UNWINDING OF A DNA TRIPLE HELIX BY THE WERNER AND BLOOM
SYNDROME HELICASES
Robert M. Brosh Jr.1, Alokes Majumdar1, Shital Desai1, Ian D. Hickson2, Vilhelm
A. Bohr1, and Michael M. Seidman1,3
1. Laboratory of Molecular Genetics, National Institute on Aging, National
Institutes of Health, Baltimore, MD 21224
2. Imperial Cancer Research Fund Laboratories, Institute of Molecular
Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS,
United Kingdom
3. Corresponding author, LMG, Box 2, GRC, 5600 Nathan Shock Dr.,
Baltimore, MD 21224 email: [email protected], 410-558-8565,
FAX 410-558-8157
JBC Papers in Press. Published on November 10, 2000 as Manuscript M006784200 by guest on A
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ABSTRACT
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’->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
BS and WS 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 were
dependent on nucleoside triphosphate hydrolysis, and required 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.
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INTRODUCTION
Despite the obvious importance of genetic stability, the mammalian
genome has an abundance of sequences that are potentially destabilizing.
Elements which 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 (9,10)). 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) 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),
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and the Xenopus homologue, FFA-1, is required for the formation of replication
foci (23). In addition, recent work suggests a role for the WRN helicase in
telomere maintenance (24-26). 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.
While 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
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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 (48) (reviewed in (49,50), 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 four 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
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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.
MATERIALS AND METHODS
Proteins Recombinant hexa-histidine tagged human WRN protein was
overexpressed in Sf9 insect cells and purified as described elsewhere (20).
Recombinant hexa-histidine tagged human BLM protein was overexpressed in S.
cerevisiae and purified as previously described (61). UvrD (DNA helicase II) was
kindly provided by Dr. Steven Matson (University of North Carolina at 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 methyl cytosines. This modification
stabilizes the triplexes at physiological pH (62). The 28-mer oligonucleotide 5’-
TCCCAGTCACGACGTTGTAAAACGACGG-3’ was from Gibco-BRL. M13mp18
ss DNA was from New England Biolabs. Ribonucleotides, deoxyribonucleotides,
and ATPγS were from Boehringer Mannheim.
Triplex substrates The plasmid psupF5 contains a duplex sequence that serves
as a target for TC30 and related oligonucleotides. Cleavage of the plasmid with
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NdeI released fragments of 4 and 0.6 kb. The triplex site lies 1800 bases from
one end of the large fragment. Triplexes were prepared by incubation of 3
pmoles of 5’ 32P-labeled oligonucleotide (based on the molecular weights of the
reaction components) overnight at room temperature with 6 pmoles 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 determined in a Perkin Elmer
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 oligos in the buffer described above. The
complexes were then separated from free oligo by ethanol precipitation from 2.5
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M ammonium acetate. They were suspended in 50 mM NaCl, 10 mM Tris-HCl,
pH 7.0, 10 mM MgCl2, 1 mM DTT, 0.1 mM spermine, 100 µg/ml BSA and
digested with Xba I and Pst I 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
32P-labeled TC30. The annealing mixture was incubated at 90 oC 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 non-complementary 5’ tail. The labeled
annealed DNA duplexes were electrophoresed on a 12% polyacrylamide (19:1,
acrylamide: bisacrylamide) 1X Tris-borate, EDTA gel and the duplex fragment
was excised. The DNA fragment was purified using the QIAEX II polyacrylamide
gel extraction kit (Quiagen) and eluted with 10 mM Tris-HCl (pH 8.0). The 28-bp
M13mp18 partial duplex substrate was constructed with a 28-mer
complementary to positions 6296 to 6323 in M13mp18. The 28 bp M13mp18
partial duplex substrates were constructed as previously described (63) with the
following modifications. The 28-mer oligonucleotides were labeled at their 5’
ends using T4 polynucleotide kinase and [γ32P]ATP and annealed to M13mp18
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ssDNA circles. Partial duplex DNA substrates were purified by gel filtration
column chromatography using Bio-Gel A-5M resin (Bio-Rad).
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 approximately 15 fmoles. The amount of the M13 partial
duplex molecules was 4 fmoles. Reactions were initiated by the addition of WRN
or BLM protein and incubated at 24 oC for 30 min. At the end of the incubation, a
10 µl aliquot of loading buffer (40% glycerol-0.9% sodium dodecyl sulfate (SDS)-
0.1% bromophenol 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 oC. 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 x 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.
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Helicase data shown are representative of at least three independent
experiments.
RESULTS
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 >16 nM. The specific ATPase activity of WRN and
BLM enzymes using M13mp18 ssDNA as the DNA effector were determined to
be 219 + 14 min-1 and 1163 + 358 min-1 respectively, consistent with previously
published values (35,64).
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, while 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,
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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, while 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 oligos. 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), (our unpublished results). We asked if the helicases would
be active with DNA triplexes with a 3’, or 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
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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 ATPγS was substituted for the other triphosphates there
was no unwinding with either enzyme.
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 effect 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
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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 ATPγS. 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.
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
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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 which 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 6A, B).
DISCUSSION
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
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(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 (67,70-72) (66),
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
suggest 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 (73,74) (75), (76,77), (66,78,79), (80)((81), (82). A possible linkage
between helicase structure and substrate preference has been proposed in a
recent study which 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
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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 non-forked 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 he 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 have observed activity by both enzymes on duplex substrates which do
not have forks (16,20,21,84) (35,38). 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
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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, of 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 (Brosh et al., in preparation). 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
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triplex forming potential are widely distributed in the human genome (1,86,87)
(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.
FIGURE LEGENDS
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) oligo on the supF5 plasmid
with the triplex target. These complexes, as well as the supF5 plasmid without
an oligo (lane 4) or the supF12 plasmid, which lacks the TC30 triplex site, with
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the 3’ tailed oligo (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 ug of
each sample was loaded on each lane. A HindIII digest of phage λ DNA was run
as a molecular weight marker.
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 (x), was incubated with the indicated concentrations of
WRN protein under the standard helicase conditions described in Materials and
Methods. Incubation was at 24oC for 30 minutes. The inset shows the gel
pattern with the 3’ tailed substrate. Heat denatured control (filled triangle).
Fig. 3. Activity of the WRN and BLM helicases on a triplex with a 6 base pair
extension. Heat denatured substrate (filled triangle).
Fig. 4. Activity of the WRN and BLM enzymes with a triplex with no duplex
extension. A. WRN helicase; B. BLM helicase.
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 in Materials and Methods. Incubation
was at 24oC for 30 minutes. Heat denatured control (filled triangle).
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
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indicated concentrations of WRN (A) or BLM (B) enzyme. Heat denatured
control (filled triangle).
Acknowledgements
We thank Dr. Nitin Puri for oligonucleotide synthesis and analysis of the thermal
stability of the triplex and duplex substrates.
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Fig. 1B
1 2 3 4 5 6
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0
20
40
60
0 20 40 60 80
WRN (nM)
% D
ispl
acem
ent
WRN (nM) 0 7 14 21 28 56
Fig. 2A
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0
20
40
60
80
100
0 20 40 60 80
BLM (nM)
% D
ispl
acem
ent
BLM (nM) 0 2 4 8 16 32 64
Fig. 2B
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Fig. 3
WRN (nM) 0 7 14 21 28 56 BLM (nM) 8 32 64
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0 7 14 21 28 56WRN (nM)
Fig. 5A
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BLM (nM) 0 2 4 8 16 32 64
Fig. 5B
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WRN (nM) 0 0.9 1.8 3.5 7 14 21 28 ATPγS
Fig. 6A
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BLM (nM) 32 0 ATPγS
Fig. 6B
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and Michael M. SeidmanRobert M. Brosh ., Jr, Alokes Majumdar, Shital Desai, Ian D. Hickson, Vilhelm A. Bohr
Unwinding of a DNA triple helix by the werner and bloom syndrome helicase
published online November 10, 2000J. Biol. Chem.
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