From the Gottstein Memorial Cancer Research
Laboratory, Departments of Pathology and Biochemistry, University
of Washington, Seattle, Washington 98195, the ¶ Department of
Biochemistry and Molecular Biophysics, Washington University School of
Medicine, St. Louis, Missouri 63110, and the
Unit of
Biochemistry, Bruce Rappaport Faculty of Medicine, Technion-Israel
Institute of Technology, P. O. Box 9649, Haifa 31096, Israel
Received for publication, January 11, 2001, and in revised form, February 2, 2001
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ABSTRACT |
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Werner syndrome (WS) is an inherited disorder
characterized by premature aging and genomic instability. The protein
encoded by the WS gene, WRN, possesses intrinsic 3' Werner Syndrome (WS),1
characterized by premature aging and genomic instability (1), is a
result of mutations in the WS gene. The polypeptide encoded by the WS
gene, WRN, contains a central seven-motif domain shared by DNA
helicases of the RecQ family (2). This family of DNA helicases is
represented by Escherichia coli RecQ (3),
Saccharomyces cerevisiae Sgs-1 (4), Schizosaccharomyces pombe Rqh1 (5), Xenopus
laevis FFA-1 (6), and human RecQL (7), BLM (8), and RecQ4 and
RecQ5 proteins (9). Multiple RecQ DNA helicases have also been
identified in Drosophila melanogaster (10) and
Arabidopsis thaliana (11). WRN is distinct from other
members of the RecQ helicase family in that it also includes an
N-terminal exonuclease domain (12-14). Indeed, recombinant WRN protein
has been shown to possess, in addition to an ATP-dependent
3' WRN helicase exhibits several characteristic features. 1) Unwinding of
double-stranded DNA requires a 3' single-stranded DNA tail, which
presumably serves as a helicase loading DNA stretch (17, 18). 2) WRN
exhibits low processivity such that the enzyme is capable of unwinding
only short duplex regions <25 nt in length. 3) The processivity of WRN
can be increased by the single-stranded DNA-binding protein, human
replication protein A (19); in its presence, WRN unwinds duplex DNA
tracts as long as 800 nt (20). 4) WRN can unwind alternate DNA
structures, including DNA tetraplexes (21), four-way Holliday junctions
(22), and triplex DNA (23).
A large body of evidence implicates WRN and its family members in
replication. The prolonged S-phase of WS cells (24, 25), their
sensitivity to the S-phase-specific topoisomerase I inhibitor camptothecin (26), and the more recent demonstrations of a physical and
functional interaction between WRN and the major replicative DNA
polymerase, pol Guanine-rich DNA sequences readily form tetraplex structures in
vitro under physiological-like conditions (30-32). Tetraplex formations of DNA are maintained by guanine quartets that are held
together by Hoogsteen hydrogen bonds and stabilized by monovalent alkali cations. A direct demonstration for the existence of tetraplex DNA structures in cells is still lacking. However, their formation in vitro by biologically important G-rich sequences, such as
telomeric DNA and the immunoglobulin class switch region, has led to
speculations on their involvement in telomere transactions (32, 33) and in homologous recombination (30, 31) in vivo. Of interest is
the formation of hairpin (34-37) and tetraplex structures (38-40) by
the d(CGG) trinucleotide repeat sequence whose expansion in the
FMR1 gene leads to fragile X syndrome. Hairpin and tetraplex structures of this sequence have been shown to perturb movement of DNA
polymerases during in vitro DNA synthesis (40-43). Stalling of replicative DNA polymerases could result in polymerase slippage and
expansion of the repeat sequence.
Here we report that DNA synthesis by several eukaryotic DNA polymerases
is blocked by hairpin and bimolecular G'2 tetraplex structures of a
d(CGG)7 tract in template DNA. Addition of WRN helicase,
however, allows pol Materials and Enzymes
[ Recombinant hexa-His-tagged WRN protein was purified to >90%
homogeneity by the protocol published by Shen et al. (16). Approximate concentrations of WRN protein were determined from Coomassie-stained SDS-polyacrylamide gels using bovine serum albumin as
a standard. Molar amounts of WRN were calculated based on its being a
monomer (~165 kDa). RecQ helicase was kindly provided by Dr. Stephen
Kowalczykowski (University of California, Davis, CA), and UvrD helicase
was a gift from Dr. Lawrence Grossman (Johns Hopkins University,
Baltimore, MD). S. cerevisiae DNA pol Preparation of Tetraplex DNA
High performance liquid chromatography-purified 18-mer
primer (5'-d(GCCGGGGCCGGCCGCCGC)-3') was 5'-end-labeled with
[ The labeled primer (500 pmol) was mixed with an equivalent amount of
unlabeled template DNA in 50 mM Tris-HCl buffer, pH 8.0, 10 mM MgCl2. The mixture was boiled for 5 min at
100 °C, and the denatured oligomers were allowed to anneal by slow
cooling to room temperature. Unlabeled primer was hybridized in
parallel to unlabeled template DNA in an identical manner. The labeled primer-template was mixed with unlabeled primer-template to a final DNA
concentration of 60 µM in the presence of 300 mM KCl in a volume of 16 µl. The mixture was incubated at
4 °C for 15-18 h to allow formation of tetraplex DNA. Thereafter,
the concentration of KCl was lowered to 30 mM by the
addition of 25 mM Tris-HCl, pH 8.0, 20% glycerol.
Approximately 30-µl aliquots of the DNA mixture were loaded in
individual lanes of a non-denaturing 6% polyacrylamide gel in TBE
buffer (45 mM Tris borate buffer, pH 8.3, 1.25 mM EDTA) containing 30 mM KCl. The samples were
electrophoresed at 4 °C at a constant current of 35 mA to resolve
tetraplex forms of the oligomer from residual duplex and
single-stranded DNA. Electrophoretically retarded tetraplex DNA was
visualized by autoradiography and cut out from the gel. The excised gel
slices were suspended in cold TE buffer (10 mM Tris-HCl, pH
8.0, 1 mM EDTA) containing 100 mM KCl and
vortexed at 4 °C overnight. Following separation of gel residue by
centrifugation, the extracted DNA was precipitated with ethanol and
resuspended in TE buffer, 20 mM KCl. Aliquots of the
recovered DNA were stored frozen at Preparation of Duplex Hairpin-containing DNA
32P-5'-End-labeled 18-mer primer was hybridized to a
2-fold molar excess of gel-purified, unlabeled 61-mer template, as
described above. The primed hairpin template was used without further
purification in primer extension assays.
Assays
DNA Polymerase-catalyzed Primer Extension--
Hairpin or
tetraplex-containing DNA template (0.5 pmol) was copied by indicated
concentrations of DNA polymerases in the absence or presence of known
amounts of DNA helicases. DNA synthesis was carried out in reaction
mixtures that contained, in a final volume of 10 µl: 40 mM Tris-HCl buffer, pH 7.5, 20 mM KCl, 5 mM MgCl2, 5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, and 0.2 mM each of dATP, dGTP,
dCTP, and dTTP. Reaction mixtures for the extension of primed
hairpin-containing template did not include KCl. Following incubation
at 37 °C for 15 min, the primer extension reactions were terminated
by rapid cooling on ice and addition of denaturing loading buffer (45).
The samples were boiled for 5 min, and aliquots were electrophoresed
through 14% polyacrylamide-urea gels. The gels were dried and primer
extension products were visualized by autoradiography or quantitated by
PhosphorImager analysis (Molecular Dynamics).
DNA Helicase Activity--
Helicase activity was measured in
primer extension reaction mixtures except that 1 mM ATP was
present in place of the four dNTP substrates. Radiolabeled tetraplex
DNA substrate (0.3-0.5 pmol) was incubated with known amounts of WRN,
E. coli RecQ, or E. coli UvrD at 37 °C for 15 min. The unwinding reaction was terminated by the addition of 2.5 µl
of a solution containing 40% glycerol, 50 mM EDTA, 2%
SDS, and 3% each bromphenol blue and xylene cyanol. Unwinding of
tetraplex DNA was monitored by electrophoresis of reaction aliquots
through a non-denaturing 12% polyacrylamide gel in 0.5× TBE, 20 mM KCl at 4 °C under a constant current of 35 mA,
followed by autoradiography, as described (21).
The d(CGG)7-containing Synthetic Template Forms a
Bimolecular Tetraplex Structure--
DNA tracts containing repeats of
the d(CGG) trinucleotide fold into hairpin structures (34-37), which,
in the presence of alkali cations, assume tetrahelical conformations
(38-40). Both the hairpin and tetraplex structures of
d(CGG)n DNA stretches have been shown to block
synthesis by DNA polymerases in vitro (41-43) and in
vivo (46).
We characterized the requirements for the formation of a tetraplex
structure by the d(CGG)7-containing template and determined its stoichiometry. A 15-h incubation of 60 µM
32P-5'-labeled d(CGG)n-containing
primer-template in the presence of 300 mM KCl at 4 °C
resulted in the formation of tetraplex structures, as evidenced by the
appearance of a band with retarded electrophoretic mobility relative to
that of the DNA duplex on non-denaturing gels (data not shown). To
determine the stoichiometry of the tetraplex generated under these
conditions, we used two oligomers of different length, each containing
seven d(CGG) repeats. The 32P-5'-labeled
d(CGG)7-containing 61-mer template annealed at its 3'
terminus to a complementary unlabeled 18-mer primer,
32P-5'-d(CGG)7 oligomer or a 1:1 equimolar
mixture thereof, were incubated at 4 °C for 15 h in the
presence of 300 mM KCl to promote tetraplex formation.
Following incubation, half of each reaction mixture was denatured while
the other half was maintained at 4 °C. Aliquots of each mixture were
diluted to 20 mM KCl and electrophoresed through
non-denaturing polyacrylamide gels to resolve single-stranded DNA from
duplex and tetraplex complexes. As shown in Fig.
1A, electrophoretically
retarded bands, representing respective multi-molecular complexes, were
generated by the primer-d(CGG)7 template and the d(CGG)7 oligomer. Previous CD measurements (47) and
dimethyl sulfate protection analyses (38) demonstrated that these
complexes were DNA tetraplexes. Most notably, however, an additional
band with mobility intermediate to the tetraplex complexes formed by each individual oligomer was observed in the 1:1 equimolar mixture of
the two oligomers. As a result of their different stabilities, different amounts of tetraplex complexes were formed in the 1:1 mixture
of d(CGG)7 and primed d(CGG)7-containing
template (Fig. 1A). The single hybrid complex is a tetraplex
composed of one molecule each of the
primer-d(CGG)7-containing template and the d(CGG)7 oligomer. Hence, mixtures containing only
primer-d(CGG)7 template generate a bimolecular tetraplex of
this DNA. The bimolecular tetraplex complex formed between two
molecules of primed-32P-template, schematically illustrated
in Fig. 1B and henceforth designated as G'2 primer-template,
was extracted from the gel (see "Experimental Procedures") and
served as a substrate for WRN helicase as well as a template for DNA
synthesis by DNA polymerases.
The Bimolecular d(CGG)7-containing Primer-Template
Tetraplex Is Unwound by WRN--
Purified G'2
primer-32P-5'-labeled template was incubated at 37 °C
with increasing amounts of WRN and electrophoresed through a
non-denaturing gel to monitor unwinding of the DNA tetraplex. As
illustrated in Fig. 2, WRN efficiently
unwound the tetraplex structure in the presence of Mg2+ and
ATP, as previously reported for different
d(CGG)n tetraplex substrates (21). Similar
unwinding results were obtained using G'2 primer-template radiolabeled
at the primer stem (data not shown). Unwinding was also observed when
ATP in the reaction mixture was substituted with 0.2 mM
each of the four dNTPs, as used in subsequent polymerase-catalyzed
primer extension reactions (results not presented).
WRN Enables Polymerase
We next asked whether WRN, by virtue of its ability to unwind
d(CGG)n tetraplex structures, would allow pol
We also inquired whether the order of addition of pol WRN Helicase Activity Is Essential to Alleviate Pausing by
Polymerase WRN Does Not Alleviate Pausing by DNA Polymerases Other than pol
To address this question, we initially carried out primer extension
assays, identical to those presented in Fig. 3, with pol
We next examined the ability of WRN to enable two other major
replicative DNA polymerases, pol Not All Helicases Alleviate Pausing by pol
Next, we carried out primer extension assays with pol WRN Alleviates Pausing by DNA Polymerase
Simultaneous addition of WRN to reactions containing pol The DNA metabolic processes that WRN participates in are still not
clear. However, several lines of evidence point to its involvement in
DNA replication. In particular, WS cells exhibit S-phase defects,
including a decreased frequency of DNA initiations and a reduced rate
of chain elongation (24, 49). Furthermore, these cells are sensitive to
the S-phase-specific topoisomerase I inhibitor, camptothecin (26). The
functional and physical interaction of WRN with a major replicative DNA
polymerase, pol We used bimolecular tetraplex or hairpin formations of the
trinucleotide repeat sequence d(CGG)n as model
template secondary structures. A d(CGG)n
trinucleotide was first identified in the 5'-untranslated region of the
FMR1 gene (50-52). The ability of
d(CGG)n tracts to fold into hairpins (34-37) and to assemble into quadruplex structures (38-40) was implicated in
the expansion of this sequence that leads to fragile X syndrome. Hairpin and tetraplex structures of d(CGG)n have
also been shown to block the progression of several DNA polymerases both in vitro (41-43) and in vivo (46).
We constructed a synthetic d(CGG)7-containing primed DNA
template that folds spontaneously into a hairpin structure, or forms a
bimolecular G'2 tetraplex structure in the presence of K+
ions (Fig. 1). In line with previous reports, we too demonstrate that
the template G'2 d(CGG)7 hairpin and tetraplex structures impose a strong barrier to DNA synthesis by three eukaryotic
replicative DNA polymerases: Addition of WRN markedly alleviates pausing by pol The ability to complete synthesis past the G'2 tetraplex
d(CGG)7 replicative barrier and to generate full-length
product DNA chains in the presence of WRN appears, by far, to be
limited to pol The specificity of alleviating tetraplex DNA-induced stalling of DNA
polymerase is not only limited to the polymerase used, but also to the
helicase utilized for unwinding. Our data show that, similarly to WRN,
E. coli RecQ can unwind G'2 d(CGG)7 and allow
pol Data presented in Figs. 5 and 6 indicate that unwinding of the
tetraplex structure by itself is not sufficient to allow traversal of
the tetraplex domain by DNA polymerases. Instead, the results suggest a
requirement for a concerted action of DNA unwinding by WRN and DNA
synthesis by polymerase. The two processes may be coupled through a
direct interaction of these proteins. Indeed, a physical interaction
between WRN and human pol Unwinding of the tetraplex structure by WRN generates double-stranded
DNA primer-template (Fig. 2). If unwinding of the tetraplex is not
coupled to extension of the primer, the single-stranded d(CGG)n tract would refold spontaneously into a
hairpin structure. Such hairpin structures effectively impede
progression of synthesis by pol The capacity of WRN to resolve tetraplex structures of
d(CGG)n and to alleviate pausing by pol In conclusion, we have shown that DNA templates containing
d(CGG)n hairpin or tetraplex structures impede
DNA synthesis by three major replicative DNA polymerases: pol 5' DNA helicase and 3'
5' DNA exonuclease activities. WRN helicase resolves alternate DNA structures including tetraplex and triplex DNA, and
Holliday junctions. Thus, one function of WRN may be to unwind secondary structures that impede cellular DNA transactions. We report
here that hairpin and G'2 bimolecular tetraplex structures of the
fragile X expanded sequence, d(CGG)n,
effectively impede synthesis by three eukaryotic replicative DNA
polymerases (pol): pol
, pol
, and pol
. The constraints
imposed on pol
-catalyzed synthesis are relieved, however, by WRN;
WRN facilitates pol
to traverse these template secondary structures
to synthesize full-length DNA products. The alleviatory effect of WRN
is limited to pol
; neither pol
nor pol
can traverse
template d(CGG)n hairpin and tetraplex
structures in the presence of WRN. Alleviation of pausing by pol
is
observed with Escherichia coli RecQ but not with UvrD
helicase, suggesting a concerted action of RecQ helicases and pol
.
Our findings suggest a possible role of WRN in rescuing pol
-mediated replication at forks stalled by unusual DNA
secondary structures.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5' DNA helicase activity, an intrinsic 3'
5' DNA exonuclease
activity (15, 16).
(27, 28), all support the notion that WRN is
involved in some aspects of DNA replication. If this is the case, a
principle function of WRN helicase may be to resolve alternate DNA
structures ahead of the replication fork that would normally impede the
progression of DNA polymerases, analogous to the function of the dda
helicase in bacteriophage T4 (29).
to traverse these template secondary structures
and to synthesize full-length DNA. Further, we demonstrate that the
ability of WRN to alleviate polymerase stalling at these secondary
structures is specific and limited to pol
.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (~3000 Ci/mmol) was purchased from
PerkinElmer Life Sciences. High performance liquid
chromatography-purified and crude oligodeoxynucleotide primer and
template, respectively, were synthesized by Operon Technologies.
Ultrapure deoxyribonucleoside triphosphates (dNTPs) were purchased from
Promega Corp. Bacteriophage T4 polynucleotide kinase was supplied by
New England Biolabs.
and pol
* were
purified to homogeneity as described (44); concentrations of pol
and pol
* were determined spectrophotometrically at A280. Human DNA polymerase
-primase complex
(pol
) and human DNA polymerase
(pol
) were the generous
gifts of Dr. Teresa Wang (Stanford University, Stanford, CA) and Dr.
Stuart Linn (University of California, Berkeley, CA), respectively.
-32P]ATP by T4 polynucleotide kinase as described
(45) and boiled to inactivate the kinase. Unincorporated
[
-32P]ATP was removed from the reaction mixture by
precipitating the labeled primer DNA with ethanol. Complementary 61-mer
template (5'-d(TATGCCGGCGGCGGCGGCGGCGGCGGATGTAATGCCTCGTCTTGCGGCGGCCGGCCCCGGC)-3') was purified by electrophoresis through a denaturing 7 M urea, 8% polyacrylamide gel (45).
80 °C until use.
Concentrations of the isolated tetraplex DNA were estimated from the
amount of radioactivity recovered.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Stoichiometry and structure of
d(CGG)7 tetraplex-containing primer-template DNA.
A, unlabeled 18-mer primer was annealed to complementary
32P-5'-labeled d(CGG)7-containing 61-mer
template. The annealed primer 32P-5'-template,
32P-5'-d(CGG)7, or a 1:1 mixture thereof, was
incubated at 4 °C for 15 h in 10-µl reaction mixtures that
contained 9 µM DNA and 300 mM KCl. At the end
of the incubation period, half of each reaction mixture was boiled for
10 min to denature formed tetraplex complexes and half was kept at
4 °C. Aliquots of the DNA samples were diluted to 20 mM
KCl, and tetraplex complexes were resolved from double-stranded
(ds) primer-template by electrophoresis at 4 °C through a
12% polyacrylamide gel in 0.5× TBE buffer, 20 mM KCl.
Boiling of G'2 primer-32P-template resulted in complete
denaturation of the electrophoretically retarded tetraplex complex
(lane 1). However, heat treatment left the
double-stranded primer-template intact. As revealed by boiling of G'2
32P-primer-template, only a minor amount of labeled primer
was released, whereas the major fraction of DNA remained as an intact
double strand (results not shown). The heat resistance of the
primer-template is presumably due to the high melting temperature of
the G-C rich 18-mer primer and/or its rapid reannealing following
denaturation. ss, single-stranded. B, schematic
of the bimolecular tetraplex primer-template complex. Downstream to the
primer-template stem of each of the two coupled DNA molecules is a
17-nucleotide-long single-stranded template tract followed by a
tetraplex domain formed by two joined d(CGG)7 template
hairpins and a 5-nucleotide-long single-stranded tail. Four guanine
quartets in a d(GGCGG) tract schematically represent the template G'2
d(CGG)7 tetraplex structure.
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Fig. 2.
WRN unwinds tetraplex primer-template
DNA. WRN helicase assay mixtures (see "Experimental
Procedures") contained 300 fmol G'2 tetraplex structures of
primer-32P-5'-template and increasing amounts (4-40 fmol)
of WRN helicase. The mixtures were incubated at 37 °C for 15 min,
and, after termination of the unwinding reaction, tetraplex DNA was
resolved from double-stranded (ds) DNA by electrophoresis at
4 °C through a 12% polyacrylamide gel in 0.5× TBE buffer, 20 mM KCl. Control reactions that did not contain WRN were
either boiled for 10 min prior to electrophoresis to denature the G'2
tetraplex DNA or were incubated at 4 °C or 37 °C to monitor
spontaneous destabilization of the tetraplex primer-template. Heat
denaturation of G'2 tetraplex primer-template resulted in its complete
conversion to double-stranded primer-template rather than to
single-stranded template (lane 1). As noted in
the legend to Fig. 1A, this is most likely due to the high
melting temperature of the primer and/or its reannealing to the
template following denaturation.
to Traverse past a Template
d(CGG)7 Tetraplex Structure--
We monitored the ability
of polymerase
to copy a G'2 d(CGG)7
tetraplex-containing template strand. The primer-tetraplex template
complex was isolated as described under "Experimental Procedures"
and incubated with pol
in the presence of all four dNTPs at
37 °C. Products of the extension reaction were electrophoresed through denaturing polyacrylamide gels and visualized by
autoradiography. We observed that pol
was able to incorporate dNTPs
up to the start of the tetraplex structure (Fig.
3, lane 4; data not
shown). Notably, pol
alone failed to generate full-length DNA
product chains; strong pause sites were observed within the first of
the seven repeat sequences at template nucleotide positions 37 and 38. The inability of pol
to extend the primer beyond the pause sites
indicates that the G'2 d(CGG)7 tetraplex structure
effectively blocked progression of pol
along the template
strand.
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Fig. 3.
WRN enables pol to
traverse a template d(CGG)7 tetraplex structure.
Gel-purified G'2 d(CGG)7 tetraplex containing
32P-5'-primer-template DNA (0.5 pmol) was copied by pol
(~0.7 fmol) or pol
* (~0.9 fmol) in the absence or presence of
WRN helicase (1 and 6 fmol). Following incubation at 37 °C for a
total length of 20 min, the reactions were terminated by the addition
of denaturing loading buffer. The samples were boiled, and aliquots
were electrophoresed through denaturing polyacrylamide gels as
described (45). Lane 1,
32P-5'-primer-template; lanes 2 and
3, WRN without added DNA polymerase; lanes
4 and 11, pol
and pol
*, respectively,
minus WRN; lanes 5, 6, 12,
and 13, WRN added together with polymerase; lanes
7, 8, 14, and 15, reactions
pre-incubated with polymerase for 10 min prior to the addition of WRN
for another 10 min; lanes 9, 10,
16, and 17, reactions pre-incubated with WRN for
10 min before incubation with polymerase for 10 min.
Arrowheads correspond to the full-length product of 61 nt.
Positions of primer and template pause sites are as indicated.
to traverse the template tetraplex domain. Primer extension
reactions were carried out as described above with the exception that
two different amounts of WRN were added to the mixtures along with pol
. As seen in Fig. 3 (lanes 5 and
6), WRN allowed pol
to extend the primer beyond the
pause sites adjacent to the tetraplex structure and generate
full-length DNA product. Using end-labeled single-stranded template DNA
as a molecular size marker, we determined that the lower of the two
bands indicated in Fig. 3 by arrowheads, corresponds to the
61-nt product. The upper band with retarded mobility is most likely to
represent an altered conformation of the full-length product since
electrophoresis of reaction products through a denaturing
urea-formamide gel resulted in the appearance of only a single 61-nt
band (data not shown). The ladder of bands observed below the primer
corresponds to degradation products generated by the 3'
5'
exonucleolytic activity of WRN as evidenced by their accumulation in
reaction mixtures that contained WRN without polymerase (Fig. 3,
lanes 2 and 3).
and WRN
affects the ability of pol
to traverse the template tetraplex structure. When DNA synthesis was carried out first by pol
for 10 min at 37 °C and WRN was added subsequently, no full-length product
was observed; the profile of extension products resembled that obtained
with pol
alone (Fig. 3, lanes 7 and
8). These results suggest that binding of the 3'-primer
terminus and synthesis up to the start of the repeat sequence by pol
may have prevented binding and unwinding of the tetraplex by WRN
helicase. On the other hand, when the primer-template was pre-incubated
with WRN followed by the addition of pol
, synthesis past the
tetraplex structure was as robust as in reactions where WRN and pol
were incubated simultaneously (Fig. 3, lanes 9 and 10). However, the amount of paused products was less in
these reactions relative to those in which WRN and pol
were added
simultaneously. This is most likely due to binding and degradation of
the 3' primer terminus by WRN during the pre-incubation step (note the
predominant
1 products of degradations in lanes
9 and 10) preventing pol
from binding and
extending the primer.
at d(CGG)7 Template Tetraplex
Region--
Results presented above demonstrated that WRN can unwind
the tetraplex structure assumed by a d(CGG)n
repeat-containing DNA. We therefore determined whether the DNA helicase
activity of WRN was essential to allow pol
to traverse the template
tetraplex tract and to synthesize full-length product chains. To
address this question, we copied the tetraplex-containing template DNA by pol
in the presence of K577M mutant WRN protein. Substitution of
the lysine residue in the Walker A motif of the ATPase domain with
methionine eliminates NTP/dNTP hydrolysis by WRN to generate a
functional helicase-minus protein (16, 17). We confirmed that in fact,
K577M WRN failed to unwind the tetraplex substrate (data not shown),
although it retained in full its exonuclease activity (Fig.
4, lanes 4 and
5). We show that, in contrast to wild-type WRN (Fig. 4,
lanes 7 and 8), K577M WRN, at molar
concentrations equivalent to those of wild-type WRN, had no effect on
the ability of pol
to extend the primer stem beyond the pause site
(Fig. 4, lanes 9 and 10). Although the
primer was depleted, much of it was degraded by K577M WRN exonuclease
rather than extended by pol
. Thus, in order to enable pol
to
synthesize DNA past template tetraplex-induced pause sites, WRN must
maintain an active tetraplex DNA unwinding function.
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Fig. 4.
A helicase-deficient WRN protein does not
enable pol to traverse a template
d(CGG)7 tetraplex structure. End-labeled primed
d(CGG)7 tetraplex-containing template DNA was extended by
pol
(~0.7 fmol) in the absence or presence of equivalent amounts
(1 or 6 fmol) of wild-type (WT) or K577M mutant WRN protein.
The reactions were incubated at 37 °C for 15 min and processed as
described in the legend to Fig. 3. Lanes under the
Pol designation correspond to
32P-5'-primer-template DNA incubated in the absence of pol
with or without WRN.
at a d(CGG)7 Tetraplex Structure--
We recently
demonstrated that WRN uniquely stimulates DNA synthesis by pol
using a template that did not require unwinding by WRN. By contrast,
evidence presented above indicates that copying of a template
containing a d(CGG)n tetraplex domain by pol
necessitated disruption of the tetraplex by WRN helicase. We inquired,
therefore, if the permissive effect of WRN on tetraplex traversal by
pol
is also observed with other DNA polymerases.
*, the
two-subunit pol
complex lacking Pol32p (44, 48). Like pol
, pol
* also strongly paused at template nucleotide position 37, with
little extension beyond this point (Fig. 3, lane
11). However, neither pre-incubation of the reaction mixture with WRN (Fig. 3, lanes 16 and 17) nor
its addition simultaneously with the polymerase (Fig. 3,
lanes 12 and 13) allowed pol
* to traverse the template d(CGG)7 G'2 tetraplex. These results
contrast strikingly with the data obtained with pol
and WRN, and
are consistent with our original report that Pol32p is an essential component in mediating the functional interaction between WRN and pol
.
and pol
, to traverse the template tetraplex structure. As observed with pol
, the
d(CGG)7 tetraplex was an effective barrier to synthesis by
both pol
and pol
(Fig. 5).
Notably, however, these two polymerases stalled before the start of the
tetraplex. This was evident by the appearance of major pause sites at
nucleotide positions 34-37 corresponding to DNA sequences just before
and within the first trinucleotide repeat. When pol
was incubated
with WRN, at concentrations identical to those used with pol
, no
alleviation of pausing was discernible. Although a few faint
read-through product DNA chains were present, no full-length products
could be observed. Similar results were obtained with lower
concentrations of pol
where only 10-20% of the primer was
extended (results not shown). Although essentially similar, results
obtained with pol
differed slightly from those of pol
, in that
WRN did allow pol
to synthesize a small amount of full-length
product. However, the proportion of full-length DNA chains was only
~20% of the amount synthesized by pol
in the presence of WRN.
Thus, to a first approximation, it appears that the ability of WRN to
allow polymerases to traverse the tetraplex DNA structure may be
limited to pol
.
View larger version (39K):
[in a new window]
Fig. 5.
WRN does not allow DNA pol
or pol
to fully traverse
the template d(CGG)7 tetraplex structure.
32P-5'-Primer-G'2 d(CGG)7 template was extended
by pol
, pol
, or pol
without or with equivalent amounts of
WRN at 37 °C for 15 min as described. Primer extension products were
visualized by autoradiography following electrophoresis through a
denaturing 14% polyacrylamide gel.
at a Template
d(CGG)7 Tetraplex Region--
To determine whether
helicases other than WRN are also capable of allowing pol
to
traverse the template tetraplex region, we used two E. coli
helicases: RecQ, the prototypical RecQ family member homologue of WRN,
and UvrD. First, we investigated whether these two helicases could
unwind tetraplex DNA. We observed that RecQ, at molar concentrations
comparable to those of WRN, resolved the template G'2
d(CGG)7 tetraplex to generate duplex primer-template DNA
(data not shown). Likewise, UvrD also unwound this tetraplex. However,
unwinding was very inefficient requiring 100-500-fold higher molar
amounts of protein relative to RecQ or WRN to attain complete unwinding
(data not shown).
as described,
with either RecQ or UvrD substituting WRN. As demonstrated in Fig.
6, RecQ allowed pol
to traverse the
template d(CGG)7 G'2 tetraplex domain and to generate
full-length product DNA chains, characteristic of reactions containing
WRN. By contrast, UvrD did not alleviate stalling by pol
. Thus, it
appears that at least some RecQ family members that efficiently unwind
d(CGG)n tetraplex DNA structures preferentially
allow pol
to synthesize DNA beyond tetraplex-induced pause
sites.
View larger version (38K):
[in a new window]
Fig. 6.
UvrD helicase can not substitute for WRN in
extending tetraplex-containing template DNA by pol
. 32P-5'-Primer-G'2
d(CGG)7 tetraplex template was extended by pol
(~0.7
fmol) in the absence or presence of indicated amounts of WRN, RecQ, or
UvrD DNA helicases. Reaction conditions were as described under
"Experimental Procedures." Aliquots of the reaction mixtures were
electrophoresed through a 14% polyacrylamide urea gel and subjected to
autoradiography to visualize primer extension products.
at a Template
d(CGG)7 Hairpin Structure--
DNA containing
d(CGG)n repeats has been shown to fold
spontaneously into hairpin structures (34-37) that also block the
progression of synthesis by DNA polymerases (43). Extension of the
32P-5'-end-labeled 18-mer primer hybridized to the
hairpin-containing template was carried out with all three replicative
DNA polymerases as described under "Experimental Procedures."
Results of such an experiment, shown in Fig.
7, demonstrated that the
d(CGG)7 hairpin impeded progression of synthesis by pol
,
, and
. The major product chains terminated before and
within the first trinucleotide repeat sequence (pol
and pol
),
or immediately after the first repeat (pol
). Trace amounts of
full-length product chains were observed with the two processive DNA
polymerases, pol
and pol
.
View larger version (63K):
[in a new window]
Fig. 7.
WRN allows pol to
traverse a template d(CGG)7 hairpin structure.
32P-5'-End-labeled 18-mer primer was hybridized to the
d(CGG)7 hairpin-containing template DNA. The
primer-template (0.5 pmol) was extended by pol
, pol
, or pol
in the absence or presence of indicated amounts of WRN as described
under "Experimental Procedures." Reaction aliquots were
electrophoresed through a denaturing 14% polyacrylamide gel, and
primer extension products were visualized by autoradiography of the
dried gel.
had no
significant effect on the extension profile. Notably, no full-length
product chains accumulated in the presence of WRN. WRN did have a
minimal effect on reactions containing pol
, as evidenced by a
slight increase in the amount of full-length 61-nt product with 6 fmol
of WRN. By far, however, WRN had the most significant effect on
reactions carried out with pol
, allowing a larger fraction of the
extension products to reach the full-length size.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(27, 28) lends additional support for a role of WRN
in replication. However, the finding that stimulation of pol
activity by WRN occurs in the absence of the pol
accessory factor,
proliferating cell nuclear antigen (28), suggested that WRN may not
participate in processive DNA replication. This observation, together
with the finding that WRN can unwind alternate DNA structures (21-23), has led us to hypothesize that WRN may be involved in proliferating cell nuclear antigen-independent replication restart at forks blocked
by DNA damage or stalled by DNA secondary structures. In this report we
tested this hypothesis in part, by monitoring the effect of WRN on the
progression of synthesis by pol
through replication-impeding
hairpin and tetraplex DNA structures.
,
, and
(Figs. 3 and 4).
Extension of a primer by all three DNA polymerases stalls either just
before or within the first repeat of the trinucleotide sequence with no
product DNA chains discernible beyond this point. Even when the
concentration of polymerase is increased such that >90% of the primer
is utilized, the initiated DNA chains pause near the start of the
tetraplex region (data not shown).
at the tetraplex
domain (Fig. 3); a significant fraction of the product constitutes
61-nt-long full-length DNA chains. Several lines of evidence indicate
that alleviation of pol
pausing is a result of the tetraplex
d(CGG)n unwinding activity of WRN. First, WRN is
able to efficiently unwind the template d(CGG)7 G'2
tetraplex in the presence of dNTPs under conditions employed in primer
extension reactions (data not shown). This is consistent with our
previous results demonstrating that dNTPs can substitute for ATP in
WRN-catalyzed unwinding reactions (19). Second, the helicase deficient
K577M mutant WRN protein that is unable to unwind DNA (16, 17), also
fails to relieve tetraplex-induced stalling of pol
(Fig. 4). Third,
by changing the order of addition of WRN and pol
, we demonstrate
that alleviation of polymerase pausing requires that unwinding of the
tetraplex precedes synthesis or occurs simultaneously with DNA
synthesis by pol
(Fig. 3).
. WRN does not allow pol
or the two-subunit pol
enzyme, pol
*, to traverse the template tetraplex structure
(Figs. 3 and 5), and only a trace amount of full-length DNA products is observed in reactions containing WRN and pol
(Fig. 5).
to synthesize past the tetraplex, albeit less efficiently than
WRN (Fig. 6). This is not totally unexpected since RecQ and WRN belong
to the same family of DNA helicases (2). Further, these results are
consistent with the finding that replicative bypass of hairpin
structures in E. coli can occur via a RecQ
helicase-dependent pathway (53). In contrast to RecQ and
WRN, E. coli UvrD that can also unwind the
d(CGG)7 tetraplex does not alleviate pol
stalling at
this secondary structure. Based on these results, we propose that DNA
helicases of the RecQ family may serve to resolve tetraplex secondary
structures in DNA templates copied by pol
.
has been reported recently (27). The lack
of a permissive effect of WRN on replication of tetraplex DNA template
by pol
* indicates that the Pol32p subunit of pol
is required to
couple synthesis with unwinding. These results extend our previous work
that implicated Pol32p as an essential component in the functional
interaction between WRN and pol
(28). It should be noted that the
human pol
subunit (p50) that has been shown to interact physically
with WRN (27) is not the same subunit that is required for stimulation of pol
activity by WRN. Although seemingly discrepant, these findings are not mutually exclusive. It is conceivable that WRN physically associates with the p50 subunit of human pol
but requires the p66 subunit (homologue of S. cerevisiae Pol32p)
(54) for mediating its stimulatory effect on pol
activity.
and pol
(Fig. 7). Therefore, we
surmise that, although WRN can unwind the tetraplex structure,
synthesis by pol
and pol
cannot keep pace with unwinding,
allowing formation of hairpin structures that block the progression of
these polymerases. In contrast, synthesis by pol
, tightly coupled
to unwinding of the hairpin (Fig. 7) or tetraplex structure (Fig. 3) by
WRN, results in traversal of the DNA secondary structures and syntheses of full-length DNA product chains.
at
these template formations might be of biological significance.
d(CGG)n trinucleotide repeats are not restricted
to the untranslated region of the FMR1 gene. Computational
analyses have revealed a statistical over-representation of
d(CGG)n tracts in the human genome in genes
other than FMR1 (55, 56). Therefore, it is conceivable that
hairpins and tetraplexes formed by such
d(CGG)n-rich DNA may be the preferred target of
WRN helicase. Exposure of single-stranded regions of DNA during
replication of d(CGG)n tracts may result in the
formation of hairpins and tetraplexes. If these structures cannot be
resolved by WRN helicase, they would impede fork progression, induce
polymerase stalling and prolong the S-phase (25), as observed in WS
cells lacking WRN protein (57). Further, stalled forks could usher the
collapse of the replisome triggering recombination pathways that could
result in the generation of large DNA deletions that are also
characteristic of WS cells (58).
, pol
, and pol
. The constraints imposed on DNA synthesis can be
relieved by the action of a DNA helicase that can displace tetraplex
structures. However, we demonstrate that alleviation of polymerase
stalling shows specificity with respect to both the polymerase and the helicase. The combination of a RecQ helicase that can unwind
d(CGG)n hairpin and tetraplex structures, and
pol
allows for synthesis of full-length reaction products.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health NCI Grants CA77852 and CA80993 (to L. A. L.), a grant from the Cancerfonden of the Swedish Cancer Society (to E. J.), National Institutes of Health Grant GM58534 (to P. M. J. B.), Conquer Fragile X Foundation Inc., United States-Israel Binational Science Fund, Technion Vice President for Research, and a grant from the Fund for Promotion of Research in the Technion (to M. F.).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 may be addressed. Tel.: 206-543-6015; Fax: 206-543-3967; E-mail: laloeb@u.washington.edu.
** To whom correspondence may be addressed. Tel.: 972-4-829-5328; Fax: 972-4-851-0735; E-mail: mickey@tx.technion.ac.il.
Published, JBC Papers in Press, February 8, 2001, DOI 10.1074/jbc.M100253200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
WS, Werner syndrome;
WRN, Werner syndrome protein;
pol , DNA polymerase
;
pol
, DNA
polymerase
;
pol
, DNA polymerase
;
nt, nucleotide(s).
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