Published online 1 October 2015
Nucleic Acids Research, 2015, Vol. 43, No. 20 10055–10064
doi: 10.1093/nar/gkv1008
Quadruplex formation by both G-rich and C-rich DNA
strands of the C9orf72 (GGGGCC)8•(GGCCCC)8
repeat: effect of CpG methylation
Bita Zamiri1,† , Mila Mirceta2,3,† , Karol Bomsztyk4 , Robert B. Macgregor, Jr.1 and Christopher
E. Pearson2,3,*
1
Received July 20, 2015; Revised September 05, 2015; Accepted September 22, 2015
ABSTRACT
INTRODUCTION
Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG
methylated with unknown consequences. Typically,
quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and
electrophoresis, we characterized the structures
formed by (GGGGCC)8 and (GGCCCC)8 strands
with and without 5-methylcytosine (5mCpG) or 5hydroxymethylcytosine (5hmCpG) methylation. All
strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in
K+ , Na+ or Li+ ), but no feature typical of i-motifs.
C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position,
hence C9orf72 quadruplexes still formed when N7deazaGuanine replace all Guanines. 5mCpG and
5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift
analysis, bound C-rich but not G-rich strands, with
a binding preference of unmethylated > 5hmCpG >
5mCpG, where methylated DNA-protein complexes
were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich
sequences interspersed with G-residues, one must
consider quadruplex formation and that methylation
of quadruplexes may affect epigenetic processes.
The most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is
a (GGGGCC)n•(GGCCCC)n repeat expansion in the
C9orf72 gene (1,2). In unaffected individuals, the most common repeat size is 2–19 units, while the pathogenic repeat size is 250–1600 repeats. ALS-FTD is one of more
than 40 neurological, neurodegenerative, and neuromuscular diseases caused by gene-specific repeat expansions,
such as myotonic dystrophy type 1 (DM1) and fragile X
mental retardation syndrome (FXS). C9orf72-expansions
show somatic repeat instability, aberrant CpG methylation,
and lead to toxic-RNAs, toxic, RAN-peptides, and lossof-function, pathological mechanisms, many of which have
been reported in DM1 and FXS (3,4).
Epigenetic modifications occur at the C9orf72 gene with
increased cytosine DNA methylation at CpG sites both in
the promoter and within the repeat of many but not all
C9orf72 expansion carriers (5–10). The CpG island and
promoter upstream, but not downstream of the GGGGCC
repeat is methylated in 36% of expansion carriers. However,
methylation within the C9orf72 repeat is present in all individuals with greater than 90 repeats (10). Reports have
not distinguished whether the C9orf72 methylation is the
canonical 5-methylcytosine (5mCpG) or the more recently
recognized 5-hydroxymethylcytosine (5hmCpG). C9orf72
methylation correlated with decreased levels of C9orf72
mRNA, RNA foci and RAN-dipeptide aggregates (6,7,9).
The expanded and methylated C9orf72 repeat may differentially bind specific proteins. The expanded C9orf72 repeats,
but not non-pathogenic repeats, have been reported to associate with a specific set of modified histones, which coincides with reduced C9orf72 mRNA expression (5). However, no study has assessed both C9orf72 DNA methylation
state and protein interaction. Although the role of epige-
* To
†
whom correspondence should be addressed. Tel: +1 416 813 8256; Fax: +1 416 813 4931; Email: [email protected]
These authors contributed equally to the paper as first authors.
C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
[email protected]
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
Graduate Department of Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto,
Ontario M5S 3M2, Canada, 2 Program of Genetics & Genome Biology, The Hospital for Sick Children, Toronto,
Ontario M5G 1L7, Canada, 3 Program of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A1,
Canada and 4 UW Medicine South Lake Union, University of Washington, Seattle WA 98109, USA
10056 Nucleic Acids Research, 2015, Vol. 43, No. 20
MATERIALS AND METHODS
DNA oligonucleotide synthesis and Labeling
All the DNA oligodeoxyribonucleotides were purchased as
purified from ACGT Corp., Toronto, Canada. The ODNs
were dialyzed versus Milli-Q water and lyophilized. The
concentrations were determined by recording their absorbance at 260 nm using molar extinction coefficients calculated using the nearest-neighbour method (21). Oligos
were then dissolved in the desired buffer to give concentrations ranging from 5 ␮M to 40 ␮M depending on the experiment. Oligonucleotides were end-labeled using [␥ -32 P]ATP.
Equal sample amounts, based on Cerenkov counting (5000
cpm/sample), were electrophoresed on 4% or 8% polyacrylamide gels at 15 V/cm for 60 min or 9 V/cm for 120 min, respectively. We also carried out electrophoresis under denaturing conditions. In this case, the samples were heat denatured in formamide, frozen on dry ice until loading and electrophoresed on pre-heated denaturing 6% 8M urea sequencing gels, run at 150 V/cm. Some degree of heterogeneity is
evident as both faster and slower migrating species that were
resistant to denaturation (Supplementary Figure S1). We,
and others have reported heat- and denaturation-resistant
structures formed by G- and C-rich sequences that aberrantly migrate faster or slower on denaturing gels (17,18,22–
24).
CD spectroscopy
CD spectra were recorded at room temperature using an
Aviv model 62 DS spectropolarimeter using 5- and 20-␮M
samples dissolved in the indicated buffers. The pH was adjusted to 7.5 with HCl. All the samples were heated to 95o C
and allowed to cool to room temperature overnight. Three
CD spectra, over the wavelength range of 320–220 nm were
recorded in a 1-mm cuvette and averaged.
UV spectroscopy
Melting experiments were carried out on a Cary model 300
Bio spectrophotometer. Depending on the concentration,
the samples were assessed in a 1-mm or 1-cm pathlength
cuvette. Oligos were dissolved in 10 mM Tris containing either 100 mM NaCl, KCl or LiCl, pH 7.5. Melting was monitored at 280 nm (for all oligos but unmodified and G-rich
5mCpG in KCl) or 290 nm. The heating rate was 1o C/min.
All experiments were done in triplicate.
Electrophoretic mobility shift assays
Reactions were carried out in a 20-␮l volume containing
∼80 fmol of [␥ -32 P]ATP end-labeled DNA with the indicated amounts of hnRNPK (Abnova). Binding reactions
contained a final buffer concentration of 10 mM Tris (pH
7.4), 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl2 ,
0.05% non-idet P40. DNA was preincubated with the buffer
described above for 30 min prior to the addition of protein
and poly(dI-dC). Reactions were incubated at room temperature for 20 min, loaded onto 4% native polyacrylamide
gels, electrophoresed at 200 V for 1 h in 1× Tris borate|
EDTA (0.089M Tris base, 0.089 M boric acid, 0.002 M
EDTA), dried and autoradiographed. The extent of binding was empirically assessed, using ImageQuant software,
by comparing the relative intensity of the band corresponding to the bound complex with that corresponding to the
unbound material.
RESULTS
Quadruplex formation by both C9orf72 repeat strands and
the effect of CpG methylation
We determined the structural effect of three different modifications, 5mCpG, 5hmCpG and N7-deazaG, on both the
G-rich and C-rich strands of the C9orf72 repeat by CD
spectroscopy and UV melting, in the presence of various
ions (Figures 1 and 2). In KCl, the unmodified G-rich strand
exhibits a CD spectrum with an intense positive peak at 290
nm, a negative peak around 260 nm a positive peak around
240 nm (Figure 1B). This spectrum is typical of antiparallel G-quadruplexes (25). In LiCl, the intensity at 290 nm
decreases and the negative peak at 260 nm turns into a positive shoulder with a small negative peak around 240 nm.
In LiCl, the population is shifted into a higher percentage
of parallel quadruplex structures reflected by the 260-nm
shoulder (Figure 1A). Methylating the cytosines at CpG
sites of the G-rich strand results in a more intense 260 nm
peak. In KCl the spectra of the unmodified and 5mCpG
G-rich strand are similar. When cytosines of CpG sites are
5-hydroxymethylated, in all salt conditions, the CD spectra exhibits a positive peak at 285 nm, a shoulder around
260 nm with a negative peak around 240 nm. Positive peaks
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
netics in C9orf72-associated ALD/FTD is not yet understood, characterizing the structural and biological affects
of C9orf72 repeat methylation may shed light on the mutagenic and pathogenic nature of the repeat.
Unusual DNA, RNA and R-loop (RNA–DNA) structures have been suggested to be involved in diseaseassociated repeat instability, or pathogenesis (11–13). Typically, G-rich strands are thought to form four-stranded Gquadruplexes, and C-rich strands have been demonstrated
to form intercalated i-motif structures at acidic pH (14,15).
Formation of G-quadruplex structures has been reported in
the RNA (GGGGCC)n repeat (16–18). The G-rich DNA
strand of the C9orf72 repeat can form quadruplexes (13).
Several proteins bind preferentially to quadruplex RNA
and DNA (19). Numerous proteins that bind quadruplexes
have been reported to bind the C9orf72 quadruplex RNAs
or RNA foci, including ASF/SF2, nucleolin, and hnRNPK
(13,17–20). There is little knowledge of protein interactions
with the C9orf72 DNA, and of the structure of the C-rich
strand.
Here, we examined the structures formed by the G-rich
and C-rich strands of the C9orf72 repeat with various modifications, including 5mCpG, 5hmCpG and N7-deazaG. We
show that both the C-rich and G-rich strands of the C9orf72
repeat can form four-stranded structures. We also demonstrate that 5mCpG, 5hmCpG and N7-deazaG modifications affect secondary structure. Furthermore, hnRNPK
binds to the C-rich DNA repeat and binding is affected by
methylation status.
Nucleic Acids Research, 2015, Vol. 43, No. 20 10057
at 285 nm suggest duplex base stacking (25). The stability of the cyclic structure of G•G•G•G-tetrads, a critical
component of G-quadruplexes, depends upon the ability of
the N7 of guanine to accept a hydrogen bond (26). We directly tested the importance of this by replacing guanine
residues with N7-deazaguanine, where the N7 position of
guanine has been replaced by CH, which is not a hydrogen bond acceptor. Previous use of N7-deazaG revealed a
strong dependence of G4-quadruplex (26). Here, modifying the N7 position of all guanines in the G-rich strands, in
all salt conditions, results in a broad positive peak around
270 nm and negative peak around 250 nm showing complete structural transition. This spectrum has been associated with G•C•G•C-tetrad formation (27). These results
support the suggestion that the peaks at 290 and 260 nm observed for the unmodified and 5mCpG G-rich strands arise
from quadruplex formation. All G-rich sequences were also
studied in the presence of LiCl or NaCl (Supplementary
Figures S2 and S3). Although LiCl is generally considered
too small to stabilize G-quadruplexes, interestingly the CD
spectrum of the C9orf72 oligos in the presence of LiCl is
very similar to that in NaCl (Supplementary Figures S2 and
S3). Quadruplex formation in the presence of LiCl has previously been reported for the FRAXA and telomeric repeats
(28,29).
The CD spectra of the C-rich strands are shown in Figure 1C and D. Spectra for the unmodified, 5mCpG, and
5hmCpG C-rich strands were qualitatively similar in LiCl,
with a positive peak at 280 nm, a negative peak at 250
nm and positive shoulder at 260 nm. Also in LiCl, an
isodichroic point is seen at 260 nm. A positive 260-nm
shoulder was not evident for the unmodified strand in KCl,
rather a negative 260-nm peak is evident which may due
to the stabilizing effect of KCl, turning the shoulder into a
more prominent peak. Methylation or hydroxymethylation
of CpGs do not alter the CD spectra significantly. Both
5mCpG, and 5hmCpG of the C-rich strands decreased the
intensity of the 280-nm peak, but retained the overall shape
of the spectra. The CD spectrum of the N7-deazaG C-rich
strands lost the 260-nm shoulder in LiCl, and retained the
280-nm and 250-nm peaks. This suggests that the 260-nm
peak arises from quadruplex formation even in the C-rich
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
Figure 1. CD spectra of indicated C9orf72 DNAs. All experiments were done at 5 ␮M strand concentration, in 10 ␮M Tris (pH 7.5), and 100 ␮M of the
indicated salt.
10058 Nucleic Acids Research, 2015, Vol. 43, No. 20
strands, which is probably due to the high fraction of guanines present. Tetrabutylammonium (TBA) is too large to
stabilize G-quartet formation (30). Interestingly, in the presence of TBA, while the 280-nm peak of the unmodified,
5mCpG, 5hmCpG C-rich strands was retained, the 260-nm
shoulder disappeared. For N7-deazaG C-rich strands, the
CD spectra were the same in TBA, KCl and NaCl. Similar to the G-rich strands, all spectra were similar in LiCl
and NaCl, suggesting that the conformation does not depend strongly on the nature of the cation (Supplementary
Figures S2 and S3).
We performed UV melting on the same samples, under
identical conditions as above. The unmodified and 5mCpG
G-rich strands exhibit the highest thermal stability in all
three ions (Figure 2A, Supplementary Figure S2). Interestingly, the unmodified and 5mCpG, G-rich strands exhibited
lower stability in KCl than in NaCl or LiCl. This is likely due
to the fact that in in KCl there is a single folding unit of antiparallel quadruplexes and the melting curve reflects these
species (measured at 290 nm), while in NaCl and LiCl the
CD spectra suggests multiple folding forms and the melting profiles may reflect denaturation of other folding units
(recorded at 280 nm). The 5hmCpG G-rich strands all have
lower stability than their unmodified forms, but higher than
the N7-deazaG strands. For the C-rich strands the melting
temperatures are lower than for the G-rich strands (Figure 2C and D). The unmodified and 5mCpG oligos all show
concentration-dependent melting temperatures, suggesting
possible multimolecular structure formation. Both the Grich and the C-rich N7-deazaG-substituted oligos are more
stable in LiCl than in than NaCl or KCl.
Methylation status of the C9orf72 repeat DNA affects its
electrophoretic migration
To further evaluate the structural heterogeneity and complexity of the various forms of the (GGGGCC)8 and
(GGCCCC)8 oligonucleotides, we assessed their electrophoretic migration on native polyacrylamide gels follow-
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
Figure 2. UV melting curves of indicated C9orf72 DNAs. All experiments were done at 5 ␮M strand concentration, in 10 ␮M Tris (pH 7.5), and 100 ␮M
of the indicated salt. Melting temperature units are ◦ C.
Nucleic Acids Research, 2015, Vol. 43, No. 20 10059
The (GGCCCC)8 strand is preferentially bound by hnRNPK
and affected by methylation
Toward addressing a biological effect of C9orf72 repeat
modifications, we assessed the binding of hnRNPK. To
date, there is no evidence on the effect of DNA methylation at the C9orf72 repeat and protein interactions. Using
recombinant hnRNPK, we observed preferential binding of
the protein to the C-rich repeat DNA (Figure 4). Unmethylated C-rich DNA was bound with complexes resolved as
two distinct slower migrating species. The other modifications gave rise to broad smears of species with increasing
amounts of hnRNPK, with most of the DNA-protein complex unable to enter the gel, likely due to increased structural complexity. Notably, the 5hmCpG form was preferentially bound (38%) over the 5mCpG form (14%), while
the unmethylated form most efficiently bound (68%). Previously, protein interactions with r(GGGGCC)n and other
repeat sequences reported the retention of the nucleic acidprotein complex in the well (18,31,32). Treatment with proteinase K and SDS broke down these complexes, allowing
the DNA to migrate similarly to the control without protein. The unmethylated G-rich DNA was also able to bind,
although very minimally, to the protein, creating a complex
that migrated faster than the DNA-protein complexes of Crich DNA. We did not observe binding to the modified Grich sequences. These results imply that methylation status
and type can alter the biological recognition of the C9orf72
repeats.
DISCUSSION
We demonstrate that both the C-rich and G-rich DNA
strands of the C9orf72 repeat can form a heterogenous population of structures, including those with features of fourstranded structures. It is accepted that a given G-rich and
C-rich sequences can form numerous structural conformations (33). Previously, it was reported for shorter G-rich
C9orf72 repeats (≤4 repeat units) that these form high levels of structural variants (34) – our findings are consistent with their the suggestion that such structural variation may be reflected in vivo and may be considerable for
longer repeat tracts. That the C-rich strand can assume
a quadruplex is distinct from many other studies, most
of which have focused upon the G-rich strand, with little attention to the complementary C-rich strand (telomere repeats is one example). In cases where the complementary C-rich strand has been studied, it has often been
reported that these form i-motifs under non-physiological
pH. We propose that the four-stranded species formed by
C-rich C9orf72 strands are stabilized by G•C•G•C-tetrads
and C•G•C•G-tetrads, which would sandwich C•C•C•Ctetrads (a predicted structure is shown in Figure 5A). Our
findings suggest that for C-rich strands, which also contain G-residues, one must consider four-stranded quadruplex formation.
We propose that both the C-rich and G-rich C9orf72
quadruplexes involve G•C•G•C-tetrads and C•G•C•Gtetrads (Figure 5A). Mixed G•C•G•C-tetrads are structurally distinct from G•G•G•G-tetrads. We propose that
the distinct CD peak or a shoulder at 260 nm that we, and
others observed for both C9orf72 DNA strands (13) reflects
the presence of G•C•G•C-tetrads. Notably, G•C•G•Ctetrads can assume at least four different inter-base interactions (27,35–46). Unlike G•G•G•G-tetrads, which are
stabilized by hydrogen bonding with the N7 position of
the guanine residues (26), some, but not all G•C•G•Ctetrads form independent of the N7-G, with hydrogen bonding of the cytosine amino protons either with the O6 oxygen of guanine (35,46). Other G•C•G•C-tetrad conformations do utilize hydrogen bonding with the N7 nitrogen of guanine (35,46). The absence of an absolute requirement of the N7-G may explain the ability of the
C-rich C9orf72 strand to form four-stranded structures
at physiological pH. The limited effect of N7-deazaG on
the CD spectrum of the C-rich strand, which is not predicted to involve G•G•G•G-tetrads, may be due to some
of the GCGC-tetrads formed by the C-rich strand involve
the N7 of G residues and hence are affected by the N7deazaG. Moreover, unlike G•G•G•G-tetrads, which require cation coordination for stability, G•C•G•C-tetrads
do not require cations for stability (35–44,46), which
may explain the insensitivity of C9orf72 quadruplex formation to LiCl or TBA. It is notable that some G4quadruplexes, including that formed by the FRAXA CGG
repeat, are extremely stable even in the presence of lithium
ions (28,29). A recent study of an oligo with a repeat of
(GGGGCC)3GG(Br)GG with an 8-bromoguanine substitution, did not detect G•C•G•C-tetrads, which may be due
to the preferential adoption of a syn-glycosidic conformation by 8-bromoguanines (47). Similarly, an NMR study of
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
ing pre-incubation in various salt solutions. An unusual pattern of electrophoretic species was evident for each, with
slight variations in migration existing between the different salts (Figure 3). In general, each oligo migrates as a
series of closely-spaced bands, with the C-rich repeats migrating slightly slower than their G-rich counterparts. A
slower migrating, but minor species, was evident for the Grich strand in all of its forms, with the exception of the N7deazaG variant. This second band migrated similarly for the
samples incubated with LiCl and NaCl, which was faster
than the samples that were in the KCl or no salt condition.
This slower migrating band disappears in denaturing gel
conditions (Supplementary Figure S1). The 5hmCpG and
N7-deazaG oligos migrate much slower than the unmethylated and 5mCpG forms. The greatest heterogeneity is displayed by the 5 -hydroxymethylated CpG forms of the Gand C-rich repeats, as evidenced by the broad smear of electrophoretic species. Curiously, unlike the 5mCpG, which
migrated faster than the unmethylated forms, the 5hmCpG
migrated the slowest. Furthermore, the 5hmCpG oligos
are distinctly different from the unmethylated and 5mCpG
methylated oligos in terms of their migration. The 5hmCpG
oligos migrate much slower in comparison, suggesting they
form different structures than the unmethylated and canonical 5mCpG modification. The N7-deazaG modified DNA,
which is unable to form G-quadruplex structures, exhibits
much more structural heterogeneity than its unmethylated
counterparts, as evidenced by the broad smear.
10060 Nucleic Acids Research, 2015, Vol. 43, No. 20
Figure 4. Methylation type influences DNA-protein interactions. Human recombinant hnRNPK band-shift with the [␥ -32 P] ATP end-labeled
d(GGGGCC)8 or d(GGCCCC)8 DNAs (∼80 fmol) and 0, 50, 200 or 400 ng of purified recombinant hnRNPK. DNAs are either not CpG methylated,
or contain 5-methylcytosine or 5-hydroxymethylcytosine bases at each CpG within the sequence. Samples were pre-incubated with the protein in a buffer
solution containing 10 mM Tris (pH 7.4), 50 mM KCl, 1 mM DTT, 2.5% glycerol, 5 mM MgCl2 , and 0.05% Non-idet P40. Samples were electrophoresed
on a 4% native polyacrylamide gel for 1 hour at 200 volts (15V/cm). Lanes were densimetrically assessed using ImageQuant software to quantify the
percent bound (%).
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
Figure 3. Methylation type and salt influence electrophoretic migration patterns. Migration of [␥ -32 P] ATP end-labeled DNA (frozen and thawed at
room temperature) in native 8% polyacrylamide gel. [␥ -32 P] ATP end-labeled (GGGGCC)8 and (CCCCGG)8 containing the indicated modifications were
dissolved in 50 mM Tris–HCl (pH 7.4) with the indicated salt, incubated for 20 min and then electrophoresed for 2 h at 120 V (9 V/cm).
Nucleic Acids Research, 2015, Vol. 43, No. 20 10061
short tracts of (GGGGCC)n with either 1, 2 or 4 repeats,
did not reveal G•C•G•C-tetrads (34). It is possible that the
involvement of G•C•G•C-tetrads may require longer tracts
of at least eight C9orf72 repeat units.
Our data imply that the C-rich C9orf72 quadruplex can
involve C•C•C•C-tetrads (Figure 5A). These may be stabilized via stacking interactions that arise from sandwiching them between sets of G•C•G•C-tetrads or G•G•G•Gtetrads. Sandwiched C•C•C•C-tetrads in the FRAXA
(CGG)n repeat were proposed to be stabilized by adjacent
G•G•G•G-tetrads (48) and elsewhere (49).
Most quadruplexes reported involve G•G•G•G-tetrads.
However, some G-rich oligos with limited numbers of
C residues can form quadruplexes involving G•C•G•Ctetrads and C•C•C•C-tetrads (27,35,36,42,46,50,51). For
example, the disease-associated fragile X (CGG)n repeat
can form quadruplexes involving G•C•G•C-tetrads and
C•C•C•C-tetrads (35,36,42,48). Notably, the FRAXA CD
spectra also revealed a 260 nm peak/shoulder (48). It is
widely accepted that quadruplex structures will involve G4
tetrads composed of four interacting guanine residues. Such
an expectation has led to the prediction that any sequence
conforming to the sequence consensus G≥3N1–7G≥3N1–
7G≥3N1–7G≥3 will form G4 quadruplexes. However,
quadruplexes may also be stabilized by G•C•G•C-tetrads;
thus, C-rich sequences with interspersed G residues can
also form stable, four-stranded structures (35–45). Our
finding here that the C-rich C9orf72 strand presents as a
quadruplex-forming sequence does not conform to the expected consensus. Thus, while it is reasonable to expect
that any sequence that conforms to the G≥3N1–7G≥3N1–
7G≥3N1–7G≥3 consensus will form a quadruplex, the reverse is not necessarily true, in that all quadruplex-forming
sequences may not conform to the consensus. Interestingly,
G•C•G•C-tetrad-containing quadruplexes have long been
proposed to act in various biological processes including the
junction of replication forks (38) and homologous pairing
of DNAs by the intercoiling of two Watson-Crick paired
helices, which may facilitate recombination (52).
Methylation of DNA is an important epigenetic mark associated with altered gene expression, replication and genetic instability. Both methylcytosine and hydroxymethylcytosine at CpG sites of each repeat unit could alter the
structures of both C9orf72 strands. The increased and decreased melting temperatures of the 5mCpG and 5hmCpG
forms may serve a regulatory role when DNA strands must
separate, as during DNA transcription, replication, repair,
or recombination. Increased and decreased thermal stability have been reported for 5mCpG and 5hmCpG sites at
non-repeated sequences (53–57). The altered thermal stability of the methylated repeats may affect the propensity of the C9orf72 repeats to form unusual DNA structures. The faster and slower electrophoretic migration of
the 5mCpG and 5hmCpG forms we observed (Figure 3)
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
Figure 5. Proposed quadruplex structure in the C9orf72 8-repeat sequence and biological implications regarding R-loop formation. (A) Model of quadruplex structure forming in the (GGGGCC)8 and (GGCCCC)8 strands with various tetrad assemblies. Green colored boxes represent methylatable cytosines
within the repeat sequence. (B) Intramolecular quadruplexes can form by either the G-rich or C-rich DNA strands of the C9orf72 repeat and may stabilized
R-loops. They can also reflect the ‘pearls’ in a ‘pearls-on-a-string’ model of slipped-quadruplexes. See text.
10062 Nucleic Acids Research, 2015, Vol. 43, No. 20
here, could reflect the displaced DNA strand in the RNADNA hybrids (R-loop) (Figure 5B), previously shown to
form on either of the C9orf72 strands (13,17). R-loops
can be biophysically stabilized by quadruplex formation
of the displaced G-rich DNA strand at non-repetitive sequences (67). Notably, our observation herein, that the Crich C9orf72 DNA can also assume a quadruplex structure suggests that it may stabilize R-loops having RNAs hybridized to the G-rich strand. Thus, R-loop formation on
either DNA strand of the C9orf72 repeats could be stabilized by quadruplex formation by either of the displaced
DNA strands. Similarly, the intramolecular quadruplexes
formed by the G-rich and C-rich C9orf72 DNA strands that
we report here, could reflect the ‘pearls’ in a ‘pearls-on-astring’ type model of slipped-quadruplexes (Figure 5B). Alternately, a ‘bubble’ of separated strands, where each forms
a series of quadruplexes may stabilize strand separation
for some function, possibly the initiation of DNA replication, as several studies reveal that metazoan replication
initiation sites coincide with quadruplex-forming sequences
(68–72). Since transcription could potentially occur in the
64% of C9orf72 expansion carriers that do not have adjacent promoter CpG methylation but have a methylated repeat, the kind of methylation could modulate the formation of the above structures. Although the role of epigenetics
in C9orf72-associated ALD/FTD is not understood, characterizing the structural and biological affects of C9orf72
repeat methylation may shed light on the mutagenic and
pathogenic nature of the repeat.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
CEP conceived, designed, and coordinated the study. BZ,
MM, RBM and CEP wrote the manuscript. BZ performed
and analyzed the experiments in Figures 1 and 2. RBM designed and interpreted the results of Figure 1. MM performed and analyzed the experiments in Figures 3 and 4.
KB provided intellectual input regarding hnRNPK. All authors reviewed the results and approved the final version of
the manuscript.
FUNDING
Weston Brain Institute (to C.E.P.); Amyotrophic Lateral Sclerosis Association of America (to C.E.P.); Natural Science and Engineering Research Council of Canada
(to C.E.P., R.B.M.); National Institutes of Health [R01
DK083310, R21 GM111439, R33 CA191135 to K.B.].
Funding for open access charge: Weston Brain Institute
Grant to Christopher E. Pearson.
Conflict of interest statement. None declared.
REFERENCES
1. DeJesus-Hernandez,M., Mackenzie,I.R., Boeve,B.F., Boxer,A.L.,
Baker,M., Rutherford,N.J., Nicholson,A.M., Finch,N.A., Flynn,H.,
Adamson,J. et al. Expanded GGGGCC hexanucleotide repeat in
noncoding region of C9ORF72 causes chromosome 9p-linked FTD
and ALS. Neuron, 72, 245–256.
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
may reflect increased and decreased compaction of the
DNA. The methylation state of non-repetitive sequences
has been shown to enhance or impair the ability to form
unwound DNA, bent-DNA, cruciforms, Z-DNA, quadruplexes and triplex–DNA (58–60). We propose that 5mCpG
of the C9orf72 repeats stabilizes the quadruplex, similar to
its effect upon FRAXA repeats (29), while 5hmCpG destabilizes the quadruplexes. Unusual structure formation by
the C9orf72 repeats may contribute to pathogenesis and
their genetic length instability, as demonstrated for R-loops
(13,17). In fragile X patients, an absence of CpG methylation of the CGG/CCG repeats has been linked to enhanced
somatic contractions of the expanded repeats in patient tissues (61–63). It will be interesting to assess the possible link
of methylation to C9orf72 repeat instability. Our findings
suggest that methylation state, as well as the form of methylation, may alter C9orf72 repeat structure. Distinguishing
the kind of CpG methylation at the C9orf72 promoter and
repeat, in specific tissues may reveal functional roles of this
epigenetic mark.
The expanded and methylated C9orf72 repeat may differentially bind specific proteins. Specific histone modifications have been reported at the expanded C9orf72 repeats,
but not non-pathogenic repeats (5,6); however, the DNA
methylation status in these studies was not assessed. We
show CpG methylation-sensitive binding of hnRNPK to
C9orf72 repeat DNAs. hnRNPK bound preferentially to
the C-rich DNA strand, consistent with previous studies
showing an association of hnRNPK with the C-rich RNA
foci in cells with C9orf72 expansions (13,64). However, direct binding of hnRNPK to the C-rich C9orf72 RNA was
not observed (64). Our finding that hnRNPK bound to the
C-rich DNA strand, is consistent with the reported preference of hnRNPK to bind DNA over RNA (65). Our data
shows that hnRNPK binding is affected by CpG methylation of the C9orf72 repeats. Protein complexes with the
r(GGGGCC)n and r(CUG)n repeat sequences have been
reported to be retained in the wells of electrophoresis gels
(18,31). We postulate that the methylated C9orf72 DNA–
protein complexes are retained in the wells because they
form multimeric DNA-protein structures incapable of entering the gels as opposed to a structural alteration of the
DNA. This is supported by the observation that treatment
of the complexes with proteinase K permits the proteinfree DNA to co-migrate with the starting DNA. The implications of this differential binding is unknown, however,
the observation that DNA methylation can alter both the
biophysical traits and the interaction of proteins supports
the concept that this epigenetic modification may be mediated by structural and protein selective avenues. hnRNPK
functions in many genetic processes, including transcription, translation, chromatin remodeling and DNA repair
(66).
Considerable evidence supports the formation of unusual
DNA, RNA or RNA-DNA structures by disease associated repeats, structures that participate in mutagenic and
pathogenic processes. It is very likely that our preparations
contain mixtures of different folded forms, antiparallel, parallel quadruplexes along with hairpins, as previously suggested (13). The intramolecular structures formed by the
G-rich and C-rich C9orf72 DNA strands that we report
Nucleic Acids Research, 2015, Vol. 43, No. 20 10063
21. Cantor,C.R., Warshaw,M.M. and Shapiro,H. (1970) Oligonucleotide
interactions. 3. Circular dichroism studies of the conformation of
deoxyoligonucleotides. Biopolymers, 9, 1059–1077.
22. Shida,T., Yokoyama,K., Tamai,S. and Sekiguchi,J. (1991)
Self-association of telomeric short oligodeoxyribonucleotides
containing a dG cluster. Chem. Pharm. Bull. (Tokyo), 39, 2207–2211.
23. Hirao,I., Naraoka,T., Kanamori,S., Nakamura,M. and Miura,K.
(1988) Synthetic oligodeoxyribonucleotides showing abnormal
mobilities on polyacrylamide gel electrophoresis. Biochem. Int., 16,
157–162.
24. Hirao,I., Nishimura,Y., Naraoka,T., Watanabe,K., Arata,Y. and
Miura,K. (1989) Extraordinary stable structure of short
single-stranded DNA fragments containing a specific base sequence:
d(GCGAAAGC). Nucleic Acids Res., 17, 2223–2231.
25. Vorlickova,M., Kejnovska,I., Bednarova,K., Renciuk,D. and Kypr,J.
(2012) Circular dichroism spectroscopy of DNA: from duplexes to
quadruplexes. Chirality, 24, 691–698.
26. Murchie,A.I. and Lilley,D.M. (1992) Retinoblastoma susceptibility
genes contain 5 sequences with a high propensity to form
guanine-tetrad structures. Nucleic Acids Res., 20, 49–53.
27. Viladoms,J., Escaja,N., Frieden,M., Gomez-Pinto,I., Pedroso,E. and
Gonzalez,C. (2009) Self-association of short DNA loops through
minor groove C:G:G:C tetrads. Nucleic Acids Res., 37, 3264–3275.
28. Lee,J.Y., Yoon,J., Kihm,H.W. and Kim,D.S. (2008) Structural
diversity and extreme stability of unimolecular Oxytricha nova
telomeric G-quadruplex. Biochemistry, 47, 3389–3396.
29. Fry,M. and Loeb,L.A. (1994) The fragile X syndrome d(CGG)n
nucleotide repeats form a stable tetrahelical structure. Proc. Natl.
Acad. Sci. U.S.A., 91, 4950–4954.
30. Fan,H.Y., Shek,Y.L., Amiri,A., Dubins,D.N., Heerklotz,H.,
Macgregor,R.B. Jr and Chalikian,T.V. (2011) Volumetric
characterization of sodium-induced G-quadruplex formation. J. Am.
Chem. Soc., 133, 4518–4526.
31. Yuan,Y., Compton,S.A., Sobczak,K., Stenberg,M.G.,
Thornton,C.A., Griffith,J.D. and Swanson,M.S. (2007)
Muscleblind-like 1 interacts with RNA hairpins in splicing target and
pathogenic RNAs. Nucleic Acids Res., 35, 5474–5486.
32. Warf,M.B. and Berglund,J.A. (2007) MBNL binds similar RNA
structures in the CUG repeats of myotonic dystrophy and its
pre-mRNA substrate cardiac troponin T. RNA, 13, 2238–2251.
33. Largy,E. and Mergny,J.L. (2014) Shape matters: size-exclusion HPLC
for the study of nucleic acid structural polymorphism. Nucleic Acids
Res., 42, e149.
34. Sket,P., Pohleven,J., Kovanda,A., Stalekar,M., Zupunski,V.,
Zalar,M., Plavec,J. and Rogelj,B. (2015) Characterization of DNA
G-quadruplex species forming from C9ORF72 G4C2-expanded
repeats associated with amyotrophic lateral sclerosis and
frontotemporal lobar degeneration. Neurobiol. Aging, 36, 1091–1096.
35. Zhang,N., Gorin,A., Majumdar,A., Kettani,A., Chernichenko,N.,
Skripkin,E. and Patel,D.J. (2001) Dimeric DNA quadruplex
containing major groove-aligned A-T-A-T and G-C-G-C tetrads
stabilized by inter-subunit Watson-Crick A-T and G-C pairs. J. Mol.
Biol., 312, 1073–1088.
36. Kettani,A., Kumar,R.A. and Patel,D.J. (1995) Solution structure of a
DNA quadruplex containing the fragile X syndrome triplet repeat. J.
Mol. Biol., 254, 638–656.
37. Kettani,A., Bouaziz,S., Gorin,A., Zhao,H., Jones,R.A. and Patel,D.J.
(1998) Solution structure of a Na cation stabilized DNA quadruplex
containing G.G.G.G and G.C.G.C tetrads formed by G-G-G-C
repeats observed in adeno-associated viral DNA. J. Mol. Biol., 282,
619–636.
38. Lowdin,P.O. (1969) Some aspects of the hydrogen bond in molecular
biology. Ann. N. Y. Acad. Sci., 158, 86–95.
39. Kubitschek,H.E. and Henderson,T.R. (1966) DNA rplication. Proc.
Natl. Acad. Sci. U.S.A., 55, 512–519.
40. McGavin,S. (1971) Models of specifically paired like (homologous)
nucleic acid structures. J. Mol. Biol., 55, 293–298.
41. Mitas,M., Yu,A., Dill,J., Kamp,T.J., Chambers,E.J. and Haworth,I.S.
(1995) Hairpin properties of single-stranded DNA containing a
GC-rich triplet repeat: (CTG)15. Nucleic Acids Res., 23, 1050–1059.
42. Mitas,M., Yu,A., Dill,J. and Haworth,I.S. (1995) The trinucleotide
repeat sequence d(CGG)15 forms a heat-stable hairpin containing
Gsyn. Ganti base pairs. Biochemistry, 34, 12803–12811.
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
2. Renton,A.E., Majounie,E., Waite,A., Simon-Sanchez,J., Rollinson,S.,
Gibbs,J.R., Schymick,J.C., Laaksovirta,H., van Swieten,J.C.,
Myllykangas,L. et al. (2011) A hexanucleotide repeat expansion in
C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD.
Neuron, 72, 257–268.
3. Gendron,T.F., Belzil,V.V., Zhang,Y.J. and Petrucelli,L. (2014)
Mechanisms of toxicity in C9FTLD/ALS. Acta Neuropathol., 127,
359–376.
4. Pearson,C.E., Nichol Edamura,K. and Cleary,J.D. (2005) Repeat
instability: mechanisms of dynamic mutations. Nat. Rev. Genet., 6,
729–742.
5. Belzil,V.V., Bauer,P.O., Prudencio,M., Gendron,T.F., Stetler,C.T.,
Yan,I.K., Pregent,L., Daughrity,L., Baker,M.C., Rademakers,R.
et al. (2013) Reduced C9orf72 gene expression in c9FTD/ALS is
caused by histone trimethylation, an epigenetic event detectable in
blood. Acta Neuropathol., 126, 895–905.
6. Belzil,V.V., Bauer,P.O., Gendron,T.F., Murray,M.E., Dickson,D. and
Petrucelli,L. (2014) Characterization of DNA hypermethylation in
the cerebellum of c9FTD/ALS patients. Brain Res., 1584, 15–21.
7. Liu,E.Y., Russ,J., Wu,K., Neal,D., Suh,E., McNally,A.G., Irwin,D.J.,
Van Deerlin,V.M. and Lee,E.B. (2014) C9orf72 hypermethylation
protects against repeat expansion-associated pathology in ALS/FTD.
Acta Neuropathol., 128, 525–541.
8. Russ,J., Liu,E.Y., Wu,K., Neal,D., Suh,E., Irwin,D.J., McMillan,C.T.,
Harms,M.B., Cairns,N.J., Wood,E.M. et al. (2015) Hypermethylation
of repeat expanded C9orf72 is a clinical and molecular disease
modifier. Acta Neuropathol., 129, 39–52.
9. Xi,Z., Zinman,L., Moreno,D., Schymick,J., Liang,Y., Sato,C.,
Zheng,Y., Ghani,M., Dib,S., Keith,J. et al. (2013) Hypermethylation
of the CpG island near the G4C2 repeat in ALS with a C9orf72
expansion. Am. J. Hum. Genet., 92, 981–989.
10. Xi,Z., Zhang,M., Bruni,A.C., Maletta,R.G., Colao,R., Fratta,P.,
Polke,J.M., Sweeney,M.G., Mudanohwo,E., Nacmias,B. et al. (2015)
The C9orf72 repeat expansion itself is methylated in ALS and FTLD
patients. Acta Neuropathol., 129, 715–727.
11. Reddy,K., Schmidt,M.H., Geist,J.M., Thakkar,N.P., Panigrahi,G.B.,
Wang,Y.H. and Pearson,C.E. (2014) Processing of double-R-loops in
(CAG).(CTG) and C9orf72 (GGGGCC).(GGCCCC) repeats causes
instability. Nucleic Acids Res., 42, 10473–10487.
12. Axford,M.M., Wang,Y.H., Nakamori,M., Zannis-Hadjopoulos,M.,
Thornton,C.A. and Pearson,C.E. (2013) Detection of slipped-DNAs
at the trinucleotide repeats of the myotonic dystrophy type I disease
locus in patient tissues. PLoS Genet., 9, e1003866.
13. Haeusler,A.R., Donnelly,C.J., Periz,G., Simko,E.A., Shaw,P.G.,
Kim,M.S., Maragakis,N.J., Troncoso,J.C., Pandey,A., Sattler,R. et al.
(2014) C9orf72 nucleotide repeat structures initiate molecular
cascades of disease. Nature, 507, 195–200.
14. Phan,A.T. and Mergny,J.L. (2002) Human telomeric DNA:
G-quadruplex, i-motif and Watson-Crick double helix. Nucleic Acids
Res., 30, 4618–4625.
15. Brooks,T.A., Kendrick,S. and Hurley,L. (2010) Making sense of
G-quadruplex and i-motif functions in oncogene promoters. FEBS J.,
277, 3459–3469.
16. Fratta,P., Mizielinska,S., Nicoll,A.J., Zloh,M., Fisher,E.M.,
Parkinson,G. and Isaacs,A.M. (2012) C9orf72 hexanucleotide repeat
associated with amyotrophic lateral sclerosis and frontotemporal
dementia forms RNA G-quadruplexes. Sci. Rep., 2, 1016.
17. Reddy,K., Zamiri,B., Stanley,S.Y., Macgregor,R.B. Jr and
Pearson,C.E. (2013) The disease-associated r(GGGGCC)n repeat
from the C9orf72 gene forms tract length-dependent uni- and
multimolecular RNA G-quadruplex structures. J. Biol. Chem., 288,
9860–9866.
18. Zamiri,B., Reddy,K., Macgregor,R.B. Jr and Pearson,C.E. (2014)
TMPyP4 porphyrin distorts RNA G-quadruplex structures of the
disease-associated r(GGGGCC)n repeat of the C9orf72 gene and
blocks interaction of RNA-binding proteins. J. Biol. Chem., 289,
4653–4659.
19. Leon-Ortiz,A.M., Svendsen,J. and Boulton,S.J. (2014) Metabolism of
DNA secondary structures at the eukaryotic replication fork. DNA
Repair (Amst.), 19, 152–162.
20. Zhang,K., Donnelly,C.J., Haeusler,A.R., Grima,J.C., Machamer,J.B.,
Steinwald,P., Daley,E.L., Miller,S.J., Cunningham,K.M., Vidensky,S.
et al. (2015) The C9orf72 repeat expansion disrupts
nucleocytoplasmic transport. Nature, 525, 56–61.
10064 Nucleic Acids Research, 2015, Vol. 43, No. 20
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
and cytosine methylation greatly enhances the effect. Biochemistry,
32, 5870–5880.
Diekmann,S. (1987) DNA methylation can enhance or induce DNA
curvature. EMBO J., 6, 4213–4217.
Hodges-Garcia,Y. and Hagerman,P.J. (1992) Cytosine methylation
can induce local distortions in the structure of duplex DNA.
Biochemistry, 31, 7595–7599.
Zacharias,W. (1993) Methylation of cytosine influences the DNA
structure. EXS, 64, 27–38.
Nichol Edamura,K., Leonard,M.R. and Pearson,C.E. (2005) Role of
replication and CpG methylation in fragile X syndrome CGG
deletions in primate cells. Am. J. Hum. Genet., 76, 302–311.
Wohrle,D., Salat,U., Glaser,D., Mucke,J., Meisel-Stosiek,M.,
Schindler,D., Vogel,W. and Steinbach,P. (1998) Unusual mutations in
high functioning fragile X males: apparent instability of expanded
unmethylated CGG repeats. J. Med. Genet., 35, 103–111.
Taylor,A.K., Tassone,F., Dyer,P.N., Hersch,S.M., Harris,J.B.,
Greenough,W.T. and Hagerman,R.J. (1999) Tissue heterogeneity of
the FMR1 mutation in a high-functioning male with fragile X
syndrome. Am. J. Med. Genet., 84, 233–239.
Cooper-Knock,J., Higginbottom,A., Stopford,M.J., Highley,J.R.,
Ince,P.G., Wharton,S.B., Pickering-Brown,S., Kirby,J.,
Hautbergue,G.M. and Shaw,P.J. (2015) Antisense RNA foci in the
motor neurons of C9ORF72-ALS patients are associated with
TDP-43 proteinopathy. Acta Neuropathol, 130, 63–75.
Tomonaga,T. and Levens,D. (1995) Heterogeneous nuclear
ribonucleoprotein K is a DNA-binding transactivator. J. Biol. Chem.,
270, 4875–4881.
Bomsztyk,K., Denisenko,O. and Ostrowski,J. (2004) hnRNP K: one
protein multiple processes. Bioessays, 26, 629–638.
Duquette,M.L., Handa,P., Vincent,J.A., Taylor,A.F. and Maizels,N.
(2004) Intracellular transcription of G-rich DNAs induces formation
of G-loops, novel structures containing G4 DNA. Genes Dev., 18,
1618–1629.
Valton,A.L., Hassan-Zadeh,V., Lema,I., Boggetto,N., Alberti,P.,
Saintome,C., Riou,J.F. and Prioleau,M.N. (2014) G4 motifs affect
origin positioning and efficiency in two vertebrate replicators. EMBO
J., 33, 732–746.
Hoshina,S., Yura,K., Teranishi,H., Kiyasu,N., Tominaga,A.,
Kadoma,H., Nakatsuka,A., Kunichika,T., Obuse,C. and Waga,S.
(2013) Human origin recognition complex binds preferentially to
G-quadruplex-preferable RNA and single-stranded DNA. J. Biol.
Chem., 288, 30161–30171.
Cayrou,C., Coulombe,P., Puy,A., Rialle,S., Kaplan,N., Segal,E. and
Mechali,M. (2012) New insights into replication origin characteristics
in metazoans. Cell Cycle, 11, 658–667.
Lombrana,R., Almeida,R., Alvarez,A. and Gomez,M. (2015)
R-loops and initiation of DNA replication in human cells: a missing
link? Front. Genet., 6, 158.
Bartholdy,B., Mukhopadhyay,R., Lajugie,J., Aladjem,M.I. and
Bouhassira,E.E. (2015) Allele-specific analysis of DNA replication
origins in mammalian cells. Nat. Commun., 6, 7051.
Downloaded from http://nar.oxfordjournals.org/ at University of Washington Libraries on December 8, 2015
43. Leonard,G.A., Zhang,S., Peterson,M.R., Harrop,S.J., Helliwell,J.R.,
Cruse,W.B., d’Estaintot,B.L., Kennard,O., Brown,T. and
Hunter,W.N. (1995) Self-association of a DNA loop creates a
quadruplex: crystal structure of d(GCATGCT) at 1.8 A resolution.
Structure, 3, 335–340.
44. Salisbury,S.A., Wilson,S.E., Powell,H.R., Kennard,O., Lubini,P.,
Sheldrick,G.M., Escaja,N., Alazzouzi,E., Grandas,A. and Pedroso,E.
(1997) The bi-loop, a new general four-stranded DNA motif. Proc.
Natl. Acad. Sci. U.S.A., 94, 5515–5518.
45. Lim,K.W., Alberti,P., Guedin,A., Lacroix,L., Riou,J.F., Royle,N.J.,
Mergny,J.L. and Phan,A.T. (2009) Sequence variant (CTAGGG)n in
the human telomere favors a G-quadruplex structure containing a
G.C.G.C tetrad. Nucleic Acids Res., 37, 6239–6248.
46. Escaja,N., Gomez-Pinto,I., Pedroso,E. and Gonzalez,C. (2007)
Four-stranded DNA structures can be stabilized by two different
types of minor groove G:C:G:C tetrads. J. Am. Chem. Soc., 129,
2004–2014.
47. Brcic,J. and Plavec,J. (2015) Solution structure of a DNA quadruplex
containing ALS and FTD related GGGGCC repeat stabilized by
8-bromodeoxyguanosine substitution. Nucleic Acids Res., 43,
8590–8600.
48. Chen,F.M. (1995) Acid-facilitated supramolecular assembly of
G-quadruplexes in d(CGG)4. J. Biol. Chem., 270, 23090–23096.
49. Meyer,M., Hocquet,A. and Suhnel,J. (2005) Interaction of sodium
and potassium ions with sandwiched cytosine-, guanine-, thymine-,
and uracil-base tetrads. J. Comput. Chem., 26, 352–364.
50. Patel,P.K., Bhavesh,N.S. and Hosur,R.V. (2000) NMR observation of
a novel C-tetrad in the structure of the SV40 repeat sequence
GGGCGG. Biochem. Biophys. Res. Commun., 270, 967–971.
51. Zhang,D., Huang,T., Lukeman,P.S. and Paukstelis,P.J. (2014) Crystal
structure of a DNA/Ba2 +G-quadruplex containing a
water-mediated C-tetrad. Nucleic Acids Res., 42, 13422–13429.
52. Wilson,J.H. (1979) Nick-free formation of reciprocal heteroduplexes:
a simple solution to the topological problem. Proc. Natl. Acad. Sci.
U.S.A., 76, 3641–3645.
53. Renciuk,D., Blacque,O., Vorlickova,M. and Spingler,B. (2013)
Crystal structures of B-DNA dodecamer containing the epigenetic
modifications 5-hydroxymethylcytosine or 5-methylcytosine. Nucleic
Acids Res., 41, 9891–9900.
54. Thalhammer,A., Hansen,A.S., El-Sagheer,A.H., Brown,T. and
Schofield,C.J. (2011) Hydroxylation of methylated CpG dinucleotides
reverses stabilisation of DNA duplexes by cytosine 5-methylation.
Chem. Commun. (Camb.), 47, 5325–5327.
55. Rodriguez Lopez,C.M., Guzman Asenjo,B., Lloyd,A.J. and
Wilkinson,M.J. (2010) Direct detection and quantification of
methylation in nucleic acid sequences using high-resolution melting
analysis. Anal. Chem., 82, 9100–9108.
56. Severin,P.M., Zou,X., Schulten,K. and Gaub,H.E. (2013) Effects of
cytosine hydroxymethylation on DNA strand separation. Biophys. J.,
104, 208–215.
57. Hardin,C.C., Corregan,M., Brown,B.A. 2nd and Frederick,L.N.
(1993) Cytosine-cytosine+ base pairing stabilizes DNA quadruplexes