Báo cáo Y học: Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy potx

PRIORITY PAPER
Structural heterogeneity of pyrimidine/purine-biased DNA sequence
analyzed by atomic force microscopy
Mikio Kato
1,2
, Chad J. McAllister
2
, Shingo Hokabe
1
, Nobuyoshi Shimizu
3
and Yuri L. Lyubchenko
2
1
Department of Life Science, Osaka Prefecture University College of Integrated Arts and Sciences, Sakai, Japan;
2
Department of Microbiology, Arizona State University, Tempe, USA;
3
Department of Molecular Biology,
Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan
We report here the direct evidence for the formation of
alternative DNA structures in a plasmid DNA, termed
pTIR10, containing a 0.23-kb pyrimidine/purine-biased
(Pyr/Pur) stretch isolated from the rat genome. Long
Pyr/Pur sequences are abundant in eukaryotic genomes, and
they may modulate the biological activity of genes and
genomes via formation of various types of triplex-related
structures. The plasmid DNA in sodium acetate buffer
(pH 4.35) was deposited on APS-modified mica, and after
drying it was imaged with an atomic force microscope in air.
Various types of thick protrusions have been observed on

pTIR10 DNA. Structural parameters (width and height) of
DNA molecules suggest that the alternative structures
observed here are variations on the theme of an intra-
molecular triplex. The biological relevance of the structural
features within Pyr/Pur stretches is discussed.
Keywords: alternative DNA structure; atomic force micros-
copy (AFM); H-DNA; intramolecular triplex DNA; poly-
pyrimidine/ polypurine sequence.
There is a wealth of evidence indicating that short
pyrimidine/purine-biased (Pyr/Pur) mirror symmetry
sequences adopt an intramolecular triplex conformation
(H-DNA) [1,2]. Intramolecular triplexes play important
roles in genome functions; e.g., triplex formation causes
pausing of polymerases during replication [3] and tran-
scription [4], and enhances homologous recombination
[5,6]. Structural transition between B-DNA and triplex
DNA may provide some target site for protein recogni-
tion, as specific proteins are involved in homologous
recombination mediated by triplex DNA [7]. Pyr/Pur
regions of several hundred base pairs long are more
abundant in eukaryotic genomes than expected from their
base composition [8]. Pyr/Pur sequences in the intergenic
regions in the genome are suggested to modulate replica-
tion timing through the pausing or stalling of DNA
polymerase, as they are often observed in the regions
where replication timing switches [9]. The barrier region
for replication-fork movement in human ribosomal RNA
genes is also known to contain several simple repetitive
sequences including Pyr/Pur tracts [10]. Long Pyr/Pur
sequences cloned in the plasmid were sensitive to single

strand-specific S1 nuclease, and led to the appearance of
retarded, but diffused, bands in agarose gel electrophoresis
under acidic conditions, suggesting the occurrence of
alternative DNA structure and the presence of heteroge-
neity in DNA conformations in vitro [11]. Characterization
of the potential for forming unusual DNA structure is
critical for understanding how the region works in the
genome. Traditional chemical and enzymatic probe tech-
niques were not efficient, however, in unraveling the
structural organization of such long DNA sequences as
the result was the sum of various conformers in solution.
Recently, we have successfully characterized the intramo-
lecular triplex structure formed in supercoiled DNA
within 46 bp of Pyr/Pur mirror symmetry by atomic force
microscopy (AFM) accompanied by an appropriate
sample preparation procedure [12]. Here, we apply the
same technique to visualize directly structural features of
supercoiled DNA containing long Pyr/Pur sequence
isolated from the rat genome. The studies revealed the
formation of alternative local DNA structures of different
shapes.
MATERIALS AND METHODS
DNA
A pUC19 derivative, pTIR10, containing  0.23 kb of Pyr/
Pur region within a 0.5-kb insert isolated from the rat
genome (GenBank accession number U22965 [11]) was used
in this work. Southern blot analysis revealed that the
pTIR10 sequence hybridized efficiently with fragments of
rat and human genomic DNA, meaning that similar
sequences were abundant in the genomes (M. Kato &

M. Yuasa, unpublished observation). Supercoiled DNA
was isolated from Escherichia coli strain JM107 transfor-
mants by FlexiPrep Kit (Pharmacia). The electrophoretic
mobility of pTIR10 DNA is shown in Fig. 1. In the acidic
buffer, migration of pTIR10 was retarded and the molecules
were diffused while it migrated normally in the neutral
Correspondence to M. Kato, Department of Life Science,
Osaka Prefecture University College of Integrated Arts and Sciences,
1-1 Gakuencho, Sakai 599-8531, Japan.
Tel./Fax: + 81 72 254 9746,
E-mail: or

Abbreviations: AFM, atomic force microscopy; Pyr, pyrimidine; Pur,
purine.
(Received 25 April 2002, revised 15 June 2002, accepted 20 June 2002)
Eur. J. Biochem. 269, 3632–3636 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03063.x
buffer, suggesting that pTIR10 molecules at acidic condi-
tions are conformationally heterogenous and/or dynamic.
AFM observation
The samples for AFM studies were prepared at acidic pH
favoring intramolecular triplex (H-DNA) formation
(50 m
M
sodium acetate, pH 4.35) or at neutral pH (10 m
M
Tris/HCl/1 m
M
EDTA, pH 7.5). An aliquot (5 lL) of DNA
solution (0.3–0.6 lgÆmL
)1

) was deposited on mica function-
alized with aminopropyl silatrane (APS-mica) as described
previously [13]. The AFM imaging procedure has been
described elsewhere [14]. Images were acquired by MM
SPM NanoScope III system (Veeco/Digital Instruments,
Santa Barbara, CA, USA) operating in Tapping Mode in air
Fig. 1. Electrophoretic mobility of pTIR10 DNA. Left panel, electro-
phoresis on 1% agarose in 40 m
M
Tris/acetate/5 m
M
sodium acetate/
1m
M
EDTA (pH 7.5); right panel, electrophoresis on 1% agarose in
30 m
M
sodium acetate/1 m
M
EDTA (pH 4.6). Lane 1, pUC19 DNA;
lane 2, pTIR10 DNA; lane 3, pTIR10 DNA linearized by HindIII
digestion; lane M, HindIII-digested lambda phage DNA size marker.
Fully supercoiled molecules, linear molecules, open circles and super-
coileddimermoleculesofpUC19aremarkedwitha,b,c,andd,
respectively, in the left panel. The faint DNA band at the bottom of
lanes 2 and 3 (marked with asterisk) might be the supercondensed
structure reported previously [25,26].
Fig. 2. AFM images of pTIR10 and pUC19 DNA. Structural irregu-
larities are indicated on large-scale images by arrows and the enlarged
rescanned images of the molecules are inserted. (A) pTIR10 DNA

prepared at acidic pH; (B) pTIR10 DNA prepared at neutral pH; (C)
pUC19 DNA prepared at acidic pH. Thick protrusions were observed
in pTIR10 samples (molecules 1–4). In the samples of pUC19, a few
molecules retained a sharp-turn part (molecule 5) but it was distinct
from the thick protrusions observed in pTIR10 samples.
Ó FEBS 2002 AFM studies on alternative DNA structures (Eur. J. Biochem. 269) 3633
at ambient conditions using silicon probes from MikroMash
Inc. (Estonia). The length and height measurements were
performed with the
FEMTOSCAN
software (Advanced Tech-
nologies Center, Moscow, Russia).
RESULTS AND DISCUSSION
Examples of AFM images of Pyr/Pur-containing super-
coiled DNA (pTIR10) are shown in Fig. 2A,B, and those of
control plasmid (pUC19) are shown in Fig. 2C. The most
abundant among these structural features observed in
pTIR10 DNA samples were thick protrusions (indicated
by arrows in Figs 2A,B and 3A). Generally, more than 60%
of the population retained this stem structure in pTIR10
samples prepared at acidic pH, and about 20% of the DNA
molecules prepared at neutral pH had the stem structure,
whereas the stem structure was rarely formed in pUC19
DNA samples. We have examined 43 molecules in total of
pUC19 DNA prepared at acidic pH as a control and only
one molecule had a stem part. Structural parameters for the
stems (the width and the height) in comparison with the
same parameters for regular DNA regions are defined as
shown in Fig. 4D and listed in Table 1. Fiber diffraction
analysis has proposed that the helix diameter of double-

stranded DNA is about 2 nm in B-form DNA [2]. Due to
the convolution effect of the probe tip [15,16], apparent
width of DNA obtained by the AFM will be larger than
actual size (Fig. 4D, right panel). In the present results,
differences in the parameters between the stem part and
regular DNA are close to those obtained for short
intramolecular triplexes (H-DNA) earlier [12] suggesting
that the structures observed are intramolecular triplexes.
Efficient formation of the stem structures at acidic pH also
supported the involvement of protonated bases that are
required for H-DNA. It is noticeable that heterogeneity in
size and shape of the stems occurred in the samples prepared
at acidic pH. All types of local DNA structures obtained at
acidic pH are shown in Fig. 3. An example of short stems is
shown in Fig. 3A. Relatively long stems were often curved
as shown in Fig. 3B. In the formation of intermolecular
triplex structure, nontriplex forming sequence conjugated to
the triplex forming oligonucleotide caused bending of target
DNA at the junction [17]. The curved triplex stems observed
in the present study might be caused by the presence of any
mismatch in the triplex stem. Two clearly separated stems,
twin stem structures, were also observed, and one example is
shown in Fig. 3C. The two stems can be very close and form
P-shaped and Y-shaped structures shown in Fig. 3D and E,
respectively. We have identified 45 molecules having the
triplex stems. Seven of these retained twin stems or
noncanonical triplex stems (P-andY-shaped structures)
and the rest had a single stem. Structural parameters for the
twin stems or noncanonical triplex stems (14 stems in total)
were similar to those for single stems. The average stem

width is 6.86 nm (SD ¼ 0.49, n ¼ 14) and the average stem
height is 1.43 nm (SD ¼ 0.21, n ¼ 14), whereas the average
width and height of regular DNA regions are 5.69 nm
(SD ¼ 0.71, n ¼ 7) and 0.90 nm (SD ¼ 0.09, n ¼ 7),
respectively. The average length of stem part is 10.61 nm
(SD ¼ 2.41, n ¼ 14), and the average of the total stem
length in one molecule is 21.21 nm (SD ¼ 3.78, n ¼ 7). The
observations suggest that the two stem structures on
pTIR10 DNA formed independently within one large
region due to the large size of the Pyr/Pur region. Moreover,
as the junction of the triplex stem and outgoing arms may be
highly flexible [18], certain twin stems are arranged in a tail-
to-tail manner forming P-andY-shaped structures.
Shimizu et al. [19] demonstrated that considerable structur-
al diversity can exist in long (GA)
n
repeats; proposed higher
order unusual DNA structure models containing multiple
formation of triplex stems. A model for the formation of
Fig. 3. High-resolution AFM images of the most representative families
of the irregularities found in pTIR10 DNA. (A) short triplex-like stem;
(B) long curved stem; (C) twin stems; (D) P-shaped structure; (E)
Y-shaped structure. Thick protrusions are indicated by arrows. Scale
bar (100 nm) is given at the bottom of panel e and common to all
panels.
Table 1. Structural parameters of pTIR10 DNA molecules defined by
AFM. Mean values are reported in nm; SD is given in parentheses.
Stem
length Width Height
No. of

molecules
Acidic sample deposition
Triplex stem 14.55 (4.28) 8.14 (0.94) 1.52 (0.25) 38
a
B-DNA
b
NA 6.03 (0.93) 0.98 (0.15) 45
Neutral sample deposition
Triplex stem 10.90 (1.43) 7.92 (1.12) 1.41 (0.33) 19
B-DNA
b
NA 6.21 (0.90) 1.03 (0.25) 19
a
The molecules having the single stem structure were used for
determining stem parameters.
b
Width and height for B-DNA were
obtained by measuring outgoing arms proximal to the stem part.
3634 M. Kato et al. (Eur. J. Biochem. 269) Ó FEBS 2002
alternative DNA structures is shown in Fig. 4. The stem
structures observed in the samples of pTIR10 DNA
prepared at neutral pH were shorter than those obtained
in the samples prepared at acidic pH, and heterogeneity in
size and shape was not seen under neutral conditions.
Long (> 200 bp) Pyr/Pur stretches are widely repre-
sented at intergenic regions in the genomes of eukaryotic
organisms. In the present work, one of the long Pyr/Pur
sequences isolated from the rat genome is shown to adopt
various types of alternative DNA structures including
multiprotrusions, supposedly involving intramolecular tri-

plex structure. Intermolecular interaction between two
intramolecular triplexes of Pur/Pur/Pyr has been reported
for (GAA/TTC)
n
triplet repeat in the first intron of the
human frataxin gene (sticky DNA) [20], and formation of
the sticky DNA directly correlates to the inhibitory effect
on transcription [21,22]. As the triplex-forming Pyr/Pur
regions exist in the genome widely and abundantly, certain
Pyr/Pur loci may offer the triplex stems for inter- and
intra-chromosomal interactions to modulate transcription
and replication. Two independent triplex stems in the twin
stem structure may be able to interact with each other to
stabilize the alternative DNA structure in certain Pyr/Pur
sequences. Conformational variability of triplex structures
can provide tuning of the interaction of the triplex-
forming regions. In addition, triplex formation absorbs the
negative supercoils of the flanking regions, and the local
changes in the DNA superhelicity can affect the global
shape of the topological domain [23,24]. These regions
with the potential for forming various types of intra-
molecular triplex may function in some processes of
genome regulation such as replication rate and timing,
recombination and chromosome folding by modulating
the local structure of DNA regions and the global
topology of chromatin domains.
ACKNOWLEDGEMENTS
This work was supported in part by the fund from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (MEXT)
(to M. K.), the fund for Research for the Future Program from the

Japan Society for the Promotion of Science (JSPS) (to N. S.), and the
National Institute of Health grant GM 62235 (to Y. L. L.).
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