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.. Keywords: altern
Trang 1P R I O R I T Y P A P E R
Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy
Mikio Kato1,2, Chad J McAllister2, Shingo Hokabe1, Nobuyoshi Shimizu3and Yuri L Lyubchenko2
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
M A T E R I A L S A N D M E T H O D S
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: mkato@rishiri.cias.osakafu-u.ac.jp or
mikio_kato@mac.com
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) FEBS 2002 doi:10.1046/j.1432-1033.2002.03063.x
Trang 2buffer,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 mMsodium acetate,pH 4.35) or at neutral pH (10 mM
Tris/HCl/1 mMEDTA,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/
1 m 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-coiled dimer molecules of pUC19 are marked with a, b, c, and d,
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.
Trang 3at ambient conditions using silicon probes from MikroMash
Inc (Estonia) The length and height measurements were
performed with theFEMTOSCANsoftware (Advanced
Tech-nologies Center,Moscow,Russia)
R E S U L T S A N D D I S C U S S I O N
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- and Y-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 (SD¼ 0.49, n ¼ 14) and the average stem height is 1.43 nm (SD¼ 0.21, n ¼ 14),whereas the average width and height of regular DNA regions are 5.69 nm (SD¼ 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 (SD¼ 2.41, n ¼ 14),and the average of the total stem length in one molecule is 21.21 nm (SD¼ 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- and Y-shaped structures Shimizu et al [19] demonstrated that considerable
structur-al diversity can exist in long (GA)nrepeats; proposed higher order unusual DNA structure models containing multiple formation of triplex stems A model for the formation of
Fig 3 High-resolutionAFM 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-DNAb 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.bWidth and height for B-DNA were
obtained by measuring outgoing arms proximal to the stem part.
Trang 4alternative 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
A C K N O W L E D G E M E N T S
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.).
R E F E R E N C E S
1 Soyfer,V.N & Potaman,V.N (1996) Triple-Helical Nucleic Acids Springer-Verlag,New York.
2 Sinden,R.R (1994) DNA Structure and Function Academic Press, New York.
3 Baran,N.,Lapidot,A & Manor,H (1991) Formation of DNA triplexes accounts for arrest of DNA synthesis at d(TC)n tracts Proc Natl Acad Sci USA 80,105–109.
4 Grabczyk,E & Fishman,M.C (1995) A long purine-pyrimidine homopolymer acts as a transcriptional diode J Biol Chem 270, 1791–1797.
5 Rooney,S.M & Moore,P.D (1995) Antiparallel,intramolecular triplex DNA stimulates homologous recombination in human cells Proc Natl Acad Sci USA 92,2141–2144.
6 Benet,A.,Molla,G & Azorin,F (2000) d (GA.TC) n microsatellite DNA sequences enhance homologous DNA
Fig 4 Schematic explanation for formation of the alternative DNA structures in long Pyr/Pur sequence and definition of the structural parameters (A) Model for typical intramolecular triplex; (B) model for curved long triplex; (C) model for twin stems; (D) definition of the structural parameters Open circles indicate Watson-Crick type duplex and the dots indicate the third strand interaction The free single strand runs from point 1 to point 2 (marked in A),and is omitted in the illustrations for the sake of clarity The long stems may be curved by the misalignment of third strand (B) Although mirror symmetry may not be necessary for the formation of base triads in the triplex stem [27],some mismatches may occur between the duplex and the third strand in the Pyr/Pur sequence of pTIR10 Multiprotrusion may occur in sufficiently long Pyr/Pur tracts (C) The junction of triplex stem and outgoing duplex arms (marked with arrows) may be flexible and the length of the linker duplex between the two stems may be variable so that the P-shaped and Y-shaped structures can be formed Here is shown a model for twin stems in which both stems conform to the same triplex isomer,but a pair of different isomers is also possible (e.g one is H-y3 and another is H-y5).
Trang 5recombination in SV40 minichromosomes Nucleic Acids Res 28,
4617–4622.
7 Datta,H.J.,Chan,P.P.,Vasquez,K.M.,Gupta,R.C & Glazer,
P.M (2001) Triplex-induced recombination in human cell-free
extracts J Biol Chem 276,18018–18023.
8 Behe,M.J (1995) An overabundance of long oligopurine tracts
occurs in the genome of simple and complex eukaryotes Nucleic
Acids Res 23,689–695.
9 Watanabe,Y.,Tenzen,T.,Nagasaka,Y.,Inoko,H & Ikemura,T.
(2000) Replication timing of the human X-inactivation center
(XIC) region: correlation with chromosome bands Gene 252,163–
172.
10 Little,R.D.,Platt,T.H.K & Schildkraut,C.L (1993) Initiation
and termination of DNA replication in human rRNA genes Mol.
Cell Biol 13,6600–6613.
11 Kato,M (1993) Polypyrimidine/polypurine sequence in plasmid
DNA enhances formation of dimer molecules in Escherichia coli.
Mol Biol Rep 18,183–187.
12 Tiner,W.J.,S.r.Potaman,V.N.,Sinden,R.R & Lyubchenko,Y.L.
(2001) The structure of intramolecular triplex DNA: Atomic force
microscopy study J Mol Biol 314,353–357.
13 Shlyakhtenko,L.S.,Hsieh,P.,Grigoriev,M.,Potaman,V.N.,
Sinden,R.R & Lyubchenko,Y.L (2000) A cruciform structural
transition provides a molecular switch for chromosome structure
and dynamics J Mol Biol 296,1169–1173.
14 Shlyakhtenko,L.S.,Potaman,V.N.,Sinden,R.R & Lyubchenko,
Y.L (1998) Structure and dynamics of supercoil-stabilized DNA
cruciforms J Mol Biol 280,61–72.
15 Lyubchenko,Y.L.,Jacobs,B.L.,Lindsay,S.M & Stasiak,A.
(1995) Atomic-force microscopy of nucleoprotein complexes.
Scanning Microscopy 9,705–727.
16 Yang,J.,Mou,J.,Yuan,J.Y & Shao,Z (1996) The effect of
deformation on the lateral resolution of atomic force microscopy.
J Microsc 182,106–113.
17 Cherny,D.I.,Fourcade,A.,Svinarchuk,F.,Nielsen,P.E.,Malvy,
C & Delain,E (1998) Analysis of various sequence-specific
tri-plexes by electron and atomic force microscopies Biophys J 74, 1015–1023.
18 Htun,H & Dahlberg,J.E (1989) Topology and formation of triple-stranded H-DNA Science 243,1571–1576.
19 Shimizu,M.,Hanvey,J.C & Wells,R.D (1990) Multiple non-B-DNA conformations of polypurineÆpolypyrimidine sequences in plasmids Biochemistry 29,4704–4713.
20 Sakamoto,N.,Chastain,P.D.,Parniewski,P.,Ohshima,K., Pandolfo,M.,Griffith,J.D & Wells,R.D (1999) Sticky DNA: self association properties of long GAAÆTTC repeats in RÆRÆY triplex structures from Friedreich’s ataxia Mol Cell 3, 465–475.
21 Sakamoto,N.,Ohshima,K.,Montermini,L.,Pandolfo,M & Wells,R.D (2001) Sticky DNA,a self-associated complex formed
at long GAA.TTC repeats in intron 1 of the frataxin gene,inhibits transcription J Biol Chem 276,27171–27177.
22 Sakamoto,N.,Larson,J.E.,Iyer,R.R.,Montermini,L.,Pan-dolfo,M & Wells,R.D (2001) GGA.TCC-interrupted triplets in long GAA.TTC repeats inhibit the formation of triplex and sticky DNA structures,alleviate transcription inhibition,and reduce genetic instabilities J Biol Chem 276,27178–27187.
23 Yang,Y.,Westcott,T.P.,Pedersen,S.C.,Tobias,I & Olson, W.K (1995) Effects of localized bending on DNA supercoiling Trends Biochem Sci 20,313–319.
24 Brukner,I.,Belmaaza,A & Chartrand,P (1997) Differential behavior of curved DNA upon untwisting Proc Natl Acad Sci USA 94,403–406.
25 Huang,X & Chen,X (1990) Supercondensed structure of plas-mid pBR322 in E coli topoisomerase II mutant J Mol Biol 216, 195–199.
26 Kato,M & Furuno,A (1992) Unusual structure of plasmid DNA formed in transformants of Escherichia coli Res Microbiol 143,665–669.
27 Klysik,J (1995) An intramolecular triplex structure from non-mirror repeated sequence containing both Py:PuÆy and Pu:PuÆPy triads J Mol Biol 245,499–507.