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INTRODUCTION |
DNA helicases of the RecQ family unwind DNA with a 3'
5'
directionality and require ATP and Mg2+ for catalysis. All
members of the RecQ family of proteins share seven sequence motifs
common to helicases, including the characteristic DexH box (1).
Prokaryotic and yeast helicases of this family include
Escherichia coli RecQ, Saccharomyces cerevisiae
Sgs-1p, and Schizosaccharomyces pombe Rqh-1p. Three RecQ
homologues have been identified in human cells: BLM, a helicase that is
mutated in cells of Bloom's syndrome patients (2);
WRN,1 a helicase mutated in
Werner syndrome (3); and RecQL, a helicase of unknown function (4). The
in vivo functions of most helicases of the RecQ family are
not fully understood. It appears, however, that these enzymes take part
in diverse DNA transactions such as replication, repair, and
recombination. E. coli RecQ is believed to initiate
homologous recombination and to suppress illegitimate recombination (5,
6). RecQ is also thought to be involved in the reassembly of
replication forks after their disruption by UV irradiation (7, 8).
Phenotypes resulting from mutations in the human homologues of RecQ
also indicate their involvement in multiple DNA transactions. Bloom's
syndrome, caused by mutations in BLM, is associated with high
spontaneous frequencies of lymphatic and other malignancies and a high
frequency of somatic mutations (9, 10). Cells of Bloom's syndrome
patients display increased sister chromatid exchanges, augmented
sensitivity to DNA damaging agents, and defective DNA replication (11).
Werner syndrome resulting from mutations that inactivate WRN is
expressed by aging in early adulthood and genetic instability (12).
Cells of Werner syndrome patients exhibit large DNA deletions,
chromosomal rearrangements, prolongation of the S phase, and an
increased sensitivity to the genotoxic agent 4NQO (13-17). The
helicases responsible for the distinct pathologies of Bloom's and
Werner syndromes also differ. Whereas both enzymes display 3'
5'
DNA helicase activity, only WRN possesses an integral 3'
5'
exonuclease activity (18-20).
The compound phenotypes of Bloom's and Werner syndromes and the
multiplicity of pathologies associated with these diseases complicate
the search for the in vivo roles of the responsible helicases. One possibility is that these helicases are involved in the
resolution of secondary structures of DNA. Of special interest are
tetrahelical structures of guanine-rich sequences (G-DNA) that readily
form in vitro under physiological-like conditions (21-23).
These tetraplex structures have at their core guanine quartets that are
stabilized by non-Watson-Crick hydrogen bonds and are coordinated by
alkali cations. Although the existence in vivo of these
quadruplex structures has yet to be directly demonstrated, indirect
evidence indicates that they might take part in homologous
recombination (21, 22) and in telomere transactions (23, 24). One
possible adverse effect of tetraplex G-DNA formation is in the
perturbation of the progression of DNA polymerases. Readily formed
in vitro, tetraplex structures of the sequence
d(CGG)n have been suggested to play a role in polymerase
slippage in vivo and expansion of the trinucleotide repeat
(25-27). The expanded repeat tract impedes the transcriptional and
replication machineries in cells of fragile X syndrome patients.
Here we report that a bimolecular tetraplex structure of the
d(CGG)n repeat sequence (G'2 d(CGG)n) is unwound by WRN
more efficiently than double-stranded DNA. By contrast, WRN fails to
unwind G'2 bimolecular tetraplex structures of a telomeric sequence or
G4 tetramolecular forms of an immunoglobulin switch region sequence.
These findings contrast the recently reported ability of BLM helicase
to unwind tetrahelical structures of the guanine-rich consensus repeat
from the murine S
2b immunoglobulin switch region and the
Oxytricha telomeric repeat (28). The different sequence or
structure specificities of unwinding of tetrahelical DNA by WRN and BLM
may be relevant to the distinctly different phenotypes of Werner and
Bloom's syndromes.
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EXPERIMENTAL PROCEDURES |
Materials and Enzymes--
Isotopically 5'-labeled
[
-32P]ATP (~3000 Ci/mmol) was provided by Amersham
Pharmacia Biotech and New England Nuclear. Bacteriophage T4
polynucleotide kinase was a product of Promega. Synthetic DNA oligomers, listed in Table I, were
supplied by Operon Technologies. Filter paper, DE-81 and 3, was
purchased from Whatman. Amresco supplied Acryl/bisacrylamide, (19:1 or
30:1.2).
N,N,N',N'-Tetramethylenediamine, bromphenol blue, and xylene cyanol FF were the products of
International Biotechnologies, Inc. Sep-Pak cartridges were purchased
from the Waters Division of Millipore. Full-length cDNA construct
of Werner Syndrome helicase (GenBankTM accession number
L76937), containing an N-terminal six-histidine residue tag was cloned
and expressed in Spodoptera frugiperda cells as recently
described (3). WRN protein was purified from cell extracts to apparent
homogeneity by successive steps of chromatography through columns of
DEAE cellulose, phosphocellulose and Ni2+ binding affinity
as we described (18).
Preparation of Double-stranded and Quadruplex DNA
Oligomers--
DNA oligomers were purified by electrophoresis through
an 8.0 M urea, 15% polyacrylamide denaturing gel
(acrylamide:bisacrylamide, 19:1) as described (29) except that salt and
acrylamide residues were removed from the extracted DNA by Sep-Pak
column. Purified DNA oligomers that were 5' end-labeled with
32P in a T4 polynucleotide kinase catalyzed reaction (30)
were maintained in their single-stranded conformation by being stored as 0.25 mM solution in water and by boiling immediately
prior to use. Labeled 20-mer/46-mer partial DNA duplex was prepared by
heating at 90 °C for 5 min and slowly cooling to room temperature a
mixture of 2 µM 5'-32P-labeled 20-mer and 4.4 µM unlabeled 46-mer DNA in 10 mM Tris-HCl buffer, pH 8.0, 5 mM MgCl2. A partial duplex
32P-5'-labeled
hook(CGG)7·hook(CCG)7 was prepared by
similarly annealing an equimolar mixture of the oligomers (4 µM each). Bimolecular quadruplex structures of
32P-5'-labeled (CGG)7, 5'-tail
(CGG)7, and 3'-tail (CGG)7 and tetramolecular quadruplex structures of oligomers Q and 5'-tail Q were prepared by
incubating at 54 °C for 20-24 h each oligomer or a mixture thereof
(2.5-5.0 µg of DNA) in 10 µl of TE buffer (10 mM
Tris-HCl buffer, pH 8.0, 1 mM EDTA) containing 300 mM NaCl. The reaction was terminated by rapidly cooling the
samples and adding 50 µl of ice-cold solution of 60 mM
KCl. Tetraplex forms of the oligomers were resolved from residual
single strands by electrophoresis under 80-100 V and at 4 °C
through a nondenaturing 12% polyacrylamide gel
(acrylamide:bisacrylamide, 30:1.2) in 0.5 × TBE buffer, (1.25 mM EDTA in 45 mM Tris borate buffer, pH 8.3)
containing 50 mM KCl and 50 mM NaCl.
Electrophoretically retarded quadruplex DNA bands were visualized by
autoradiography and excised. The cut gel slices were suspended in 100 µl of TE buffer containing 20 mM KCl and rotated at
4 °C overnight. The extracted DNA was precipitated by ethanol and
resuspended in TE buffer that contained 20 mM KCl. Nondenaturing gel electrophoresis analysis showed that 75-90% of the
DNA maintained its tetraplex structure for up to a month when stored at
4 °C in TE buffer. Bimolecular tetraplex structures of
32P-5'-labeled telomeric sequence oligomers TeR2 or 5'-tail
Ter2, were similarly prepared except that incubation of the DNA at
54 °C for 20-24 h was conducted in the presence of 500 mM KCl. Tetraplex structures of the telomeric sequences
were resolved by electrophoresis through a nondenaturing 12%
polyacrylamide gel in 0.5 × TBE buffer, 50 mM KCl. To
assess concentrations of labeled quadruplex forms of the various
oligomers, the specific radioactivity of source oligomers was
determined, and concentrations of the isolated tetraplexes were deduced
from measurements of their radioactivity.
DNA Helicase Activity Assay--
DNA helicase reaction mixtures
contained in a final volume of 10 µl: 40 mM Tris-HCl
buffer, pH 7.4, 4 mM MgCl2, 20 mM
KCl, 5 mM dithiothreitol, 1 mM ATP, 10 µg of
bovine serum albumin, 300 fmol of 32P-5'-labeled
20-mer/46-mer partial DNA duplex or tetraplex DNA. After adding
specified amounts of WRN, the reaction mixtures were incubated at
37 °C for 10 or 15 min. The DNA unwinding reaction was terminated by
rapidly cooling the samples and adding 2 µl of a solution of 40%
glycerol, 50 mM EDTA, 2% SDS, 3% bromphenol blue, and 3%
xylene cyanol. Displaced DNA single strands were resolved from
remaining double-stranded or tetraplex DNA by electrophoresis at
4 °C and under 80-120 V through a nondenaturing 12% polyacrylamide gel in 0.5 × TBE buffer, 20 mM KCl. Resolved DNA
bands were visualized by exposing to autoradiographic film gels that
were dried on Whatman 3 filter paper. Amounts of double-stranded or
tetraplex DNA and of displaced single strands were quantified by
exposing gels dried on Whatman DE81 filter paper to phosphorimager
plate (Fuji).
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RESULTS |
Formation of Tetraplex DNA Structures--
The trinucleotide
repeat sequence d(CGG)n has been shown to readily fold into
hairpin structures (31-34) and to assemble into tetrahelical
formations (25-27). Tetraplex structures of d(CGG)n are bonded
by guanine-guanine non-Watson-Crick hydrogen bonds and are stabilized
by monovalent alkali cations (25-27). In investigating the unwinding
of d(CGG)n tetraplex structures by WRN, we first characterized
these tetraplexes by defining requirements for their formation and
determining their stoichiometry. As seen in Fig.
1A, 32P-5'-labeled
d(CGG)7 incubated at 54 °C in the presence of
Na+ yielded an electrophoretically retarded form whose
amount increased exponentially as a function of the oligomer
concentration. The second order kinetics of formation of the slowly
migrating form of d(CGG)7, as indicated by the linearity of
a log/log plot of results presented in Fig. 1A (not shown),
suggests that it was a multi-molecular complex. To determine the
stoichiometry of this structure, 32P-5'-labeled
d(CGG)7, 3'-tail d(CGG)7, or an equimolar
mixture thereof was incubated at 54 °C in the presence of 300 mM NaCl. Slowly migrating structures of the oligomers were
resolved from remaining single strands by nondenaturing gel
electrophoresis. As seen in Fig. 1B, an equimolar mixture of
the two oligomers yielded a third hybrid species in addition to the
electrophoretically retarded forms of d(CGG)7 and 3'-tail
d(CGG)7. Similar results were obtained when
d(CGG)7 was incubated together with 5'-tail d(CGG)7 (data not shown). The presence of three retarded
bands in the mixtures of d(CGG)7 and 5'- or 3'-tail
d(CGG)7 indicated that the slowly migrating complexes were
G'2 bimolecular structures. Additional results presented in Fig.
1B show that only negligible amounts of G'2 structures of
d(CGG)7, 3'-tail d(CGG)7, or 5'-tail d(CGG)7 were generated in the absence of Na+.
To assess the role of hydrogen bonding in the formation of the complexes, guanine residues in d(CGG)n were substituted by
inosines that lack a C2 amino group necessary for the formation of both
Watson-Crick and non-Watson-Crick hydrogen bonds. As seen in Fig.
1B, no complex was generated by d(CII)8 when
increasing amounts of the oligomer were incubated in the presence of
Na+. This finding, as well as our previously reported
observation that the d(CGG)7 complex resisted methylation
by dimethyl sulfate (25), suggests that the slowly migrating form of
d(CGG)7 was a bimolecular tetraplex complex stabilized by
non-Watson-Crick hydrogen bonds (Fig. 1C).

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Fig. 1.
Formation of tetrahelical d(CGG)n
complexes and their stoichiometry.
A, second order formation of a slowly migrating
d(CGG)n tetraplex complex. A constant amount of
32P-5'-labeled 3'-tail d(CGG)7 at 0.01 µM was mixed with 0.03-3.5 µM of unlabeled
3'-tail d(CGG)7 in a final volume of 10 µl of TE buffer,
300 mM NaCl. The DNA was denatured at 90 °C for 5 min,
the mixtures were incubated at 54 °C for 21 h, and the reaction
was terminated by the addition of 50 µl of ice-cold 60 mM
KCl. Single-stranded and tetraplex 3'-tail d(CGG)7 were
resolved by electrophoresis at 4 °C through a nondenaturing 12%
polyacrylamide gel containing 50 mM NaCl and 50 mM KCl in 0.5 × TBE buffer. The curve
shows results of phosphorimager quantification of the accumulation of
the electrophoretically retarded complex as a function of concentration
of 32P-5'-labeled 3'-tail d(CGG)7. An
autoradiogram of the gel is shown in the inset.
B, stoichiometry of the d(CGG)7 tetraplex
complex and requirements for its formation. Mixtures that contained 4.7 µM of 32P-5'-labeled d(CGG)7 or
3'-tail d(CGG)7 or a 1:1 mixture thereof in 10 µl of TE
buffer, 300 mM NaCl were incubated at 54 °C for 21 h, and DNA single strands were resolved from their slowly migrating
complexes by electrophoresis as in A above. Typical
electrophoretic migration of the DNA oligomers and their complexes is
shown in lanes 1-3. Requirements for complex formation:
mixtures containing 4.7 µM of 32P-5'-labeled
d(CGG)7, 3'-tail d(CGG)7, or 5'-tail
d(CGG)7 were incubated at 54 °C for 21 h in TE
buffer with no salt. Nondenaturing gel electrophoresis resolution of
the three DNA preparations is shown in lanes 4-6.
Increasing amounts of d(CII)8 were incubated at 54 °C
for 21 h in TE buffer containing 300 mM NaCl.
Electrophoretic resolution of the d(CII)8 samples is shown
in lanes 7-11. C, scheme of d(CGG)n
tetraplex structures. Based on the findings in A and
B above and on previously described results (25, 26), the
bimolecular tetrahelices are depicted as dimers of two hairpins
bonded by guanine quartets. Shown are G'2 bimolecular tetraplexes
without or with a non-d(CGG) single-stranded tail at their 3' or 5'
ends (dashed lines). Only two pairs of stacked guanine
quartets are drawn in each tetraplex. The two hairpins are aligned
against each other in one of several possible orientations (26).
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Formation of electrophoretically retarded complexes of the telomeric
sequences TeR2 and 5'-tail TeR2 and of the IgG switch region sequence
oligomers Q and 5'-tail Q was also found to be cation- and DNA
concentration-dependent (results not shown). The presence
of three electrophoretically retarded bands of methylation-protected structures in a mixture of TeR2 and 5'-tail TeR2 oligomers indicated a
bimolecular stoichiometry of these complexes (data not shown). Generation of five slowly migrating dimethyl sulfate-resistant species
in a mixture of oligomer Q and 5'-tail Q suggested a tetramolecular stoichiometry of these G4 complexes (results not presented).
WRN Unwinds Tetraplex G'2 5'-Tail d(CGG)7--
Werner
syndrome DNA helicase (WRN) incubated at 37 °C for increasing
periods of time in a helicase reaction mixture progressively unwound
32P-5'-labeled G'2 5'-tail d(CGG)7 (Fig.
2A). The WRN-catalyzed unwinding reaction depended on the presence of Mg2+ and
ATP. Further, ATP could not be substituted by its nonhydrolyzable analog
-S-ATP (Fig. 2B). Similar results were obtained
for the unwinding by WRN of G'2 3'-tail d(CGG)7 (not
shown).

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Fig. 2.
WRN helicase unwinds a bimolecular
d(CGG)7 tetraplex. A, kinetics of
tetraplex unwinding. DNA helicase reaction mixtures, (see
"Experimental Procedures") containing 300 fmol of
32P-5'-labeled G'2 tetraplex 5'-tail d(CGG)7
and 20 fmol WRN helicase were incubated at 37 °C for the indicated
periods of times. A reaction mixture without WRN was incubated at
100 °C for 10 min to visualize denatured 5'-tail d(CGG)7
single strands. The DNA unwinding reaction was terminated and displaced
5'-tail d(CGG)7 single strands were resolved from remaining
G'2 5'-tail d(CGG)7 tetraplex by electrophoresis through a
nondenaturing 12% polyacrylamide gel in 0.5 × TBE buffer, 20 mM KCl (see "Experimental Procedures"). B,
requirements for unwinding of G'2 5'-tail d(CGG)7. DNA
helicase reaction mixtures containing 300 fmol of
32P-5'-labeled G'2 tetraplex 5'-tail d(CGG)7
were incubated at 37 °C for 15 min with or without 20 fmol WRN, 1 mM ATP, 4 mM MgCl2, or 1 mM -S-ATP as indicated. Displaced 5'-tail
d(CGG)n single strands were resolved from the G'2 tetraplex
form by nondenaturing gel electrophoresis as in A.
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WRN Requires a Single-stranded Tail for the Unwinding of Tetraplex
d(CGG)7--
To study the DNA substrate specificity of
WRN, increasing amounts of the enzyme were incubated under standard
helicase assay conditions with 300 fmol each of
32P-5'-labeled 20-mer/46-mer partial duplex DNA or G'2
tetraplex forms of d(CGG)7, 3'-tail d(CGG)7, or
5'-tail d(CGG)7. As seen in Fig.
3, WRN resolved G'2 structures of 5'-tail
d(CGG)7 and 3'-tail d(CGG)7 at efficiencies
that were similar to or greater than that of the unwinding of a
20-mer/46-mer partial DNA duplex. Interestingly, under these
conditions, WRN did not measurably unwind a partial double strand of
hook d(CGG)7·hook d(CCG)7, probably because of its high stability (results not presented). Thus, it appeared that WRN does not preferentially unwind all
d(CGG)n-containing DNA structures. Notably, WRN failed to
measurably unwind G'2 d(CGG)7 that lacked a single-stranded
tail (Fig. 3). Unwinding of G'2 d(CGG)7 could not be
detected even in the presence of a 1.5-fold molar excess of WRN over
this blunt-ended tetraplex (data not shown).

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Fig. 3.
DNA substrate specificity of WRN
helicase. The indicated amounts of WRN helicase were incubated at
37 °C for 10 min in a DNA helicase reaction mixture containing 300 fmol of the indicated double-stranded or tetraplex DNA substrates.
Reaction mixtures without WRN were incubated at 100 and 37 °C for 10 min to visualize single-stranded and G'2 5'-tail d(CGG)7,
respectively. The incomplete denaturation of the 20-mer/46-mer partial
duplex at 100 °C as shown, is atypical, and in most other
experiments this substrate became fully denatured. Following
termination of the reaction, displaced single strands were resolved
from double- or four-stranded DNA by nondenaturing gel electrophoresis
as described in the legend to Fig. 2.
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WRN Unwinds Tailed G'2 d(CGG)7 More Efficiently than a
Partial DNA Duplex--
To assess the efficacy of unwinding by WRN of
G'2 3'-tail d(CGG)7 relative to a 20-mer/46-mer DNA partial
duplex, increasing amounts of WRN helicase were added to 300 fmol of
either labeled DNA substrate, and proportions of displaced single
strands were quantified by phosphorimager analysis. Average results of
multiple experiments presented in Fig. 4
indicated that WRN unwound G'2 3'-tail d(CGG)7 at an
efficiency that was ~3.5-fold higher than for partial DNA duplex.
Whereas 50% of the G'2 3'-tail d(CGG)7 tetraplex became
unwound in the presence of 9 fmol WRN, the unwinding of 50% of the
20-mer/46-mer partial duplex required 32 fmol of the helicase (Fig. 4).
Similar analysis revealed that G'2 5'-tail d(CGG)7 was
resolved by WRN at a ~2-fold greater efficiency than partial duplex
DNA (results not shown).

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Fig. 4.
Relative efficiency of unwinding of
double-stranded DNA and tetraplex 3'-tail d(CGG)7 by
WRN. Increasing amounts of WRN (3.1-62.5 fmol) were added
to DNA helicase reaction mixtures each containing 300 fmol of either
32P-5'-labeled 20-mer/46-mer partial DNA duplex or
32P-5'-labeled G'2 3'-tail d(CGG)7. Following
incubation at 37 °C for 15 min and termination of the reaction,
displaced single strands were resolved from tetraplex 3'-tail
d(CGG)7 by nondenaturing gel electrophoresis as described
in the legend to Fig. 2. Amounts of single-stranded and G'2 tetraplex
3'-tail (CGG)7 were quantified by phosphorimager analysis.
Results presented are the averages of four independent
experiments.
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WRN Fails to Unwind Tetraplex Forms of Telomeric DNA and an IgG
Switch Sequence--
The ability of WRN to unwind tetraplex forms of
guanine-rich sequences other than d(CGG)n was examined.
Increasing amounts of the helicase were incubated with 300 fmol each of
blunt-ended or 5'-tailed G'2 bimolecular tetraplex forms of the
telomeric sequence TeR2 or with 300 fmol of blunt-ended or 5'-tailed G4 four-molecular tetraplex forms of the IgG switch sequence Q. As seen in
Fig. 5, no displacement of single strands
from any of the quadruplex DNA structures was detected even at a molar
excess of WRN over tetraplex DNA substrate. Control partial DNA duplex was completely unwound by WRN under the same reaction conditions (not
shown). As also seen in Fig. 5, amounts of tetraplex TeR2, 5'-tail TeR2
and oligomer Q were diminished in the presence of maximum amounts of
the helicase. This decrease was due to digestion of the DNA by the 3'
5' WRN-associated exonuclease, as demonstrated by visualizing DNA
degradation products by denaturing gel electrophoresis of the DNA
(results not shown). Hence, unlike 3'- or 5'-tailed G'2
d(CGG)7, quadruplex forms of telomeric DNA or of the IgG
switch region sequence could not be resolved by WRN.

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Fig. 5.
WRN helicase fails to unwind tetraplex forms
of TeR2 DNA and oligomer Q. The indicated amounts of WRN protein
were added to helicase reaction mixtures each containing 300 fmol of
32P-5'-labeled bimolecular G'2 tetraplex forms of TeR2 or
5'-tail TeR2 or four-molecular G4 forms of oligomer Q or 5'-tail
oligomer Q (see "Experimental Procedures"). Reaction mixtures
without WRN were incubated at 100 or 37 °C for 10 min to visualize
single-stranded or tetraplex DNA, respectively. Following incubation at
37 °C for 15 min and termination of the reaction, displaced single
strands were resolved from their respective tetrahelical DNA structures
by electrophoresis through a nondenaturing 12% polyacrylamide gel in
0.5 × TBE buffer, 20 mM KCl.
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DISCUSSION |
Results presented in this paper demonstrate WRN helicase is
capable of unwinding G'2 bimolecular tetraplex structures of the d(CGG)
repeat sequence. Unwinding of G'2 d(CGG)7 by WRN required hydrolyzable ATP and Mg2+, and the extent of the reaction
was dependent on the amount of enzyme added and time of incubation
(Fig. 2). WRN required short single-stranded tracts at the 3' or 5'
ends of the G'2 d(CGG)7 tetraplex and a blunt-ended
bimolecular d(CGG)7 tetraplex could not be unwound (Fig.
3). Unwinding by WRN of 3'- or 5'-tailed G'2 d(CGG)7 was
3.5- and 2-fold, respectively, more efficient than the unwinding of
partial DNA duplex (Fig. 4). It is notable that although
double-stranded DNA is unwound by WRN at a 3'
5' direction (3), G'2
tetraplex structures of d(CGG)n with both 3' and 5'
single-stranded tails served as efficient substrates for this helicase.
It might thus be that the directionality of disruption of guanine
quartets by WRN differs from that of unwinding of double-stranded DNA.
Specificity and Efficacy of Tetraplex d(CGG)n Unwinding by
WRN--
Two helicases, BLM (28), and SV40 T-antigen (36) have been
shown to unwind tetraplex DNA structures. However, the substrate specificity and efficiency of tetraplex unwinding by WRN differs from
the unwinding of tetraplex DNA by these helicases. Both BLM (28) and
SV40 T-antigen (36) were reported to unwind tetraplex structures of an
IgG switch region sequence. By contrast, WRN failed to unwind tetraplex
forms of this sequence (Fig. 5). In addition, whereas BLM was reported
to unwind a tetrahelical structure of Oxytricha
d(TTTTGGGG)n telomeric sequence (28), WRN was unable to
measurably unwind the G'2 tetraplex structure of the vertebrate
telomeric sequence d(TTAGGG)n (Fig. 5). However, in unwinding a
G'2 tetraplex 3'-tail d(CGG)7, WRN acted more efficiently
than either BLM or the SV40 helicase. Unwinding of G4 DNA by BLM and
SV40 T-antigen was reported to reach completion within 20-45 min at
enzyme to DNA ratios of 2:1 and 360:1, respectively (28, 36). WRN,
however, fully unwound within 15 min G'2 3'-tail d(CGG)7 at
a molar ratio of enzyme to DNA of 0.1 (Fig. 4).
Possible Biological Significance of Unwinding of Tetraplex
d(CGG)n by WRN--
The recently reported capacity of BLM to
unwind G4 structures of the immunoglobulin switch region and of
Oxytricha telomeric sequence were interpreted as indicating
a role of this helicase in the resolution of tetraplexes generated by
strand invasion during DNA recombination or replication (28). The
failure of WRN to unwind tetraplex structures of the IgG switch region
or telomeric sequences suggests that it cannot replace BLM in resolving tetraplexes of specific sequence or structure. However, WRN efficiently unwound bimolecular quadruplex structures of d(CGG)n. A d(CGG)
trinucleotide repeat was first identified in the 5'-untranslated region
of the FMR1 gene (37-39). The propensity of d(CGG)n tracts to fold into hairpin structures and to assemble into tetraplex structures was implicated in the expansion of this sequence and the
obstructed transcription and replication of FMR1 in fragile X syndrome (25, 32, 34). More recent evidence indicated that d(CGG)
repeats are not restricted to FMR1 in the human genome. Computer analysis revealed statistical over-representation of d(CGG)n tracts in the human genome and identified this sequence
in multiple known genes (40). Moreover, several expressed sequences
from human genomic library were found to bear d(CGG)n repeats
(41). Notably, trinucleotide repeats other than d(CGG) also readily
fold into secondary structures (31). It is possible that hairpin or
tetraplex structures of trinucleotide repeats form subsequent to their
exposure as single-stranded stretches during DNA replication,
transcription, or recombination. A potential function of WRN might be
to unwind such secondary structures, thus relieving replication,
transcription or recombination constraints. The slowed replication in
Werner syndrome cells and accumulation of large deletions in their DNA
might therefore be a reflection of defective removal of DNA secondary
structures resulting from lack of WRN activity. The different DNA
substrate specificities of the two human RecQ homologues, BLM and WRN,
could be relevant to the distinctly different phenotypes of the two syndromes.