From the § Lineberger Comprehensive Cancer Center,
¶ Department of Pathology, Department of
Biochemistry and Biophysics, and
Curriculum in Genetics and
Molecular Biology, University of North Carolina,
Chapel Hill, North Carolina 27599-7295
Received for publication, October 17, 2002, and in revised form, November 14, 2002
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ABSTRACT |
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Triplet repeat tracts occur throughout the human
genome. Expansions of a (GAA)n/(TTC)n repeat tract
during its transmission from parent to child are tightly associated
with the occurrence of Friedreich's ataxia. Evidence supports DNA
slippage during DNA replication as the cause of the expansions. DNA
slippage results in single-stranded expansion intermediates. Evidence
has accumulated that predicts that hairpin structures protect from DNA
repair the expansion intermediates of all of the disease-associated repeats except for those of Friedreich's ataxia. How the latter repeat
expansions avoid repair remains a mystery because (GAA)n and
(TTC)n repeats are reported not to self-anneal. To characterize
the Friedreich's ataxia intermediates, we generated massive expansions
of (GAA)n and (TTC)n during DNA replication in
vitro using human polymerase Tracts of pure repeating triplets, referred to either as
trinucleotide- or triplet repeat
(TR)1 tracts, occur
throughout the human genome (1). Expansions of TR tracts located in
specific genes cause more than 14 hereditary neuromuscular diseases
(2). TRs associated with disease include 5'-d(CAG)n/5'-d(CTG)n, (abbreviated as CAG/CTG) associated with Huntington's disease and myotonic dystrophy, among others (reviewed in Ref. 2), GCC/GGC, associated with fragile X
syndrome, and GAA/TTC repeats, associated with Friedreich's ataxia
(FRDA). The disease expansions vary from a few triplets in a coding
region of the gene (type I) to hundreds of triplets located in a
noncoding region of the gene (type II). The expansions exactly mimic
the sequence of the repeat tract and occur during a single transmission
from parent to child.
Studies in several model systems indicate that repeat instability is
dependent on the length of the repeat tract and its orientation in the
genome; a TR sequence capable of forming hairpins, constructed by
cloning to be oriented relative to an origin of replication so that it
is the template replicated by discontinuous lagging strand replication,
is predicted to suffer deletions. The same sequence, oriented so that
it is the Okazaki fragment, is predicted to suffer expansions (3-5).
The discontinuous nature of lagging strand replication is characterized
by the presence of single-stranded regions in the template and free
5'-ends in the growing strand (Okazaki fragment) (for reviews, see
Refs. 6 and 7). The template single-stranded DNA (ssDNA) presents the
opportunity for formation of secondary structures such as hairpins (8). Lagging strand replication is predicted to lead to contractions by
replication across hairpins formed in the template strand and expansion
by DNA slippage to give hairpin formation in the Okazaki fragment (4,
5). Factors that increase DNA slippage should favor expansion over
contraction. CAG, CTG, GCC, and GGC TR tracts synthesized and studied
in vitro, form hairpin and hairpin-related tetraplex
secondary structures through self-annealing (9-12). The hairpins are
predicted, by the above mechanisms, to produce contractions as well as
expansions; both have been observed to occur during replication of TR
tracts in bacteria and yeast (4, 5, 8, 13).
The disease-associated, single-stranded expansion products may be
thousands of nucleotides in length, a great potential target for DNA
repair. Secondary structure formation within the single-stranded expansion intermediate is predicted to protect the expansion from repair activities of the cell, including mismatch repair and flap endonuclease (14-20). The latter removes 5' flaps of Okazaki fragments displaced by DNA synthesis. Secondary structures formed from the self-annealing of synthetic single-stranded GAA and TTC TRs are much
less stable (21-23) than the structures adopted by single-stranded CAG, CTG, GCC, and GGC TRs (9-12); GAA and TTC do not self-anneal under physiological conditions of temperature and salt (21-23), although (GAA)15 may self-anneal at low temperature (23).
Thus, when presented with heteroduplex DNA, generated by meiotic
recombination to contain single-stranded loops with different repeat
sequence tracts, yeast repairs 10-repeat GAA and TTC single-stranded
repeat tracts during meiosis but is much less efficient at repairing 10-repeat CAG, CTG, GCC, and GGC single-stranded repeat tracts (19).
The differences in repair presumably are caused by the different
abilities of the repeat tracts to self-anneal to form hairpins.
What intermediate structures are involved in FRDA TR expansions? Mixing
together GAA and TTC TR tracts gives three-stranded structures called
triplexes (purine-purine-pyrimidine and pyrimidine-purine-pyrimidine) (22, 24) and duplexes that associate strongly with each other called
sticky DNAs (25). The predicted formation of these structures appears
to be responsible for pausing of DNA replication within the repeat
tracts observed in vitro (24, 26-28). Blockage of DNA
replication within a repeat tract induces expansion in vitro and is predicted to induce DNA slippage and thus expansion in vivo (29, 30). This leaves open the question of how the large single-stranded regions resulting from massive expansions
characteristic of FRDA manage to avoid DNA repair.
TR expansion is predicted to occur in vivo as the result of
DNA slippage of the Okazaki fragment occurring during DNA lagging strand replication of the TR tract (31, 32) and possibly DNA slippage
during DNA repair (33). In vitro models of DNA replication and repair have been used to generate type I and type II repeat expansion. Initial attempts at TR expansions during DNA replication used the complementary triplet repeats as template and primer (34-38).
Recent studies have added unique sequences flanking the TR tract to
better mimic the in vivo situation of the repeats (29). The
latter study found that priming from within the TR tract gave much
higher levels of expansion compared with priming from within the
upstream unique flanking sequence. Priming from within the TR tract
mimics the occurrence of an Okazaki fragment completely within a repeat
tract. One of the most common lesions that arise in cellular DNA is the
loss of a base to give an abasic site (39), which blocks DNA
replication (40, 41). An abasic site analog, tetrahydrofuran (THF),
synthesized at the 3' terminus of the template-strand repeat tract
greatly enhanced expansion during replication of the template,
suggesting a possible role for DNA damage in TR expansion (29). The
lesion enhanced triplet repeat expansion presumably by increasing the
opportunities for DNA slippage by keeping the growing strand end within
the repeat tract (29). Another recent in vitro model system,
which uses unique sequences flanking the TR, mimics DNA slippage at a
nick during DNA repair synthesis within the DNA construct (42). Using these in vitro replication models, type I expansions of many
triplet repeats have been generated using bacterial polymerases
(34-37). Human pol Here we report using the lagging strand replication model (29) together
with h-pol GAA and TTC Expansion Reactions--
Pol
Klenow pol I replication reactions contained, in a final volume of 40 µl, 50 mM Tris-HCl (pH 8.0), 0.4 mM
MgSO4, 5 µM primer and template, 2 mM dithiothreitol, a 2 mM concentration of
either all four dNTPs or the two dNTPs required to replicate the
template repeat tract (as indicated), and 0.1 unit/µl of Klenow
polymerase (Promega). Reactions were started by the addition of
dithiothreitol, dNTPs, and polymerase, incubated for 4 h at
37 °C, and stopped by adding EDTA to 30 mM and cooling
to 4 °C. Reaction products were resolved by 8% PAGE in 6 M urea.
The sequences of the primer and template were (TTC)3 and
5'-dACTGTGTCTGTC(GAA)10GCGACCTGATCC, respectively, for TTC
expansions and (GAA)3 and
5'-dACTGTGTCTGTC(TTC)10GCGACCTGATCC for GAA
expansions. For study of the effects of an abasic site on expansion,
the abasic site analog THF was substituted at the 5'-end of the
repeat tract to give
5'-dACTGTGTCTGTC(FAA)(GAA)9GCGACCTGATCC and
5'-dACTGTGTCTGTC(FTC)(TTC)9GCGACCTGATCC as described
elsewhere (29). F indicates placement of THF within the repeat tract.
All template strands were synthesized to contain a three-carbon spacer
(Glen Research) at their 3' terminus that cannot be removed or extended
by DNA polymerase (not shown). Primers and templates were tested
individually for their ability to support expansion under the above
reaction conditions. Only primed templates supported TRE under our
reaction conditions.
DNA Sequencing--
The replication products from an 80-µl
nonradioactive expansion reaction were separated by electrophoresis on
a 2% agarose gel. A gel slice containing expansion products between
200 and 400 bp was excised from the gel, and the products were isolated with GenElute EtBr Minus spin columns (Sigma) and then dried. The
expanded DNA was reconstituted in replication buffer (final volume 20 µl), annealed to a 12-mer primer sequence (final concentration 62 µM), and replicated for 60 min at 37 °C to give
double-stranded product. The product was purified through a G-25
MicroSpin column (Amersham Biosciences). 3'-A overhangs were added for
cloning using Taq polymerase in PCR buffer (Roche
Molecular Biochemicals). The products were cloned into pCR2.1-TOPO
using a TOPO TA cloning kit (Invitrogen) following the manufacturer's
instructions. Plasmids were isolated from positive colonies using a
QIAprep Spin miniprep kit (Qiagen). The plasmids were analyzed for
large inserts via electrophoresis on 1% agarose gels. Only DNAs with
inserts within the expected size range were sequenced. DNA sequencing
was performed by the University of North Carolina Chapel Hill Automated
DNA Sequencing Facility.
Electron Microscopy--
Nonradiolabeled DNA replication using
biotin-labeled oligodeoxyribonucleotides (oligonucleotides) was
performed as described above. The oligonucleotides were synthesized as
above to contain a biotin-labeled nucleotide at their 5' termini. DNA
replication products were prepared for EM as described elsewhere (45).
TTC expansion products were cross-linked by adding
4'-hydroxymethyltrioxsalen to 0.25 µg/µl (41-µl total reaction
volume) and exposing the mixture, on ice, to 366-nm UV light for 1 h. GAA expansion products were cross-linked by adding
cisplatin·AgNO3 to 13.3 mM (60-µl total volume) and incubating for 2 h at 37 °C.
Endonuclease Digestion--
Expansion products used for P1
digestion were generated in reactions prepared to contain 0.03 µM triplet repeat primer and template and 0.03 µM h-pol
For P1 nuclease digestion, 0.3 pmol (total DNA) of h-pol
MboII, MnlI, and BbsI (New England
Biolabs) digests were performed in digestion buffer (30-µl total
volume) containing 50 mM NaCl, 10 mM Tris-HCl
(pH 7.9), 10 mM MgCl2, and 1 mM
dithiothreitol. MnlI reactions also contained bovine serum
albumin (100 µg/ml). For h-pol GAA and TTC Repeat Expansions--
To study expansion of the FRDA
repeats GAA and TTC, we employed an in vitro replication
system previously shown to generate massive expansions of AAT, ATT, and
CAG growing strand repeats using Klenow pol I in the presence of an
abasic site analog that blocks replication (29). The analog, THF, was
placed at the downstream 5'-end of the template repeat tract (Fig.
1) (29) (see "Experimental
Procedures"). The abilities of GAA and TTC growing strand repeats to
expand (where expansion means to a length greater than predicted from
the length of the template repeat tract) varied with the polymerase
used and the composition of the repeat (Fig. 1). Expansion was
manifested by gel electrophoresis as resolved bands and, at higher
molecular weights, an unresolved smear composed of many lengths of
expansion products (Fig. 1). Klenow pol I generally gave larger
expansions than h-pol DNA Sequencing--
The GAA and TTC TR expansion products were
replicated and cloned for DNA sequencing as detailed under
"Experimental Procedures." For GAA, 78 colonies were isolated, and
the plasmids were screened by size. Thirteen of the plasmids appeared
by size to have inserts and were sequenced. Six were found by
sequencing (not shown) to have inserts that varied in size from 6 to 66 repeats of perfect GAA (6, 8, 8, 11, 47, 61, and 66 repeats). For TTC,
40 colonies were isolated, and the plasmids were screened for inserts
by size. Fifteen of the plasmids appeared to have inserts and were
sequenced. Eight were found by sequencing (not shown) to have inserts
that varied in size from 5 to 140 repeats of perfect TTC (5, 8, 10, 28, 32, 46, 62, and 140 repeats). Larger expansions were most likely not
recovered because they are reported to be unstable in Escherichia
coli (4).
Electron Microscopy--
To visualize their secondary structures,
the GAA and TTC growing strand expansion products from h-pol Endonuclease Digestion--
The secondary structures of the GAA
and TTC growing strand expansion products were also probed using
restriction enzyme MboII and single strand endonuclease P1.
MboII cleaves only dsDNA and specifically recognizes the
sequences GAAGA and TCTTC, whereas nuclease P1 cleaves ssDNA randomly.
Nuclease P1 hydrolyzes dsDNA slowly. The expansion products were
clearly susceptible to cleavage by MboII in a
time-dependent manner (Fig.
3, A and B) but not by enzymes (BbsI and MnlI) that recognize
different, but related, sequences (GAAGAC/GTCTTC and GAGG/CCTC,
respectively) (not shown). The expansion products were 96% digested by
MboII in 30 min (Fig. 3, A and B).
Boiling the GAA and TTC expansion reaction products in replication
buffer followed by incubation at room temperature gave back
MboII-susceptible products (89% digested in 30 min) (Fig.
3A, lane 8). Nuclease P1 was titrated
against random sequence 54-base pair (bp) dsDNA and the random sequence
template strand to find the concentration of nuclease P1 that best
differentiated between dsDNA and ssDNA by cleavage efficiency (not
shown). Conditions were found that cleaved 98 ± 2%
(n = 4) of the ssDNA, as measured by loss of the 54-mer
band, but only 37 ± 7% (n = 4) of the dsDNA (Fig. 4, lanes
3-6). Under these conditions, 68 ± 10%
(n = 3) of the total expanded GAA growing strand DNA
(Fig. 4) and 46 ± 8% (n = 3) of the total
expanded TTC growing strand DNA (not shown) were digested by nuclease
P1. Total expanded DNA refers to all products with higher mobility than
the fully replicated template (indicated by the arrow in
Fig. 4 at 41-base length; since priming is within the repeat
tract, the longest labeled replicated product expected on a denaturing
gel is 41-mer). Digestion of ssDNA by nuclease P1 was characterized by
almost complete loss of the ssDNA band and production of a wide range
of faster mobility products dispersed below the starting material (Fig.
4, lane 6). Nuclease P1 favors production of 5'
mononucleotides. The labeled mononucleotide products were mostly
removed by gel filtration to remove salt for gel analysis. The effects
of nuclease P1 on the expanded products did not follow this pattern. As
seen from the digestion of the GAA growing strand expansion products by
nuclease P1 (Fig. 4, lanes 1 and 2),
the expanded intermediate bands at ~90 and 120 bp are each apparently
reduced in size by less than 10 bp. Quantitating the relative densities
of the apparent 90-bp region (Fig. 4, lane 1) and
80-bp region (lane 2), assuming that all of the
density of the apparent 80-bp band came from the 90-bp band, showed
that nuclease P1 digested 23 ± 4% (n = 3) of the 80-bp region. This was significantly less than the overall
amount of digestion of expanded product (see above). Therefore, the
ability of nuclease P1 to cleave different size expansion products was
assessed by measuring identical corresponding areas of lanes
1 and 2 that encompassed 50-65- and 80-95-base
lengths, as estimated from size markers in lane
7. For GAA, 85 ± 2% (n = 3) of the
50-65-base length products was digested, whereas 64 ± 7%
(n = 3) of the 80-95-length products was digested by
nuclease P1.
Two Versus Four dNTPs--
Occasional interruptions in the
expanded products caused by misinsertions of nucleotides complementary
to the repeat could contribute to stabilization of secondary structure.
To eliminate this possibility, the above expansion reactions for GAA
and TTC were repeated using only the two required dNTPs in each
reaction. Results of expansion reactions, of DNA sequencing of the
expansions, and of MboII cleavage of the expansion products
(Fig. 3C) were similar to the experimental results when all
four dNTPs were present. This is additional evidence against the
occurrence of GAA sequences within the expanded TTC products (and
vice versa) being responsible for hairpin formation.
Self-annealing of Synthetic Oligonucleotides--
Small synthetic
GAA and TTC single-stranded DNAs do not easily self-anneal (19,
21-23). We clearly observed by EM and nuclease cleavage experiments,
however, that expanded GAA and TTC growing strand DNAs form structures
consistent with hairpins. The GAA and TTC expansion products that
formed hairpins were significantly longer than the small synthetic GAA
and TTC repeats that provided evidence against hairpin formation. We
tested synthetic oligonucleotides synthesized to give GAA and TTC
repeats 99 and 51 nucleotides long (TTC results shown in Fig.
4D). MboII cleaved both the 99-mers (GAA cleaved
17 ± 4%, n = 6; TTC cleaved 44 ± 16%,
n = 7) and the 51-mers (GAA cleaved 12 ± 6%,
n = 6; TTC cleaved 48 ± 10%, n = 4).
To provide evidence for the secondary structures of the
intermediates involved in expansion of the repeats associated with FRDA, we generated massive expansions of GAA and TTC repeats in vitro using h-pol Given the different possible ways to generate apparent expanded
molecules (22, 43), we characterized the intermediate expansion
products using DNA sequencing, EM, and endonuclease digestion. In
addition, we repeated the experiments using only the two nucleotides
required to replicate the template TR tract so that contaminating
complementary nucleotides could not be incorporated into the expansion
products at a low level. Finally, we synthesized 99- and 51-mer GAA and
TTC oligonucleotides, and we determined their abilities to reanneal
using endonuclease digestion experiments.
DNA sequencing of cloned reaction products from the GAA and TTC
expansion reactions demonstrated that the expansion products mimicked
the growing strand repeat tract. The sizes of the sequenced products
are consistent with the instability of repeat tracts in E. coli (4). We cannot rule out the possibility that sequences that
varied from the GAA/TTC repeats were preferentially deleted during
cloning in E. coli. That seems unlikely, however, since interrupted sequences are relatively stable in E. coli (46), and the repeated isolation of pure GAA/TTC sequences argues that the
in vitro expansion products mimicked the growing strand
repeat sequences.
The thicknesses and secondary structures of the DNA products determined
by EM (Fig. 2) are consistent with the EM of duplex DNA (45). The
spreading technique used to prepare the DNA for EM collapses ssDNA
(45). The 5' ends of the DNA molecules were tagged with
biotin-streptavidin that is easily visualized by EM. Most of the GAA
and TTC expanded DNA molecules seen by EM were tagged at only one end.
This is consistent with hairpin formation (Fig.
5A). The expanded growing
strand becomes double-stranded by folding back to give a hairpin. The
large size of the expanded DNA relative to the replicated template
means that the molecule will appear tagged at one end independent of
whether the primer, template, or both are biotin-labeled (Fig.
5A). The duplex DNA molecules observed increased in size
with increasing reaction time. A percentage of the GAA and TTC growing
strand expansion products observed by EM were tagged at both ends only
when the primer was biotin-labeled. One possibility is that
biotin-labeled primer molecules interacted with single-stranded loops
formed by DNA fold back to give the hairpin structures (Fig.
5A). To test this possibility, the concentration of
unlabeled template DNA in the expansion reaction was doubled to compete
for unannealed primer molecules. The percentage of double-stranded
structures observed by EM to be tagged at both ends was reduced by
almost half by the 2-fold increase in template concentration. The
reduction supports the notion that a small amount of biotin-labeled
primer annealed to the expanded end of some of the double-stranded
structures observed by EM (Fig. 5A). The annealing may be
because of the presence of a single-stranded loop. The EM results
eliminate the possibility that the observed expansion products could
come from any kind of fill-in reactions involving linear arrays of
multiple overlapping molecules (22, 43). Such reactions would have produced visual tags between the two ends of the expanded molecules when the primer and template were biotin-labeled. The products of such
reactions are also predicted to have DNA sequences that vary from true
TR expansions.
and the Klenow fragment of
Escherichia coli polymerase I. Electron microscopy, endonuclease cleavage, and DNA sequencing of the expansion products demonstrate, for the first time, the occurrence of large and growing (GAA)n and (TTC)n hairpins during DNA synthesis. The
results provide unifying evidence that predicts that hairpin formation
during DNA synthesis mediates all of the disease-associated, triplet
repeat expansions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
(h-pol
) has been used to generate type I
expansions of GCC/GGC (35) and CAG/CTG (42). Massive type II expansions of AAT, ATT, and CAG growing strand repeat tracts were achieved using
the in vitro lagging strand model (29) with Klenow pol I
in vitro. (Throughout this paper we refer to the sequence of the expanded growing strand. Thus, expanded TTC repeat means that the
TTC growing strand grew beyond the length expected from replication of
the template strand.) The DNA sequences of the type I GCC/GGC expansions (35) and the type II expansions by Klenow pol I (29, 38)
were sequenced to provide evidence that the expanded reaction products
completely mimic the growing strand TR sequence. Such demonstrations
are important because other possible DNA expansion pathways, not based
on DNA slippage, are predicted to give products during DNA replication
in vitro that vary from the growing strand TR sequence (22,
43). Thus, in vitro DNA synthesis models are beginning to
successfully mimic many facets of TR expansion in vivo.
and Escherichia coli Klenow pol I to demonstrate that large expansions occur when these polymerases copy
GAA and TTC TRs in vitro. DNA sequencing and restriction enzyme cleavage demonstrate that the expansions have the correct repeat
sequences. EM and nuclease cleavage experiments demonstrate that the
respective expanded TTC and GAA growing strand repeats form
hairpin structures and thus self-anneal.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
replication
reactions contained, in a final volume of 40 µl, a 0.28 mM concentration of either all four dNTPs or just the two
dNTPs required for replication of the template repeat tract (as
indicated), 0.015 mCi of 32P-labeled dCTP or dATP, 5 µM annealed primer-template, and 0.025 units/µl of
h-pol
(purchased from CHIMERx or purified to >95% purity as
described elsewhere (44) and shown to have similar replication kinetics
and expansion behavior to h-pol
generously provided by Sam Wilson)
and replication buffer (final concentrations: 50 mM
Tris-HCl (pH 8.0); 10 mM MgCl2, 2.5% glycerol,
20 mM NaCl, and 2 mM dithiothreitol). The
reactions, which were started by the addition of dNTPs and polymerase,
were incubated at 42 °C for the indicated times. Reactions were
stopped by the addition of EDTA to 30 mM and cooling to
4 °C.
in a final volume of 20 µl. The reactions were carried out as described above. Control dsDNA was the product of a
fill-in reaction using h-pol
, a "random" sequence DNA template (5'-ACTGTGTCTGTCAGGCTATCGATAGACAGTACTGCATACAGAGCGACCTGATCC), a primer (5'-GGATCAGGTCGC), and radiolabeled dNTPs, under replication conditions described above. Control ssDNA was made by end labeling the
random DNA template using T4 polynucleotide kinase (New England Biolabs) and [32P]ATP.
expansion
products, control dsDNA, and control ssDNA were each added to P1 buffer
containing 200 mM NaCl, 50 mM sodium acetate (pH 7.4), 1 mM ZnSO4, 5% glycerol, and 0.0016 units of P1 nuclease (Sigma) in a final volume of 30 µl. Reactions
were incubated at 37 °C for 3 min and stopped by adding EDTA to 50 mM and cooling to 4 °C. Excess salt was removed by
passing the digestion products twice through G-25 MicroSpin columns
(Amersham Biosciences) before denaturing (7.5 M urea) 12%
PAGE analysis.
expansion products, 27 pmol of
expansion products were added to 5 units of restriction enzyme in
digestion buffer and incubated at 37 °C for 30 min. Reactions were
stopped by adding EDTA to 24 mM and cooling to 4 °C.
Some samples were boiled in digestion buffer for 5 min and allowed to
reanneal at room temperature before digestion. For MboII
digestion of synthetic oligonucleotides, 99- and 51-mer GAA and TTC
repeat tracts were synthesized by the LCCC DNA synthesis facility,
gel-purified, and end-labeled with T4 polynucleotide kinase (New
England Biolabs). End-labeled oligonucleotides were incubated in
replication buffer overnight, and 0.5 pmol was digested with a total of
7.5 units of MboII (2.5 units every 1 h were added to
give a final reaction volume of 31 µl) in digestion buffer at
37 °C for 3 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
(not shown). GAA was expanded by both
polymerases without the need to incorporate a replication-blocking DNA
lesion in the TTC template strand. In fact, GAA growing strand
expansion was reduced by the presence of THF at the end of the TTC
template repeat tract (Fig. 1). TTC also expanded in the absence of a
blocking lesion but showed significantly more expansion in the presence
of the replication block in the GAA template strand (Fig. 1). As found
for other growing strand repeats (29), expansion was much greater when replication was primed from within the repeat tract compared with priming from the upstream 3' unique flanking sequence of the template strand (not shown).
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Fig. 1.
Expansion of TTC (A) and GAA
(B) growing strand repeats during DNA replication
in vitro. TTC and GAA refers to the growing
strand sequences that expand beyond the replication lengths
(arrow) predicted from the lengths of the respective
templates. A phosphor image of denaturing 8% polyacrylamide gel (6 M urea, ~50 °C) is shown. Lanes
1-5 ( THF) and lanes
6-10 (+THF), expansion times 0-60 min.
Lane 11, 100-base pair ladder. Schematics
demonstrating the template-primer construct and placement of THF are
shown under the gel. The unique flanking
sequences are indicated by open rectangles. The
solid lines indicate the template repeat tracts.
The arrows represent the TTC (A) and GAA
(B) growing strand primers. The solid
vertical rectangle indicates the placement of THF
in the template strand at the 5'-end of the repeat tract (see Ref. 29
for detailed discussion of this template-primer construct).
and
Klenow pol I were cross-linked, where indicated, by psoralen/UV light
for TTC primer expansion products and cis-platinum for GAA expansion
products. Using primer and template strands synthesized to have
biotin-labeled nucleotides at their 5'-ends, we were able to recognize
the 5'-ends of the expansion products by EM after the addition of
streptavidin (molecules containing visible dots
in Fig. 2). The products were visualized
by EM using standard methods described elsewhere (45). Similar results
were obtained without cross-linking (Fig. 2). 70 and 75% of the
respective GAA and TTC expansion products (372 total molecules
observed) were visually tagged by streptavidin at only one end of the
expanded duplex when either the primer alone or both the primer and
template strands were labeled with biotin (Fig. 2, A-D).
The other 25 ± 7% (n = 5 for TTC, where n is the number of independent experiments) and 30% ± 1%
(n = 2 for GAA) of the products were visually tagged at
both ends (Fig. 2E). No molecules visually tagged at both
ends were observed when biotin-labeled template was used with unlabeled
primer for expansion. Titrating excess primer molecules with twice the
concentration of template DNA reduced the occurrence of double
end-tagged TTC and GAA expansion products (293 molecules observed) to
13 ± 4% (n = 2) and 18 ± 6%
(n = 2), respectively.
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Fig. 2.
Electron microscopic visualization of
expansion intermediates. A, TTC growing strand
expansion using Klenow pol I, template (GAA), and primer (TTC) tagged
with biotin-streptavidin (dots at the ends of DNA
molecules), cross-linked with psoralen and UV light. B, same
as A but using h-pol . C, GAA growing strand
expansion using Klenow pol I, only primer-tagged with
biotin-streptavidin (see dots) and no cross-linking.
D, GAA growing strand expansion using h-pol
, primer
tagged with biotin-streptavidin, cross-linked with cis-platinum.
E, TTC growing strand expansion (left
panel, only primer-tagged with biotin streptavidin) and GAA
growing strand expansion (right panel, primer and
template tagged with biotin-streptavidin), both using h-pol
. For
visual tagging, see dots at both ends of DNA molecules. Both
expansion products were cross-linked. The micrographs (A-E)
are shown in reverse contrast. Bar, 100 nm equivalent to
~300 base pairs of B-form DNA.
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Fig. 3.
Digestion of triplet repeat expansion
products by MboII restriction endonuclease.
A, digestion of TTC growing strand expansion products.
Lane 1, molecular weight markers. Lane
2, no enzyme. Lanes 3-7, 5 units of
MboII. Lane 8, expansion products were
boiled, reannealed, and treated with MboII. Native 6% PAGE
resolved the digestion products. Gels were analyzed using a
PhosphorImager. The arrow indicates finished replication of
template molecule. B, quantitation of the relative
amounts of MboII digestion in A. The areas
above the arrow in lanes
3-7 were measured by a PhosphorImager and plotted as
percentage decrease (increase in digestion) relative to lane
3. C, digestion of TTC and GAA growing strand
expansion products from replication with two versus four
dNTPs. Lane 1, molecular weight markers.
Lanes 2-6, TTC growing strand expansion.
Lanes 7-11, GAA growing strand expansion. h-pol
expansions in lanes 5, 6,
10, and 11 used all four dNTPs (dATP, dGTP, dCTP,
and dTTP), those in lanes 2-4 contained
only dTTP and dCTP, and those in lanes 7-9
contained only dGTP and dATP. Lanes 2,
5, 7, and 10, no MboII.
Lanes 3, 4, 6,
8, 9, and 11 were digested with 5 units of MboII. Lanes 4 and
9, reaction products were boiled and reannealed before
digestion with MboII. The presence of two versus
four dNTPs is indicated by the numbers 2 and 4 above the gel in the dNTPs
row. D, MboII digestion of
(TTC)17 and (TTC)33. Lane
1, (TTC)17 and no enzyme. Lane
2, digestion of (TTC)17 with MboII.
Lane 3, (TTC)33 and no enzyme.
Lane 4, digestion of (TTC)33 with
MboII. Lane 5, 100-bp marker.
Radiolabeled reaction products were resolved by denaturing 8% PAGE
containing 6 M urea and analyzed by a PhosphorImager.
Repeat tracts (99-mer and 51-mer) were synthesized by the LCCC DNA
synthesis facility and gel-purified. End-labeled (using T4
polynucleotide kinase and [32P]ATP) 99- and 51-mer (0.5 pmol) were incubated in expansion buffer for 3 h and digested with
MboII (7.5 units total; 2.5 units added at the beginning of
each hour for 3 h).
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[in a new window]
Fig. 4.
Digestion of GAA growing strand expansion
products by P1 nuclease. A phosphor image is shown of 12%
denaturing polyacrylamide gel with 7.5 M urea.
Lanes 1 and 2, GAA growing strand
expansion. Lanes 3 and 4, dsDNA
control. Lanes 5 and 6, ssDNA control.
Lane 7, molecular weight markers.
Lanes 1, 3, and 5, no P1.
Lanes 2, 4, and 6, 0.0016 units of P1 added. An arrow indicates finished replication
of template molecule.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
CONCLUSION
REFERENCES
and Klenow pol I. A lagging strand
replication model was used to generate the expansions (29). The model
uses a template strand with unique sequences flanking the repeat tract and a covalent block to template expansion synthesized at the 3'
terminus. A replication-blocking lesion, found necessary with this
model to generate massive expansions of AAT, ATT, and CAG growing
strands (29), was unnecessary for, and in fact interfered with, GAA
growing strand expansion. It significantly aided TTC growing strand
expansion, but TTC showed expansion without DNA damage. This is
consistent with the proclivity of GAA/TTC to form intramolecular
triplexes that block DNA replication primed farther upstream (26-28).
The presence of the damage in the template strand may interfere with
formation of the triplex when GAA is the template repeat.
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Fig. 5.
Schematics of secondary structure consistent
with EM visualized expansion products labeled at one end
(A) and generation of triplet repeat expansion using
floating primer (B). A, the template
contains a GAA repeat tract. The TTC expanded during replication to
give a TTC expanded region that formed a hairpin. The 5'-ends of the
primer and template are labeled with biotin-streptavidin. The template
region is too small, compared with the expanded region, to resolve by
EM. B, proposed mechanism to generate type II expansions
during DNA replication in vitro. Two mechanisms are shown.
Left panel (1), slippage and 5'-tail
formation; right panel (1), one or
both ends slip to form an internal loop. Following the initial
slippage, step 2 is formation of the initial hairpin, and step 3 shows
growing hairpin formation caused by continued replication and slippage
within the TR tract.
Endonuclease digestion gave independent confirmation of the double-stranded nature of the expanded primer strand. MboII recognizes the double-stranded sequences GAAGA and TCTTC and cleaves downstream of both sequences (47). MboII cleaved both the GAA and TTC expansion products in a time-dependent manner (Fig. 3A). On the other hand, the expanded products were relatively resistant to nuclease P1 under conditions that cleaved 98% of the control ssDNA but only 37% of the control dsDNA. Moreover, the results of nuclease P1 digestions of GGA repeat DNA provided evidence that longer dsDNA expansion products (80-95 bp) are more resistant to cleavage with nuclease P1 than shorter products (50-65 bp). These results suggest that GAA hairpin formation is dependent on DNA length and may help explain why earlier studies (21-23), which used shorter DNA lengths, failed to demonstrate GAA and TTC hairpin formation under physiological conditions. To test this hypothesis, longer GAA and TTC TRs (17 and 33 repeats) were synthesized and were shown to be partially susceptible to MboII cleavage under physiological conditions of temperature and salt concentration. Thus, the endonuclease cleavage experiments, like the EM experiments, support hairpin formation within the expanded strands. GAA and TTC hairpins are most likely stabilized by GA and TC base pairs, respectively. Alternating d(GA)n and d(TC)n repeats self-anneal (48-50), and possible base-pairing schemes have been determined from crystallographic and NMR studies (50-53).
Hairpin formation occurred during DNA replication in vitro
using a primer complementary to the repeat tract. Priming within the
repeat tract gave dramatic amounts of type II triplet repeat expansion.
No type II expansion was detected when replication was primed from the
unique upstream flanking sequence. The difference in expansion
abilities argues that free 3'- and 5'-ends within the repeat tract are
important for expansion. Free 3'- and 5'-ends could arise from the
occurrence of an Okazaki fragment completely within the boundaries of a
TR tract (31, 32) (Fig. 5B). The association with free
5'-ends is consistent with the preferred occurrence of TR expansion
during DNA lagging strand replication (3-5). The requirement for a
free 5'-end suggests at least two different possible pathways for DNA
slippage. The first pathway predicts that the growing strand slips back
along the template repeat tract, resulting in a 5' flap caused by
displacement of the 5'-end when it tries to overlap the unique upstream
flanking sequence. This flap should be a substrate for flap
endonuclease-1 (14-18, 20), unless either hairpin formation
occurs to protect the 5'-end from flap endonuclease-1 (20) or flap
endonuclease-1 activity is absent. The second pathway involves an
accordion-like slippage that predicts movement of both ends of the
growing strand to form a loop within the growing strand. The loop
should be a substrate for mismatch repair (19), unless either the loop
grows large enough to form a stable hairpin structure or mismatch
repair is defective (19). The requirement for longer repeat tracts for
GAA and TTC hairpin formation suggests that in vivo
FRDA-associated premutations may be longer than those for the other
triplet repeat diseases; the longer premutagenic TR tract may enable a
single replication slippage event to generate an initial expansion long enough to form a hairpin predicted to protect the expanded
single-stranded region from repair.
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CONCLUSION |
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The results demonstrate massive expansions of GAA and TTC
trinucleotide repeats implicated in Friedreich's ataxia during DNA replication by h-pol and Klenow pol I. The results further
demonstrate that both expansions form hairpins in vitro and
thus self-anneal. The results predict that all of the triplet repeat
diseases are mediated by hairpin formation.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295. Tel.: 919-966-8208; Fax: 919-966-3015; E-mail: mdtopal@med.unc.edu.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M210643200
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ABBREVIATIONS |
---|
The abbreviations used are:
TR, triplet repeat;
pol, polymerase;
h-pol , human polymerase
;
GAA/TTC, (GAA)n/(TTC)n;
FRDA, Friedreich's ataxia;
EM, electron microscopy;
THF, tetrahydrofuran;
dsDNA, double-stranded DNA;
ssDNA, single-stranded DNA.
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