent of
unwinding reached a plateau at 28 nM protein with 50% of
the 3' tail substrate unwound. There was no significant unwinding of
the blunt and 5' tail substrates.
Similar data were obtained with the BLM helicase with only the
3'-tailed substrate unwound (Fig. 2B). Unwinding was
measurable at the lowest levels of enzyme, and more than 90% of the
substrate was unwound at higher enzyme concentrations.
Nucleotide Preference of WRN and BLM Helicases in the Triplex
Unwinding Assay--
The activity of helicases is dependent on a
nucleoside triphosphate (65). We examined the nucleotide preference of
the WRN enzyme in unwinding the 3' tail triplex substrate (data not
shown). 21 nM enzyme was incubated with the substrate in
the presence of the ribo- and deoxyribonucleoside triphosphates.
Unwinding was observed with dCTP (27%), CTP (50%), dATP (50%), and
ATP (26%). There was no significant activity in the presence of the
other triphosphates. The BLM helicase was less discriminating as
extensive unwinding (80-90%) was observed with all triphosphates with
the exception of UTP (71%) and TTP (26%). When ATP Triplex Unwinding by WRN and BLM Helicases Does Not Require a
Fork--
The substrate employed in the preceding experiments
presented different structural elements to the enzymes. These included the single strand tail, the triplex region, and the duplex adjacent to
the triplex. The nature of the junction between single strand tails and
duplex substrate of different helicases is well known to affect enzyme
activity (66-68). We wanted to know if the activity of the BLM and WRN
helicases would also be affected by the nature of the junction between
the single strand and the triplex. Accordingly, we prepared two
additional substrates. The first was based on the substrate used in the
previous experiments. The plasmid was linearized at the
BglII site (Fig. 1) and the 4-base 5' overhang filled in by
Klenow polymerase. This was incubated with the 3'-tailed third strand
to produce a triplex with a 6-base duplex extension. This substrate
maintained a long duplex region as before, and a junction of a
3'-tailed third strand, a triplex, and a short duplex extension. Both
enzymes unwound more than 85% of this substrate (Fig.
3).
The second substrate consisted of the 3'-tailed third strand complexed
to an oligonucleotide duplex such that there were no duplex extensions
beyond the triplex (3'-tailed blunt triplex). This structure was
unwound by the WRN enzyme, although less efficiently than the previous
substrate (50% unwound at 56 nM WRN helicase) (Fig.
4A). The BLM enzyme was more
active with this substrate with 80% unwound at 32 nM
enzyme (Fig. 4B). There was no activity with either enzyme
in the presence of ATP Activity of Bloom and Werner Helicases on a Duplex With and
Without a Fork--
It was of interest to know if the two enzymes
would unwind a duplex substrate containing the same strand used in the
triplex experiments. The 45-nucleotide fragment used as the third
strand in the triplex experiments was hybridized with a 30-base
oligonucleotide complementary to the triplex region. This produced a
duplex with the same 3' tail as in the triplex experiments. An amount
of substrate equivalent to that used in the triplex experiments
("Materials and Methods") was incubated with increasing amounts of
each enzyme as before. The results of the titration with the WRN enzyme
(Fig. 5A) showed little or no
unwinding at concentrations that had displaced substantial amounts of
triplex in the earlier experiments (compare with Fig. 2). There was
detectable, but weak, unwinding by the BLM enzyme (9%) at the highest
concentration of enzyme tested (64 nM) (Fig.
5B). The poor activity of the two enzymes was not a
peculiarity of this particular substrate since UvrD unwound the
substrate completely (not shown).
We then asked if the same duplex could be unwound when associated
with a fork rather than the single strand tail (see "Materials and
Methods"). In contrast to the preceding experiment, both enzymes unwound the forked substrate, with the plateau value (85%) reached by
the WRN enzyme at 7 nM and 90% unwound by the BLM enzyme
at 32 nM (Fig. 6,
A and B).
The BLM and WRN enzymes are DNA-stimulated ATPases with
3'-5'-helicase activity. They are active against short duplexes on the
M13 single strand substrate, unwind longer duplex substrates in the
presence of RPA (20), (38), and are potently inhibited by minor groove
binding compounds such as netropsin and distamycin (69).
The structure and reaction requirements for the triplex unwinding by
the WRN and BLM helicases are in accord with the previous data from
experiments with duplex substrates with regard to the requirement for a
3' single strand tail (21). The nucleotide preference analysis
indicated that the triplex unwinding was supported by both adenosine
and cytosine triphosphates similar to that found with duplex substrates
(21). The utilization of nucleoside triphosphates other than ATP or
dATP has been reported for other helicases (65).
Interactions between Helicases and Substrates--
There are
several models for DNA unwinding by helicases (66, 67, 70-72), based
on activity with duplex substrates. In some versions helicase function
is seen as an active process in which the enzyme interacts with both
single- and double-stranded DNA at a junction, with the precise nature
of the interactions and the subsequent events subject to much debate
(67, 71). The preference of the BLM and WRN helicases for the forked
duplex or the triplex substrates suggests an active engagement by the enzymes with the junction of the single strand tail and the fork or
triplex. Like the BLM and WRN enzymes, a preference, if not a
requirement, for a forked substrate is a feature shared by other helicases. This is often taken as an indication of a replicative role
for the enzyme (66, 73-82). A possible linkage between helicase structure and substrate preference has been proposed in a recent study
that suggests that the distinction between forked and non-forked substrates reflects the exclusion of the forked strand from the central
channel of hexameric helicases (83). This proposal is relevant to the
enzymes studied here as the BLM helicase has been shown to be a hexamer
(61).
The results of our experiments with the triplex and duplex substrates
can be summarized as follows: both enzymes unwound triplexes without a
requirement for single strand:double strand forks at the junction of
the single strand tail and the triplex region. On the other hand, the
enzymes were weakly active with a nonforked duplex with a single strand
tail but were both very active with the forked version of the same
duplex substrate. Thus it would seem that the stimulatory influence of
the fork junction seen with duplex substrates is unnecessary with the
triplex substrates. This suggests that some feature of the triplex can
compensate for the absence of the fork. In a triplex the third strand
lies in the major groove of an intact duplex. Perhaps an interaction with the underlying duplex as well as the third strand replaces the
interaction with the "other" strand at a fork. Alternatively, if
the WRN enzyme, like the BLM, proves to be hexameric, then the channel
exclusion hypothesis of Kaplan (83) may also explain the similar
activity with the fork and triplex substrates.
Although the enhanced activity of the WRN and BLM enzymes seen with
forked duplex substrates is consistent with reports on other helicases,
we and others (16, 20, 21, 35, 38, 84) have observed activity by both
enzymes on duplex substrates that do not have forks. With one exception
(21) all these studies employed the popular M13 partial duplex
substrate. The resolution of this apparent contradiction may reflect
the actual DNA structure required for initiation of unwinding, as well
as a particular feature of the M13 substrate. Entry into the duplex
region of a tailed (not forked) substrate by BLM and WRN proteins may
be dependent on the fraying of the duplex end, giving the enzyme an
opportunity for a requisite interaction with the complementary strand.
The M13 substrate has a long single strand "loading" region to
which enzyme can be bound even if not engaged in actual unwinding. The
WRN and BLM enzymes are distributive enzymes (20, 38). Should an active
enzyme molecule be released from the substrate prior to initiating,
then, with the M13 substrate, the released enzyme could be replaced by
a one-dimensional movement of already bound enzyme. Thus the local
concentration of replacement enzyme would be much greater with the M13
substrate and the probability of initiation much higher than for the
short tailed duplex substrate, which would require replenishment of
bound enzyme from solution. One prediction of this hypothesis is that
duplex substrates with short single strand tails, the kind examined in
Fig. 5, should be unwound if the enzyme concentration in solution is
high enough. In experiments to be presented elsewhere, we have found
that these substrates can indeed be unwound at enzyme concentrations a
hundred-fold higher than required to unwind the corresponding forked
substrate.3 This result
demonstrates that there is not an absolute block to unwinding the
tailed substrates and reconciles the apparent contradiction between the
results with M13 and the tailed duplex substrates presented here.
Genomic Instability in BS and WS Cells--
Specific functions for
WRN and BLM in vivo have yet to be identified, although
roles in replication and as suppressors of recombination are consistent
with the characteristics of cells derived from affected individuals and
recent biochemical analyses (22, 23, 85). It has been proposed that
these helicases unwind alternate DNA structures that might form during
replication and block fork progression (37, 39, 47). In that light it is of particular interest that sequences with triplex forming potential
are widely distributed in the human genome (1, 86-88). The possibility
that triplexes form in vivo is supported by the reports of
mammalian proteins that bind specifically to them (89-92) and to the
polypyrimidine (93) and polypurine single strands (94) that would
appear when the complementary strands were engaged in alternate structures.
The loss of the helicase activity of the BLM and WRN enzymes is
generally seen as the basis of the genome instability that is common to
both syndromes. Triplex structures are known to be recombinogenic (95,
96), and recently it has been shown that triplexes can arrest
progression of Holliday junctions in a model system (97). These, like
arrested replication forks, are likely to be susceptible to breakage.
In light of the data presented here, it seems reasonable to suggest
that triplex structures might be more persistent in WS and BS cells and
could contribute to the genomic instability characteristic of both syndromes.
5' polarity, and require a 3' single strand
tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4
tetraplexes, prompting speculation that failure to resolve these
structures in Bloom syndrome and Werner syndrome cells may contribute
to genome instability. The triple helix is another alternate DNA
structure that can be formed by sequences that are widely distributed
throughout the human genome. Here we show that purified Bloom and
Werner helicases can unwind a DNA triple helix. The reactions are
dependent on nucleoside triphosphate hydrolysis and require a free 3'
tail attached to the third strand. The two enzymes unwound triplexes
without requirement for a duplex extension that would form a fork at
the junction of the tail and the triplex. In contrast, a duplex formed
by the third strand and a complement to the triplex region was a poor
substrate for both enzymes. However, the same duplex was readily
unwound when a noncomplementary 5' tail was added to form a forked
structure. It seems likely that structural features of the triplex
mimic those of a fork and thus support efficient unwinding by the two helicases.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
S were from Roche
Molecular Biochemicals.
-32P]ATP and annealed to M13mp18 single-stranded DNA
circles. Partial duplex DNA substrates were purified by gel filtration
column chromatography using Bio-Gel A-5M resin (Bio-Rad).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
16 nM. The
specific ATPase activities of WRN and BLM enzymes using M13mp18
single-stranded DNA as the DNA effector were determined to be 219 ± 14 and 1163 ± 358 min
1,
respectively, consistent with previously published values (35, 64).
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Fig. 1.
Triplex DNA substrates used in these
experiments. A, the sequence of the TC30
oligonucleotide and the different triplex substrates. The target was
embedded in the supF5 plasmid (flanking sequences indicated as
- - -). B, restriction enzyme protection by triplex
formation. Triplexes were formed with the 3' tail (lane 1),
the blunt (lane 2), or 5' tail (lane 3)
oligonucleotide on the supF5 plasmid with the triplex target. These
complexes, as well as the supF5 plasmid without an oligonucleotide
(lane 4) or the supF12 plasmid, which lacks the TC30 triplex
site, with the 3'-tailed oligonucleotide (lane 5), were
digested with PstI and XbaI. Plasmid digested
with PstI alone is also shown (lane 6). Triplex
formation protects the XbaI site. An arrow
denotes the 4000-base pair fragment with the triplex site. 0.5 µg of
each sample was loaded on each lane. A HindIII digest of
phage DNA was run as a molecular weight marker.
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Fig. 2.
Unwinding of triplex DNA by WRN
(A) and BLM (B) helicases. A
triplex DNA substrate with a 3' single-stranded tail (filled
circles), 5' single-stranded tail (open circles), or no
tail (×) was incubated with the indicated concentrations of WRN
protein under the standard helicase conditions described under
"Materials and Methods." Incubation was at 24 °C for 30 min. The
inset shows the gel pattern with the 3'-tailed substrate.
Heat-denatured control, filled triangle.
S was
substituted for the other triphosphates there was no unwinding with
either enzyme.
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Fig. 3.
Activity of the WRN and BLM helicases on a
triplex with a 6-base pair extension. Heat-denatured substrate,
filled triangle.
S. These experiments showed that both enzymes
could unwind the triplex without a requirement for a single
strand:double strand fork at the junction of the third strand and the
triplex and without regard for the presence or absence of an associated
long duplex region.
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Fig. 4.
Activity of the WRN and BLM enzymes with a
triplex with no duplex extension. A, WRN helicase;
B, BLM helicase.
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Fig. 5.
Activity of the WRN and BLM helicases on the
TC30 duplex. A TC30 duplex DNA substrate with a 3' single-stranded
tail was incubated with the indicated concentrations of WRN protein
(A) or BLM protein (B) under the standard
helicase conditions described under "Materials and Methods."
Incubation was at 24 °C for 30 min. Heat-denatured control,
filled triangle.
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Fig. 6.
Activity of WRN and BLM on a forked duplex
substrate. A TC30 duplex DNA substrate with non-complementary 3'
and 5' tails was incubated with the indicated concentrations of WRN
(A) or BLM (B) enzyme. Heat-denatured control,
filled triangle.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Nitin Puri for oligonucleotide synthesis and analysis of the thermal stability of the triplex and duplex substrates. We also thank Dr. Qin Yang for useful comments.
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FOOTNOTES |
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* 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: LMG, Box 1, GRC, 5600 Nathan Shock Dr., Baltimore, MD 21224. Tel.: 410-558-8565; Fax: 410-558-8157; E-mail: seidmanm@grc.nia.nih.gov.
Published, JBC Papers in Press, November 10, 2000, DOI 10.1074/jbc.M006784200
2 R. M. Brosh, Jr., A. Majumdar, S. Desai, I. D. Hickson, V. A. Bohr, and M. M. Seidman, unpublished results.
3 R. M. Brosh, Jr., J. A. Sommers, C. von Kobbe, P. Karmakar, P. L. Opresko, I. I. Dianova, G. L. Dianov, and V. A. Bohr, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
WS, Werner syndrome;
BS, Bloom syndrome;
RPA, replication protein A;
ATPS, adenosine
5'-O-(thiotriphosphate).
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