The FASEB Journal Review

DNA triplexes and Friedreich ataxia

Robert D. Wells1
Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University System
Health Science Center, The Texas Medical Center, Houston, Texas, USA
Friedreich ataxia, the most common inherited ataxia, is caused by the transcriptional silencing
of the FXN gene, which codes for the 210 amino acid
frataxin, a mitochondrial protein involved in ironsulfur cluster biosynthesis. The expansion of the
GAATTC tract in intron 1 to as many as 1700 repeats
elicits the transcriptional silencing by the formation of
non-B DNA structures (triplexes or sticky DNA), the
formation of a persistent DNARNA hybrid, or heterochromatin formation. The triplex (sticky DNA)
adopted by the long repeat sequence also elicits profound mutagenic, genetic instability, and recombination behaviors. Early stage therapeutic investigations
involving polyamides or histone deacetylase inhibitors
are being pursued. Friedreich ataxia may be one of the
most thoroughly studied hereditary neurological disease from a pathophysiological standpoint.Wells,
R. D. DNA triplexes and Friedreich ataxia. FASEB J. 22,
16251634 (2008)
ABSTRACT

Key Words: sticky DNA GAATTC transcription silencing

non-B DNA conformations genetic instability polyamides
Friedreich ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive neurodegenerative disease that affects 1 in 50,000 individuals (1, 2).
The gene has been mapped to chromosome 9q13-q21.1
by linkage studies. The FRDA gene, FXN or X25,
contains 7 exons and encodes a 210 amino acid protein
called frataxin, a mitochondrial protein involved in
iron-sulfur cluster biosynthesis. Of the patients with
FRDA, 98% have an expanded GAATTC trinucleotide
repeat sequence (TRS) in the first intron of the FXN
gene and 2% have point mutations. Normal alleles
contain 6 34 repeats, but the expanded alleles of
patients range from 66 to 1700 repeats (3, 4). The age
of onset and the severity of the disease are inversely
correlated with the length of the GAATTC repeats
(1 6), in many cases. Patients carrying expanded
GAATTC repeats in both alleles have reduced levels of
frataxin. The somatic instability of the expanded repeats progresses throughout life (7).
The etiology of FRDA involves the loss of transcriptional activity of the FXN gene, which leads to reduced
levels of functional frataxin protein. This inhibition
may be due to the repeat expansion (15, 8 12) and
the capacity of the expanded GAATTC repeats to form
non-B DNA structures, especially triplexes (1322) and
sticky DNA (2326). Other types of non-B DNA structures that have been characterized for the polymorphic
GAATTC sequence include hairpins (22, 27, 28) and
0892-6638/08/0022-1625 FASEB

parallel DNA (28). The expansion of the GAATTC

sequence is performed by replication, recombination,
and repair in model systems (4, 29 35). After the TRS
are expanded, the repeat may influence these processes
since long repeats are known to stall replication forks in
vivo and in vitro (36 39). The pausing of replication is
attributed to the expanded TRS and its ability to form
these types of non-B DNA structures (9, 14, 21, 36).
Excellent recent reviews exist on clinical and pathophysiological issues (6, 40), experimental therapeutics
(41), evolution and instability of the TRS (30, 40, 42),
mouse models (43, 44), the involvement of triplexes
and sticky DNA (45, 46), iron dysregulation (47, 48),
oxidative stress (49), and frataxin as a protein (50).
Furthermore, a brief overview has been published (51)
of a very recent meeting of researchers in the field
covering all of these topics.
Interestingly, this laboratory published a review in
FASEB Journal on the chemistry and biology of triplexes
in 1988 (52). We stated that it is likely that these
unorthodox structures play an important role in the
function of the eukaryotic genome; obviously, we now
recognize that this prediction was correct.

DNA CONFORMATIONS ASSOCIATED WITH

LONG GAATTC TRACTS
Molecular investigations on FRDA took a giant leap
forward in 1996 when Pandolfo et al. (reviewed in refs.
1, 6) discovered that the expansion of a GAATTC
repeat sequence in intron 1 of the FXN gene was
responsible for the disease etiology in most cases.
Predictably, the attention of many investigators immediately turned toward this interesting repeat sequence
at both the DNA level as well as its mRNA. Numerous
investigations prior to 1996 (53 and references cited
therein) revealed the polymorphic nature of this sequence since its capacity to form three-stranded DNA
structures (triplexes) was well established due to its
mirror repeat RY sequence with the concomitant possibility of forming a number of base-paired structures.
The types of triplexes formed as well as the conditions
for their stabilization and their biochemical and ge1

Correspondence: Center for Genome Research, Institute

of Biosciences and Technology, Texas A&M University System
Health Science Center, The Texas Medical Center, 2121 W.
Holcombe Blvd., Houston, TX 77030-3303, USA. E-mail:
rwells@ibt.tamhsc.edu
doi: 10.1096/fj.07-097857
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netic functions have been reviewed (9, 13, 2326, 35,

46, 54). Inter- as well as intramolecular DNA triplexes
are well known (13) as are DNARNA triplexes where
the RNA strand fills the major groove in the DNA
duplex (5557). As stated in the introduction, several
other types of non-B DNA structures may be adopted by
this repeating sequence.
However, in 1999 when Sakamoto et al. (23) undertook investigations directly related to FRDA on long
tracts of (GAATTC)n (where nup to 270), they had
substantial reservations due to the relatively imprecise
types of triplexes that were formed by much shorter
lengths (n20 58) of GAATTC in plasmids (16).
Indeed, these authors were astonished to realize that
extremely well-defined types of a certain triplex were
formed by tracts of GAATTC, which were longer than
59 repeats in length (75, 90, 115, 150, and 270 repeats).
Thus, whereas we thought that this investigation might
not yield interpretable and quantitative data, exactly
the opposite occurred. Extremely unusual behavior was
observed since segments of the recombinant plasmids
were tenaciously adhering to each other, which gave
rise to the concept of sticky DNA (Fig. 1). Sticky DNA
has been the subject of intense study in this and other
laboratories over the past 8 years (9, 14, 2326, 35, 45,
46, 54). Investigations have focused on the types of
triplexes that are adopted by the long GAATTC repeats, both in vitro and in vivo, the role of the structure
as a mutagen [see Effects of Triplexes (Sticky DNA) on
Other DNA Metabolic Events], its involvement in replication, repair, and recombination-mediated genetic
instabilities, the influence of small sequence interruptions
on the structure, and the capacity of DNA sequencespecific ligands to alter the conformation to alleviate
transcriptional repression (see Therapeutic Strategies).
Sticky DNA (Fig. 1) is a long GAAGAATTC triplex
that is formed intramolecularly in vivo and in vitro
(reviewed in ref. 45) by a pair of repeating GAATTC
tracts. Two tracts of GAATTC repeats within a given
DNA interact to form a complex that can only be
broken with high temperatures (80C) and with the
addition of EDTA to remove divalent metal ions. The
net effect of these intramolecular interactions is to lock
the DNA molecule into a dumbbell-shaped structure

Figure 1. Models of an intramolecular DNA triplex and sticky

DNA structures. Reprinted with permission (45).
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(Fig. 1), which impedes virtually all biological processes

in vitro including transcription, replication, repair, and
recombination (9, 14, 2326, 35, 45, 46, 54) and
excludes nucleosome assembly (H. Ruan, M. Napierala,
R. D. Wells, and Y.-H. Wang, unpublished results).
Because of the extreme stability of sticky DNA, the two
ends of the circular plasmid behave independently in
terms of their supercoiled domains (26).
Since this sequence is important in the etiology of
FRDA, substantial attention has been devoted to the in
vivo properties of sticky DNA (26). Clearly, sticky DNA
exists and functions in bacterial systems (26, 35, 46).
Likewise, long tracts (n60, 150) of (GAATTC)n are
mutagenic in African green monkey kidney (COS-7)
cells as well as CV-1 and human embryonic kidney
HEK-293 cells (58), which implies behaviors similar to
those observed in bacteria. However, direct determinations in eukaryotic cells remain to be conducted to
ascertain the precise type of non-B structure responsible.
Extensive investigations have been conducted in living Escherichia coli to evaluate the role of the lengths
and orientations of GAATTC repeat tracts (either one
or two in number) on the supercoil relaxation (26).
This procedure has been used extensively for triplexes,
cruciforms, and Z-DNA, because when DNA structural
transitions occur in small regions of plasmids as a result
of DNA underwinding, the global negative supercoil
value of the plasmid decreases (becomes less negative).
To maintain the physiological negative supercoil density of the plasmid (0.025) in the cell, DNA gyrase
adds negative supercoils into the DNA to reestablish
the normal homeostatic supercoil levels. Hence, when
the plasmid is isolated and analyzed in vitro, the result
of this negative supercoil change on agarose gels containing chloroquine is an overly negative supercoiled
DNA with a greater negative linking number difference
(26). When a family of plasmids containing either one
or two tracts of GAATTC of varying lengths were
subjected to these analyses, a unique behavior was
observed since plasmids that have the capacity to form
sticky DNA displayed an overwound, not an underwound, primary helix. This behavior was unexpected
and indicates an apparent, but theoretically intractable,
compression of the primary helices, an unprecedented
result with any other non-B structure forming sequence
(26). Since these investigations involve measurements
of the gel mobility of intact plasmids in vitro to enable
conclusions about in vivo behaviors, this apparent compression of the bp may be due to other interactions,
which remain to be identified. These data clearly
demonstrate that sticky DNA exists in vivo and has
important functional consequences (interactions with
proteins or roles in metabolic processes), which have
not been observed previously. Further work will be
required to elucidate the details of these properties.
Sticky DNA requires GAATTC tracts, which are at
least 59 repeats (and up to 300) in length and a pair
of repeat tracts in the direct repeat orientation. If the
inserts are in the indirect (inverted) repeat orientation,
no sticky DNA is observed. Divalent metal ions (magnesium) as well as negative supercoiling are also required for the sticky DNA structure formation (26).

The FASEB Journal

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To elucidate the effect of sequence on sticky DNA

formation, various extents of GGATCC interruptions
were introduced into long repeats of GAATTC tracts in
a family of homologous plasmids. The plasmids were
prepared in vitro but then were investigated in vivo. For
plasmids that harbored more than 20% of GGATCC
interruptions in their TRS, the formation of sticky DNA
was abolished. However, the DNAs with GAATTC
repeats with less than 11% of the GGATCC interruptions formed triplexes and/or sticky DNA similar to the
uninterrupted sequences (46, 54). Thus, when considering therapeutic strategies in the future for FRDA,
site-directed mutagenesis could be an effective way to
destabilize the sticky DNA conformation (45). Interestingly, an individual with late onset ataxia was found to
be heterozygous for an unusual (GAAGGATCCTTC)65
sequence and a normal GAATTC repeat in the frataxin
gene. This repeating hexanucleotide is nonpathogenic
(59). Furthermore, this sequence alone will not adopt
the sticky DNA structure or a triple helical conformation, as expected from its base pairing capabilities.
However, if a tract of the repeating hexanucleotide
sequence is present in the same plasmid as a pure long
GAATTC tract, sticky DNA is formed (25, 26) as
expected on the basis of base pairing possibilities. The
kinds of base-paired structures that may be formed
(24 26) are highly predictable with respect to the
formation of sticky DNA.
Sticky DNA apparently occurs and functions only in
the FXN gene (in its first intron), which is one of the
20 25,000 genes in humans. Whereas the GAATTC
sequence is not uncommon (60), the extremely long
tracts that are found to be associated with FXN are
apparently unique. Closely related RY sequences can
also adopt this structure (25), as expected, and thus
future investigations will be required to evaluate the
ubiquity of this non-B DNA conformation. Note that no
direct evidence exists for triplexes or sticky DNA having
an obligatory role in the etiology of FRDA.

TRANSCRIPTION INHIBITION BY LONG

TRACTS OF GAATTC
Three plausible mechanisms exist to explain the transcriptional silencing by long GAATTC tracts in intron
1 including: 1) non-B DNA structures (triplex, sticky
DNA, slipped structures) of the TRS; 2) the formation
of a specific and persistent inhibitory d(TTC)nr(GAA)n
hybrid; and 3) heterochromatin formation.
DNA conformational effects
All molecular biological investigators in the Friedreich
ataxia field concur that the inhibition of FXN transcription is the principal and critical step affected in this
autosomal recessive disease (4). Long tracts of
GAATTC suppressed FXN gene expression (1 6, 61).
This pathogenic loss of function mechanism agrees
with the well-known recessive nature of disease and,
thus, the deficiency of the protein frataxin is explicable.
Furthermore, virtually all molecular biological investiDNA TRIPLEXES AND FRIEDREICH ATAXIA

gators have implicated non-B DNA structures, either

triplexes or sticky DNA, in the mechanism of this
inhibition (6, 8 12, 14, 21, 33, 45, 46, 54, 62, 63).
After the discovery of sticky DNA (23), in vitro
investigations were conducted (9) to directly evaluate
the transcription behaviors of this unorthodox conformation. As expected, sticky DNA was an effective
inhibitor of the synthesis of RNA, consistent with the
well-known inhibitory effect of triplexes on transcription (5557, 64 66). Surprisingly, transcriptional
inhibition was observed not only for the sticky DNA
template but also another DNA molecule used as an
internal control in an orientation-independent manner. Thus, the molecular mechanism for the transcription inhibition by sticky DNA was a sequestration of the RNA polymerases by direct binding to the
complex DNA structure. Only sticky DNA showed this
behavior (9). Furthermore, a family of GGATCCinterrupted long GAATTC repeat tracts have been
cloned and characterized (see above). As expected,
the greater the extent of the interruptions of the
GAATTC repeats, the less inhibition of in vitro
transcription was observed, consistent with the capacity of the interruptions to inhibit the formation of
sticky DNA. The interruptions introduced base mismatches into the RRY triplex, which explains its
observed chemical and biological properties (54).
Grabczyk et al. (66) have recently reported the
formation of a persistent RNADNA hybrid by transcription of the FXN GAATTC repeat sequence in E.
coli and by T7 RNA polymerase in vitro. RNADNA
hybrids were observed with repeats numbering 44 or
88 in length. During in vitro transcription of the
longer repeats, T7 RNA polymerase arrested in the
promoter distal end of the GAATTC tracts and an
extensive RNADNA hybrid was tightly linked to this
arrest. Thus, RNADNA hybrid formation appears to
be an intrinsic property of transcription through
long GAATTC repeats. An attractive model (Fig. 2)
was proposed to explain the transcription-coupled
RNADNA hybrid formation in the GAATTC repeats.
Initially, the repeating DNA d(TTC)n strand serves as
the template for synthesis of r(GAA)n to form a
moderate length of DNARNA hybrid. Due to the
stability of this hybrid, the DNA triplex is dislodged
behind the growing transcription complex to give an
even longer RNADNA hybrid. The waves of negative
supercoiling behind the translocating RNA polymerase facilitates these processes from a topological
standpoint. As stated above, the poly RY sequence of
the FXN repeat sequence enables these interactions
from a nucleic acid structural standpoint. The formation of the quasi-stable DNA triplex and DNARNA
hybrid generates a pause site at the TRS. However,
the possible involvement of a DNARNA triplex (two
strands of DNA and one strand of RNA) (56, 57) has
not been eliminated (62, 66; E. Grabczyk, personal
communication).
In 1998, the inhibitory effects of the expanded
GAATTC repeats (up to 270 U in length) were
investigated in vivo by transient transfection into
COS-7. A length and orientation-dependent inhibition of a reporter gene was observed (8). Very low
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Influences of chromatin structure

Figure 2. Transcription-coupled RNADNA hybrid formation

in a GAATTC repeat. Model for transient transcriptiondependent triplex formation leading to an RNA polymerase
pause and RNADNA hybrid formation. The purine (GAA or
R) strand of the repeat is red, the pyrimidine (TTC or Y)
strand is yellow, and the flanking DNA is gray. A) A standing
wave of negative supercoiling follows RNA polymerase. At the
transcription bubble, the nontemplate (GAA) strand is available to fold back in an RRY interaction; the template strand
is covered by RNA polymerase. B) Rotation of the helix
(curved arrow) as it winds in the third strand relaxes the
negative supercoils caused by transcription and leaves a
length of the template single-stranded. C) RNAP is impeded
at the distal template-triplex junction and the nascent transcript (green) can anneal to the single-stranded stretch of
template. D) The RNADNA hybrid displaces the much less
stable triplex structure. Reprinted with permission (66).

levels of mature mRNA were found when plasmids

with longer TRS were investigated with no accumulation of the primary transcript. Inhibitory effects
were also found in DNA replication studies with long
tracts of the TRS (8).
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A rather different mechanism of transcriptional silencing has been investigated by Saveliev et al. (67) based on
the packaging of DNA within chromatin. DNA embodied in chromatin and heterochromatin has been implicated in gene silencing. DNA wraps around histones,
creating nucleosomes in chromatin. Repetitive DNA
sequences have the propensity to package genomic
regions into inaccessible heterochromatin structures
that can lead to gene silencing (67). Prior in vitro
investigations (68) revealed that long tracts of repeating CTGCAG bind histones tightly and position
nucleosomes. Alternatively, long tracts of the fragile X
CCGCGG repeats displayed strong nucleosome exclusion (69, 70). Hence, a precedent was established in
vitro for a profound influence of TRS on chromatin
structure. The FXN TRS confer variegation of expression on a linked transgene in mice (67). Silencing was
correlated with a decrease in promoter accessibility and
was enhanced by the classical position effect variegation
modified heterochromatin protein 1. Interestingly, the
TRS-associated variegation was not restricted to classical heterochromatic regions but occurred irrespective
of chromosomal location. Hence, the mechanisms responsible for the heterochromatin-mediated silencing
might have a role in gene regulation throughout the
mammalian genome and, thus, modulate the extent of
gene silencing and possibly the severity of several TRS
diseases (67).
In contrast, direct binding measurements have been
conducted in vitro on plasmids containing long tracts of
GAATTC duplex as well as GAAGAATTC triplexes
(H. Ruan and Y.-H. Wang, unpublished results). The
duplex excludes nucleosome assembly, and the triplex
further lowers the nucleosome assembly efficiency. The
difference in assembly efficiency is amplified when
hypoacetylated histones were used, compared to assembly with hyperacetylated histones. Also, the triplex
structure destabilizes the extent of adjacent sequences
to assemble nucleosome arrays. Thus, these in vitro
investigations on the GAATTC duplex and triplex
structures reveal profound influences on local chromatin structure.
Relationship between in vitro and in vivo chromatin
structure determinations
This apparent discrepancy in FRDA systems between in
vitro binding measurements, where the GAATTC tracts
and/or triplexes inhibit nucleosome assembly, and the
in vivo genetic determinations (67), which indicate an
enhancement of heterochromatin formation by the
long GAATTC repeats, may be resolved by further
biochemical experiments in the future. Other studies
with histone deacetylase inhibitors indicate that a heterochromatin structure is present in the region surrounding the expanded GAATTC repeats and inhibits
the transcription of the FXN gene in cells from patients
(71). Heterochromatin is enriched with hypoacetylated
histones, which our results (see above) suggest could
distinguish the GAATTC duplex and the triplex from

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adjacent (flanking) sequences more than with hyperacetylated histones. Hence, the presence of the hypoacetylated histones at the expanded GAATTC site
might increase accessibility to other chromatin-remodeling proteins, which result in heterochromatin
structure and/or promoting the formation of the
GAAGAATTC triplex.
Also, the observation of a difference between heterochromatin-like structure present in vivo adjacent to the
expanded GAATTC repeat and in vitro nucleosome
exclusion of the GAATTC repeats is reminiscent of the
results with the CGGCCG repeats in the FMR1 gene of
fragile X syndrome patients (69, 72). For the fragile X
syndrome, the discrepancy between in vitro and in vivo
studies suggests that epigenetic events, such as DNA
CpG methylation, play a role in the formation of
unusual chromatin structure over the expanded
CGGCCG repeats. Although the GAATTC repeats in
FRDA do not contain intrinsic CpG dinucleotides, a
CpG island is located 1 kb upstream of the GAATTC
repeats at the boundary of exon/intron 1 of the FXN
gene (H. Ruan and Y.-H. Wang, unpublished results).
In fact, three CpG dinucleotides located at the 5 end
of the GAATTC repeat have been shown to posses
preferential methylation in FRDA patients (73). The
nucleosome exclusion of the GAATTC repeats could
increase the accessibility of these regions to various
protein machineries, such as DNA methyltransferase.
In summary, further investigations must be conducted
in FRDA systems in order to resolve the apparent
discrepancy between the conclusions derived from in
vivo genetic investigations on heterochromatin formation and the in vitro direct binding studies.
A different epigenetic study has focused on DNA
methylation in segments of the FXN gene, which flank
the GAATTC repeat tract in intron 1. Greene et al. (73)
showed that a region adjacent to the TRS was methylated in both the unaffected and affected individuals.
However, methylation was more extensive in patients.
Furthermore, three residues were almost completely
methylation-free in unaffected individuals but almost
always methylated in patients with FRDA. One of these
residues is located within an E-box, whose deletion
caused a significant drop in promoter activity in reporter assays. Elevated levels of histone H3 dimethylated on lysine 9 were found in FRDA cells, consistent
with a more repressive chromatin organization. This
type of chromatin is known to reduce transcription
elongation. Hence, this could be a mechanism where
the expanded repeats contribute to the frataxin deficit
in FRDA.
In summary, in vitro and in vivo investigations on the
role of long tracts of GAATTC on transcription are
consistent with the recessive nature of the disease and
its loss of function characteristics. Considering all of the
30 hereditary neurological diseases (74) caused by
expansions of triplet repeats (4), FRDA may be the
clearest case of a well-defined biochemical step (transcription) that is inhibited, thus causing the disease
etiology. Accordingly, this realization elevates the hope
of being able to develop effective therapeutics (see
Therapeutic Strategies) to ameliorate the disease manifestations.
DNA TRIPLEXES AND FRIEDREICH ATAXIA

EFFECTS OF TRIPLEXES (STICKY DNA) ON

OTHER DNA METABOLIC EVENTS
Mutagenesis
Triplexes are mutagenic. Substantial prior investigations (75, 76 and references cited therein) have clearly
demonstrated the mutagenic capacity of both interand intramolecular triplexes. A wide range of triplexes
is effective in inducing recombination and DNA repair
(75). Indeed, specific mutations have even been induced in mice by triplex-forming oligonucleotides
(77). Furthermore, naturally occurring triplex-forming
sequences are mutagenic in mammalian cells and generate substantial deletions, translocations, and other
mutations (78, 79). Thus, it is not surprising that
triplexes (sticky DNA) associated with the FXN gene
show mutagenic behaviors.
Whereas ample evidence demonstrates the involvement of triplexes in genetic instabilities, DNA repair
and recombination (75, 76 and references cited
therein), a new paradigm in disease etiology has been
discovered with the finding of triplexes coinciding with
breakpoints of gross deletions (reviewed in refs. 80, 81).
Triplexes as well as other non-B DNA conformations
are found at the breakpoints of a wide range of genetic
rearrangements (58, 60, 80 85). The types of genomic
rearrangements include translocations, deletions, insertions, inversions, and duplications and include the
TRS as well as regions of flanking DNA sequences. The
non-B DNA conformations are directly involved in the
repair-recombination functions, since remnants of the
triplexes and other non-B conformations are found in
the genetically rearranged products. Thus, the residual
portions of the non-B structures cleaved at their labile
contorted residues leave telltale tracks of the roles of
these unorthodox conformations (80). These rearrangements are the genetic basis for approximately 50
human diseases, including adrenoleukodystrophy, follicular lymphomas, and spermatogenic failure (reviewed in refs. 17, 80). Hence, it would not be surprising if other physiological roles are discovered in the
future for the long and stable sticky DNA triplex in
FRDA.

Genetic instabilities
The genetic instabilities of TRS (repeating GAATTC,
CTGCAG, and CGGCCG) associated with approximately 30 hereditary neurological disorders (4, 14, 39,
74, 86) occur by DNA replication, recombination, and
repair (especially methyl-directed mismatch repair and
nucleotide excision repair) as studied in pro- and
eukaryotic model systems. These biochemical-genetic
processes probably act in concert due to slippage of the
complementary strands relative to each other. The
biophysical properties of the folded-back repeating
sequence strands have a critical role in these instabilities. The non-B DNA structural elements include hairpins, slipped structures, DNA unwinding elements,
tetraplexes, triplexes, and sticky DNA. The replication
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mechanisms are influenced by pausing of the replication fork, orientation of the triplet repeat strands,
location of the repeat sequences relative to the replication origins, and flap endonucleases. The long
GAATTC tracts associated with FRDA are expanded
and deleted by mechanisms similar to the other known
TRS (4, 14, 39, 74). However, in contrast to the other
TRS involved with myotonic dystrophy, Huntingtons
disease, fragile X syndrome, etc., it seems that sticky
DNA occurs and functions only in the FXN gene, which
is one of the 20 25,000 genes in humans. Whereas
other related sequences will form sticky DNA (25),
none of these sequences has been identified yet with
disease syndromes.
Genetic instability investigations on TRS responsible
for FRDA, myotonic dystrophy, fragile X syndrome, and
Huntingtons disease from 1990 to 2003 indicated
that the TRS underwent either expansions or deletions
with few or no base pair changes to the flanking
sequences. However, more recent investigations (14,
17, 58, 80, 82, 87 89) showed that substantial deletions,
rearrangements, duplications, translocations, and inversions occurred in flanking sequences, in addition to
the repeating triplet or tetranucleotide repeats. Rearrangements of several kbp were observed and were
promoted by long tracts of GAATTC, CTGCAG,
CCAGCTGG, or CGGCCG. Thus, the presence of the
simple repeating sequence has a profound influence
on the repair-recombination properties of flanking
DNA sequences.
An insightful family of investigations (58) revealed
that the non-B DNA conformations (triplexes or sticky
DNA and slipped structures) formed by a portion of the
long repeating tracts of FRDA, myotonic dystrophy type
1, and myotonic dystrophy type 2 genes, not the sequences per se, promoted mutagenesis in the flanking
DNA regions. Studies in E. coli and three types of
mammalian (COS-7, CV-1, and HEK293) fibroblast-like
cells revealed that conditions that promoted the formation of the non-B structures enhanced the genetic
instabilities, both in the repeat tracts and in the flanking sequences of up to 4 kbp. The three strategies
utilized included: the in vivo modulation of global
negative supercoil density using topA and gyrB mutant
E. coli strains; the in vivo cleavage of hairpin loops,
which are an obligate consequence of slipped-strand
structures, cruciforms, and intramolecular triplexes, by
inactivation of the SbcC protein; and by genetic instability studies with plasmids containing long repeating
sequence inserts that do, and do not, adopt non-B DNA
structures in vitro (58). Thus, it was possible to tease
apart the effects of the sequences vs. the conformations. Clearly, the conformations are the culprits.
Even earlier investigations (87) demonstrated that
the slipped structures, cruciforms, and sticky DNA
adopted by long tracts of GAATTC, CGGCCG, and
CTGCAG repeats were responsible for the genetic
instabilities of these sequences. The influence of negative superhelical density on the genetic instabilities of
these repeat tracts was studied in vivo in topologically
constrained plasmids in E. coli. The capacity of these
DNA tracts to undergo deletions-expansions was explored with three genetic-biochemical approaches, in1630

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cluding first, the utilization of topoisomerase I and/or

DNA gyrase mutants; second, the specific inhibition of
DNA gyrase by novobiocin; and third, the genetic
removal of the HU protein, thus lowering the negative
supercoil density. All three strategies revealed that
higher negative supercoil densities in vivo enhanced
the formation of deleted repeat sequences. Higher
levels of negative supercoiling stabilize the formation of
triplexes, sticky DNA, and slipped structures at appropriate repeat tracts. Also, numerous prior genetic instability investigations invoke a role for these structures in
promoting the slippage of the DNA complementary
strands. Thus, the authors concluded that the in vivo
modulation of the DNA structure, localized to the
repeat tracts, is responsible for these behaviors. These
data agree with the conclusions (58) derived from
alternate types of mutagenic investigations.
Recombination
The triplex (sticky DNA) structures adopted by long
GAATTC sequences from intron 1 of the FXN gene
exert profound structure-dependent influences on the
recombination behaviors of these repeating sequences
(35). Intramolecular and intermolecular recombination studies showed that the frequency of recombination between the GAATTC tracts was as much as 15
times higher than the nonrepeating control sequences.
Homologous, intramolecular recombination between
GAATTC tracts and GAAGGATCCTTC repeats also
occurred with a very high frequency (0.8%). Biochemical analyses of the recombination products
demonstrated the expansions and deletions of the
GAATTC repeats. These results, together with our
previous studies (90, 91) on the CTGCAG sequences,
suggest that the recombinational hot spot characteristics may be a common feature of all triplet repeat
sequences. Unexpectedly, we found that the recombination properties of the GAATTC tracts were unique,
compared with CTGCAG repeats, because they depended on the DNA secondary structure polymorphism.
Specifically, increasing the length of the GAATTC repeats decreased the intramolecular recombination frequency between these tracts. This result was unexpected.
Also, a correlation was found between the propensity of
the GAATTC tracts to adopt the sticky DNA conformation and the inhibition of intramolecular recombination.
Investigations with novobiocin to modulate the intracellular DNA topology (i.e., the lowering of the negative
superhelical density, repressing the formation of the
sticky DNA structure) restored the expected positive
correlation between the length of the GAATTC tracts
and the frequency of intramolecular recombination.
Thus, these studies support other investigations (58,
86) demonstrating the role of the DNA structure, not
the sequence per se, in the biological behaviors.

THERAPEUTIC STRATEGIES
FRDA is an attractive disease target for experimental
therapeutics since apparently only a single gene is

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involved (at least as the initial insult), the mechanism is

a loss of function, and the cause of this loss is uniformly
acknowledged to be a reduction in the amount of
synthesis of mRNA caused by a decrease in transcription (see Transcription Inhibition by Long Tracts of
GAATTC). Whereas virtually all workers in the field
concur with these conclusions, the molecular basis for
the reduction of transcription is uncertain. Reviews on
gene-based strategies for FRDA therapeutics have been
published (92, 93).
Polyamides
Napierala et al. (35) stated that the use of pharmacological agents, capable of interfering with the formation of these unusual (DNA) structures, may up-regulate the expression of the FRDA gene, thereby
providing an effective therapeutic approach. Gottesfeld et al. (94) have shown that sequence-specific polyamides (95) alleviate transcription inhibition associated
with long GAATTC repeats. Since excellent reviews
(95, 96) on this topic are available, only the FRDA
salient issues will be discussed briefly herein. The
pyrrole-imidazole polyamides synthesized by P. Dervan
and associates are the only available class of small
molecules that are designed to recognize virtually any
predetermined DNA sequence (97 and papers cited
therein). These molecules have affinities and specificities that equal or exceed natural eukaryotic transcriptional regulatory proteins. A major strength of polyamides from a therapeutic strategy is their cell
permeability and their capability of localizing in the
nucleus in various cultured cell lines; also, they downregulate target genes in these cells. Studies with model
gene systems (95) and a variety of eukaryotic and viral
transcription factors showed that these small ligands
are potent inhibitors of protein-DNA interactions.
Burnett et al. (94) reasoned that appropriate polyamides might bind to long GAATTC tracts with high
affinity and, thereby, disrupt the intramolecular
DNADNA-associated region of the sticky DNA conformation formed by these long TRS. Fluorescent polyamide-Bodipy conjugates localize in the nucleus of a
lymphoid cell line derived from a FRDA patient. The
synthetic ligands increase transcription of the frataxin
gene in cell culture, resulting in increased levels of
frataxin protein. DNA microarray analyses indicated
that a limited number of genes were significantly
affected in FRDA cells. Thus, the polyamides may
increase transcription by altering (abolishing) the
sticky DNA conformation of the FXN gene harboring
the long GAATTC repeats; alternatively, a chromatin
opening mechanism was discussed (94).
HDAC inhibitors
Second, a quite different therapeutic strategy has been
broached by attempting to modulate gene regulation at
the epigenetic level. Gottesfeld et al. (71) have explored
the chromatin structure of the FXN gene in normal
and FRDA cell lines using antibodies to the various
modification states of the core histones and chromatin
DNA TRIPLEXES AND FRIEDREICH ATAXIA

immunoprecipitation methods. Gene silencing at expanded FXN alleles is accompanied by hypoacetylation

of histones H3 and H4, and methylation of histone H3
at lysine 9, consistent with a heterochromatin-mediated
repression mechanism. Hence, these authors conclude
that histone deacetylase (HDAC) inhibitors, compounds that reverse heterochromatin, might activate
the FXN gene. One commercial HDAC inhibitor, BML210, which partially reverses silencing in the FRDA cell
line, was identified. Based on the structure of this
compound, Gottesfeld et al. synthesized and assayed a
series of derivatives of BML-210 and identified HDAC
inhibitors that reverse FXN silencing in primary lymphocytes from Friedreichs patients. These molecules
act directly on the histones associated with the FXN
gene, increasing acetylation at particular lysine residues
on histones H3 and H4 (H3-K14, H4-K5, and H4-K12).
Since the expanded GAATTC repeats do not alter the
coding potential of the FXN gene, gene activation
should be beneficial from a therapeutic standpoint (98).

UNANSWERED QUESTIONS
Unanswered questions include the following:
What is the mechanism of transcription silencing? Is
it one of the three mechanisms described in Transcription Inhibition by Long Tracts of GAATTC or another
process?
Does sticky or triplex DNA exist and function in
humans? What is the relationship to FRDA?
What is the detailed molecular structure of sticky
DNA, a unique non-B DNA conformation, both in vivo
and in vitro?
What is the equilibrium in vivo between the triplex
(sticky) DNA structure and the orthodox B-DNA form
of the GAATTC repeats? Since sticky DNA inhibits
replication, transcription, recombination, and repair,
cells could not survive or divide if this conformation was
dominant and very long lived in vivo. Thus, what is the
lifetime of this transient structure?
What is the role, if any, of heterochromatin in FRDA
gene silencing?
What is the role of DNA methylation in FRDA?
What is the molecular biological mechanism in humans of the genetic instability of GAATTC? Is this
process conducted by replication, recombination, or
repair or a combination of all three mechanisms acting
in concert?
What is the role in FRDA etiology, if any, of mutagenesis promoted by GAATTC in DNA tracts flanking the
TRS?
Could genomic translocations (copy number variations, deletions, insertions, inversions, etc.) play a role,
at least for some patients, in the mutation spectra?
Many reviews and papers repeat the declaration that
98% of all FRDA cases are due to the expansion of the
GAATTC repeats and 2% are due to point mutations
(1). Is this the entire range of mutations in the FXN
gene or might future detailed analyses reveal the existence of other types of mutations that could be caused
by the presence of long GAATTC repeats (see Mutagenesis)? Note that the fragile X mutation spectra is
1631

broad (reviewed in refs. 82, 84), as probably promoted

by its highly mutagenic TRS.
What is the optimum therapeutic strategy and ideal
agent for treating FRDA patients?
What are the functions of other moderate-length
GAATTC repeats, which are shorter than in the FXN
intron 1, in genomes? What are the roles of triplexes
(sticky DNA) in these biological processes?
Obviously, substantial additional work remains to be
conducted to unravel these critical questions in the
future.

REFERENCES
1.

2.

3.

PROSPECTS FOR THE FUTURE

4.

Remarkable advances have been made in our understanding of the pathophysiology of FRDA in the past
decade. Indeed, it was only in 1996 when the gene was
cloned and sequenced. At the Third International
Friedreichs Ataxia Scientific Conference at the National Institutes of Health (Bethesda, MD, USA). in
November 2006, the Director of the National Institute
of Neurological Disorders and Stroke noted in her
opening remarks that there was a palpable sense of
energy, excitement, and enthusiasm over the scientific
progress made (51). One conclusion from this review
article is an agreement with this sentiment and an
acknowledgment that substantial additional work needs
to be concentrated on fundamental mechanisms responsible for the initial steps in the disease etiology.
FRDA is a conceptually tractable disease for the reasons
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targeted to downstream events. It is critical to determine the fundamental molecular biological cause for
transcription silencing (non-B DNA structures or heterochromatin or methylation or other mechanisms).
Therapeutics for FRDA may be more advanced than for
any other of the 30 hereditary neurological diseases
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Since the number of investigators conducting innovative research in this area has expanded greatly over
the past decade (51), hope is raised for viable treatment
and therapeutic strategies for the future. The development of high-throughput assays to evaluate the efficacy
of a large number of ligands that may be therapeutically successful is currently underway.
I thank the N.I.H. (ES11347), Friedreichs Ataxia Research
Alliance, Seek a Miracle, and the Robert A. Welch Foundation for support, and Drs. Marek Napierala and Albino
Bacolla for helpful discussions. I also express appreciation to
J. E. Larson for 40 years of research on non-B DNA structures.
All materials from the R. D. Wells laboratory will be transferred to Dr. Sergei Mirkin (Tufts University, Department of
Biology, Barnum 101B, 163 Packard Ave., Medford, MA
02155, USA. E-mail: sergei.mirkin@tufts.edu). The author
conveys his apologies to colleagues whose fine work could not
be cited due to a journal restriction on the number of
references and the length of the review.

1632

Vol. 22

June 2008

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Bacolla, A., Wojciechowska, M., Kosmider, B., Larson, J. E., and
Wells, R. D. (2006) The involvement of non-B DNA structures in
gross chromosomal rearrangements. DNA Repair 5, 11611170
Kosmider, B., and Wells, R. D. (2006) Double-strand breaks in
the myotonic dystrophy type 1 and the fragile X syndrome

Vol. 22

June 2008

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triplet repeat sequences induce different types of mutations in

DNA flanking sequences in E. coli. Nucleic Acids Res. 34, 5369
5382
Bacolla, A., Jaworski, A., Larson, J. E., Jakupciak, J. P., Chuzhanova, N., Abeysinghe, S. S., OConnell, C. D., Cooper, D. N.,
and Wells, R. D. (2004) Breakpoints of gross deletions coincide
with non-B DNA conformations. Proc. Natl. Acad. Sci. 101,
1416214167
Kosmider, B., and Wells, R. D. (2007) Fragile X CGGCCG
repeats are potent inducers of complex, multiple site rearrangements in flanking DNA sequences. DNA Repair 6, 1850 1863
Wojciechowska, M., Bacolla, A., Larson, J. E., and Wells, R. D.
(2005) The myotonic dystrophy type 1 triplet repeat sequence
induces gross deletions and inversions. J. Biol. Chem. 280,
941952
Orr, H. T., and Zoghbi, H. Y. (2007) Trinucleotide repeat
disorders. Annu. Rev. Neurosci. 30, 575 621
Napierala, M., Bacolla, A., and Wells, R. D. (2005) Increased
negative superhelical density in vivo enhances the genetic
instability of triplet repeat sequences. J. Biol. Chem. 280, 37366
37376
Dere, R., and Wells, R. D. (2006) DM2 CCTGCAGG repeats
are crossover hot spots that are more prone to expansions
than the DM1 CTGCAG repeats in Escherichia coli. J. Mol. Biol.
360, 2136
Meservy, J. M., Sargent, R. G., Iyer, R. R., Chan, F., McKenzie,
G. J., Wells, R. D., and Wilson, J. H. (2003) Long CTG tracks
from the myotonic dystrophy gene induce deletions and rearrangements during recombination at the ARPT locus in CHO
cells. Mol. Cell Biol. 23, 31523162
Pluciennik, A., Iyer, R. R., Napierala, M., Larson, J. E., Filutowicz, M., and Wells, R. D. (2002) Long CTGCAG repeats from
myotonic dystrophy are preferred sites for intermolecular recombination. J. Biol. Chem. 277, 34074 34086
Napierala, M., Parniewski, P., Pluciennik, A., and Wells, R. D.
(2002) Long CTGCAG repeat sequences markedly stimulate
intramolecular recombination. J. Biol. Chem. 277, 34087
34100
Hebert, M. D., and Whittom, A. A. (2007) Gene-based approaches toward Friedreich ataxia therapeutics. Cell Mol. Life Sci.
[E-pub ahead of print] doi: 10.1007/s0018-007-7293-6
Di Prospero, N. A., and Fischbeck, K. H. (2005) Therapeutics
development for triplet repeat expansion diseases. Nat. Rev.
Genet. 6, 756 765
Burnett, R., Melander, C., Puckett, J. W., Son, L. S., Wells, R. D.,
Dervan, P. B., and Gottesfeld, J. M. (2006) DNA sequencespecific polyamides alleviate transcription inhibition associated
with long GAATTC repeats in Friedreichs ataxia. Proc. Natl.
Acad. Sci. U. S. A. 103, 1149711502
Melander, C., Burnett, R., and Gottesfeld, J. M. (2004) Regulation of gene expression with pyrrole-imidazole polyamides.
J. Biotech. 112, 195220
Gottesfeld, J. M. (2007) Small molecules affecting transcription
in Friedreich ataxia, Pharmacol. Ther. 116, 236 248
Tsai, S. M., Farkas, M. E., Chow, C. J., Gottesfeld, J. M., and
Dervan, P. B. (2007) Unanticipated differences between - and
-diaminobutyric acid-linked hairpin polyamide-alkylator conjugates. Nucleic Acids Res. 35, 307316
Festenstein, R. (2006) Breaking the silence in Friedreichs
ataxia. Nature. Chem. Biol. 2, 512513

The FASEB Journal

Received for publication November 2, 2007.

Accepted for publication December 27, 2007.

WELLS