From the
Department of Environment and Natural
Sciences, Graduate School of Environment and Information Sciences, Yokohama
National University, 79-7 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan and
the
RIKEN Genomic Sciences Center, Tsurumi,
Yokohama 230-0045, Japan
Received for publication, April 9, 2003
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ABSTRACT |
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INTRODUCTION |
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We determined the structure of d(GGAGGAGGAGGA) (d(GGA)4) composed of four tandem GGA units under physiological conditions (19). d(GGA)4 folds into an intramolecular quadruplex composed of a G:G:G:G tetrad and a G(:A):G(:A): G(:A):G heptad. Four G-G segments of d(GGA)4 are aligned parallel to each other due to seven successive turns of the main chain at each of the GGA and GAGG segments. This was the first demonstration that DNA can form an intramolecular parallel quadruplex. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for the telomere DNA in the crystalline state (20). We also showed that two quadruplexes of d(GGA)4 form a dimer stabilized through the stacking interaction between the heptads of the two quadruplexes.
Our findings as to GGA triplet repeat DNA, together with the crystallographic result for telomere DNA, indicated the possibility of intramolecular higher order packing of quadruplexes for longer DNA with certain repeating units. To address this point and to elucidate the character of naturally occurring GGA repeat DNA, we studied the structure of d(GGAGGAGGAGGAGGAGGAGGAGGA) (d(GGA)8) under physiological conditions. Here, we present its unique structure. This is the first demonstration of higher order packing of quadruplexes at atomic resolution. The biological events related to GGA triplet repeat DNA can be rationalized in the light of the elucidated structure. Furthermore, our findings provide a support for the hypothetical higher order packing of quadruplexes of telomere DNA and indicate the mode of packing for it.
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EXPERIMENTAL PROCEDURES |
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CD and NMR SpectroscopiesCD spectra and thermal CD melting curves were recorded with a Jasco J-720 spectropolarimeter. The temperature of the solution was raised from 1 to 95 °C at the rate of 1 °C/min. Changes in CD intensity were monitored at 263 nm. The melting temperature was determined by use of the derivative of the melting curve.
NMR spectra were recorded with Bruker DRX600 and DRX800 spectrometers equipped with a quadruple-resonance probe with X, Y, and Z gradients. The following NMR experiments were used to assign the resonances and to obtain distance and dihedral angle constraints; NOESY,1 TOCSY, 31P-decoupled DQF-COSY, 1H-13C HSQC, 1H-15N HSQC, and 1H-31P HetCor. Spectra were processed with XWIN-NMR (Bruker), NMRPipe (21), and Capp/Pipp/Stapp (22).
Distance and Dihedral Angle ConstraintsInterproton distances were calculated from NOESY spectra with mixing times of 50 and 200 ms as described previously (19). In total, 750 distance constraints were obtained.
Dihedral angle constraints for the torsion angle were derived from
3JH5'-P and
3JH5''-P couplings
as described previously (19,
23,
24). Thus, the
torsion
angles of G2, G5, G8, G14, G17, and G20 were constrained to 180 ±
20°. For the other residues,
was left unconstrained. Dihedral angle
constraints for the
torsion angles were derived from
3JH4'-H5'
and
3JH4'-H5''
couplings as described previously
(19,
23,
24). Thus, the
torsion
angles of G2, G4, G5, G7, G8, G10, G14, G16, G17, G19, G20, and G22 were
constrained to 60 ± 20°. For the other residues,
was left
unconstrained.
Dihedral angle constraints for the and endocyclic
0,
1,
2,
3, and
4
torsion angles were derived from
3JH1'-H2',
3JH2''-H3',
and
3JH3'-H4'
couplings as described previously
(19,
2325).
The sugar pucker was determined as follows on the basis of the results of
analysis of the intensities of the H1'-H2', H2''-H3',
and H3'-H4' cross-peaks in the DQF-COSY spectrum: all G residues,
except for G13, and A12, C1'-exo conformation; all A residues
except for A12 and A24, C3'-endo conformation; A24,
O4'-endo conformation; G13, not determined due to overlapping
of resonances. The sugar conformations were confirmed independently by the
C3' and C4' chemical shift values
(23,
24,
26). The
and
endocyclic
0
4 torsion angles of all G,
except for G13, and A12 residues were moderately constrained, leaving the
sugar free to take any conformation without an energy penalty between
O4'-endo and C2'-endo including
C1'-exo in the pseudorotation cycle. In the same way, those of
all A residues, except for A12 and A24, were constrained between
C2'-exo and C4'-exo, including
C3'-endo, and those of A24 between C4'-exo and
C1'-exo, including O4'-endo. That of G13 was not
constrained.
Dihedral angle constraints for the torsion angle were derived from
3JH3'-P coupling as
described previously (19,
23,
24). Thus, the
torsion
angles of all residues, except for A24, were constrained to 120
± 45°. For A24,
was left unconstrained. No dihedral angle
constraints were used for the
and
torsion angles.
Structure CalculationStructure calculations were carried out using distance and dihedral angle constraints with a simulated annealing protocol supplied with X-PLOR, version 3.8 (27). Neither hydrogen bonding constraints nor planarity constraints were included. Twenty final structures were selected from 100 calculations on the basis of the criteria of the smallest residual energy values. None of them violated the distance constraints by more than 0.3 Å or the dihedral angle constraints by more than 6°. The structures were viewed with Insight II (MSI).
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RESULTS |
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It is known that a parallel quadruplex gives a positive CD band at 260 nm, while an antiparallel one gives a positive CD band at 295 nm either with or without a positive CD band at 260 nm (28, 29). The CD spectrum of d(GGA)8 in the presence of K+ had a positive CD band at around 260 nm without a positive CD band at around 295 nm (Fig. 1D). This suggests that d(GGA)8 forms a parallel quadruplex. The melting temperature of the structure formed by d(GGA)8 in the presence of K+ was determined to be rather high, 86 °C. This again suggests the formation of a quadruplex, because the melting temperature of a quadruplex is generally very high.
It was also found that the melting temperature does not depend on the concentration of d(GGA)8. This indicates that the structure formed is unimolecular. This conclusion was supported by the result of analysis of the line widths of NMR resonances that are sensitive to molecular weight. The line widths for d(GGA)8 (24-mer) were the same as those for d(GGA)4 (12-mer), which exists as a dimer in solution (19).
Resonance AssignmentsThe non-exchangeable 1H, 13C, and 31P resonances of d(GGA)8 were assigned as described previously (19) using standard methods (23, 30). As an example, Fig. 2A shows expansion of the NOESY spectrum allowing the sequential assignments of H1' and H6/H8 through H1'(i 1)-H6/H8(i)-H1'(i) connectivities. H2', H2'', H3', H4', and H5'/H5'' resonances were assigned by means of TOCSY and DQF-COSY spectra. These assignments were confirmed by the sequential H3'(i 1)-P(i)-H4'/H5'/H5''(i) connectivities in the 1H-31P HetCor spectrum, the assignments of 31P resonances being made at the same time (data not shown). The chemical shifts of 13C resonances were obtained from the 1H-13C HSQC spectrum. Exchangeable 1H resonances were assigned as described previously with the use of mutant oligomers in which each single G residue was replaced by an I residue (19).
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Formation of Two G:G:G:G Tetrads and Two G(:A):G(:A): G(:A):G Heptads and Their Mutual ArrangementThe formation of the G2:G5:G8:G11 and G14:G17:G20:G23 tetrads was concluded on the basis of GNH/NH2-GH8 (Fig. 2B) and GNH-GNH NOEs; the NOEs commonly observed for both tetrads are shown in Fig. 3A. The formation of the G1(:A3):G4(:A6): G7(:A9):G10 and G13(:A15):G16(:A18):G19(:A21):G22 heptads was concluded on the basis of GNH/NH2-AH8 (Fig. 2B), ANH2-GH1' (Fig. 2C), GNH2-ANH2, and GH8-AH8 NOEs, in addition to GNH/NH2-GH8 and GNH-GNH ones; the NOEs commonly observed for both heptads are shown in Fig. 3B. A12 and A24 are not involved in either the tetrads or the heptads.
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The arrangement of two heptads was determined on the basis of the strong AH2-AH1'/2'/2'' NOEs between A3 and A21, A6 and A18, and A9 and A15, and the medium to weak GH8-GH1' ones between G1 and G22, G4 and G19, G7 and G16, and G10 and G13 (Fig. 2A). Thus, the overall structure of d(GGA)8 was concluded to be as shown in Fig. 3C.
Structure of d(GGA)8The applied constraints and the structural statistics for the 20 final structures are summarized in Table I. The root mean square deviations (r.m.s.d.s) of the 20 final structures versus the mean structure for all heavy atoms were 0.35 ± 0.13 Å.
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Fig. 4A shows a stereo view of the superposition of the 20 final structures. A representative structure with the lowest energy is shown in Fig. 4B, a trace of the sugar-phosphate main chain being indicated by a tube for clarity. Two tetrad and two heptads can be seen (Fig. 4, CE), as already discussed qualitatively. A12 is located close to the upper heptad, although it is not involved in the heptad. A24 is stacked on G23.
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A turn at a G1-G2-A3 trinucleotide segment, which is composed of a sheared
G1:A3 base pair and a single G2 residue at the top, is followed by another
turn at a G2-A3-G4-G5 segment (Fig.
4B). A further succession of turns is found for each GGA
and GAGG segment except for the G11-A12-G13-G14 and G22-G23-A24 segments. In
the cases of the canonical B- and A- forms, the backbone dihedral angles are
as follows; (g),
(t),
(g+),
(t), and
(g)
(31). Deviation from the
canonical dihedral angles of
,
, and
was found for all A
residues. Deviation of
and
was found for G4, G7, G10, G11, G16,
G19, and G22. Deviation of
was found for almost all G residues. The
combination of these deviated dihedral angles results in the successive turns
of the backbone.
These successive turns cause the four G-G segments, i.e. the G1-G2, G4-G5, G7-G8, and G10-G11 segments, to be aligned parallel to each other. As a result, the G1G11 portion of d(GGA)8 forms an intramolecular parallel quadruplex (Figs. 3C and 4B). Similarly, the G13-G14, G16-G17, G19-G20, and G22-G23 segments are aligned parallel to each other, and the G13G23 portion forms another intramolecular parallel quadruplex. The two quadruplexes are packed in a tail-to-tail manner through stacking between the two heptad planes, the orientation of G-G segments of one quadruplex being opposite to that of the other quadruplex (Figs. 3C and 4B).
The stacking between the tetrad and heptad planes is shown in Fig. 4, C and D. The five-membered ring of one guanine base is stacked on the six-membered ring of the other guanine base for each G-G segment. The A3, A6, A9, A15, A18, and A21 bases are stacked on the G2, G5, G8, G14, G17, and G20 sugars, respectively, which is characteristic of the structure of a GNA trinucleotide loop and consistent with the extreme upfield shift of H4' (2.622.84 ppm) and the moderate upfield shift of H3' (4.564.66 ppm) of these G residues (32, 33). Stacking of the two heptads is shown in Fig. 4E. The G1, A3, G4, A6, G7, A9, and G10 bases are stacked on the G22, A21, G19, A18, G16, A15, and G13 ones, respectively.
All G residues take on the anti conformation with respect to the glycoside bond, while all A residues, except for A12 and A24, take on the high anti-conformation. All G residues take on the B-form sugar conformation (around C1'-exo), while all A residues, except for A12 and A24, take on the A-form sugar conformation (around C3'-endo). The high anti and C3'-endo conformations of the six A residues are supposed to be preferable for the series of turns and/or for good stacking interactions.
Weak to medium NOEs were observed between A6H2 and G7H8, A9H2 and G10H8, A18H2 and G19H8, and A21H2 and G22H8. These NOEs could be very strong, if A6, A9, A18, and A21 take on the syn conformation, forming hydrogen bonds, i.e. G4NH2-A6N1 and G4N3-A6NH2, G7NH2-A9N1 and G7N3-A9NH2, G16NH2-A18N1 and G16N3-A18NH2, and G19NH2-A21N1 and G19N3-A21NH2, respectively. Therefore, there is the possibility that there is a small fraction of the syn conformation for A6, A9, A18, and A21, being in fast exchange with the major high anti conformation on an NMR time scale.
Comparison of the Structures of d(GGA)8 and d(GGA)4The structures of d(GGA)8 and d(GGA)4 are shown schematically in Fig. 5, A and B. d(GGA)8 forms a monomeric structure, while d(GGA)4 forms a dimeric one. The structure of the G1-G11 portion of d(GGA)8 is similar to that of the upper monomer of d(GGA)4. A12 of d(GGA)8 folds back and is located close to the upper heptad, while that of d(GGA)4 is stacked on G11 and moves away from the center. The main chain of d(GGA)8 goes down further through A12, and G13 is involved in the lower heptad. The structure of the G13-A24 portion of d(GGA)8 is similar to that of the lower monomer of d(GGA)4. As a whole, the monomeric architecture of d(GGA)8 resembles the dimeric architecture of d(GGA)4.
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DISCUSSION |
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From the viewpoint of achieving maximum stacking interactions, the relative arrangement of the two heptads of d(GGA)8 can be rationalized. For example, if the lower heptad is rotated by either 90° or 180°, one A base of each heptad cannot be involved in the stacking interaction, which is energetically less stable and thus unfavorable.
Each monomer of d(GGA)4 forms an intramolecular parallel quadruplex (Fig. 5B). Matsugami et al. (19) first demonstrated that DNA can form the intramolecular parallel quadruplex. Later, an intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state (20). These structures have indicated the intramolecular higher order packing of quadruplexes. In this study, we have demonstrated for the first time that the intramolecular higher order packing of quadruplexes actually occurs and the mode of the packing at atomic resolution is discussed. The intramolecular packing of the two quadruplexes for d(GGA)8 is achieved through the stacking interaction between the heptads of each quadruplex (Fig. 5A). As a result, the two quadruplexes are arranged in a tail-to-tail manner. Alternatively, if the G13(:A15):G16(:A18):G19(:A21): G22 heptad is stacked on the G2:G5:G8:G11 tetrad, with A12 bulging out, then the two quadruplexes are arranged in a head-to-tail manner. However, this does not occur for d(GGA)8, because heptad-heptad stacking in a tail-to-tail manner is energetically much more stable and favorable than heptad-tetrad stacking in a head-to-tail manner.
When the GGA unit is further repeated, formation of the structure shown in Fig. 5A is expected for the other 24-mer region, too. The structures formed for each 24-mer region may further pack through the stacking interaction between the tetrads of each structure.
The GGA triplet repeat is abundant in eukaryotic genomes (7), and is frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites (1113). Thus, the biological significance of the GGA repeat is widely recognized. The GGA repeat shows considerable genetic polymorphism due to genetic instability (7, 34). This genetic instability is supposed to originate from DNA slippage during DNA replication and/or frequent recombination of the repeats (35). The structure elucidated in this study can be used to rationalize the occurrence of these events. In the course of replication, double-stranded DNA is locally melted to yield single-stranded DNA for use as a template for DNA synthesis. In this case, the GGA strand is expected to form the unique intramolecular structure we found, when the high concentration of K+, over 100 mM, in the nucleus is considered. The structure is very stable, as revealed by its high melting temperature of 86 °C. It was also found for d(GGA)4 that once the unique structure is formed, the addition of the complementary strand, d(TCC)4, is not effective for the formation of a duplex (19). The unique structure remained even in the presence of the complementary strand. The formed structure could cause the slippage during DNA replication, which results in a gain or loss of repeats and thus genetic instability.
Alternatively, intermolecular association of two double-stranded DNAs through the packing of two quadruplexes formed in each DNA, as shown in Fig. 5B, may also occur and facilitate the recombination. This idea is coincident with the fact that the GGA repeat is frequently located in recombination hot spot sites. The facilitated recombination may also be responsible for the genetic instability of the GGA repeat. Furthermore, the intermolecular association at the GGA repeat may play a role in the pairing of homologous chromosomes during meiosis. The idea of pairing through a quadruplex has already been proposed for the G stretch (3740). It is known that the synapsis formation between homologous chromosomes in meiosis usually begins at telomere DNA, but that it sometimes starts at loci inside a chromosome.
It is expected that the unique intramolecular structure shown in Fig. 5A is formed in the course of transcription as well, because DNA is locally melted in this case, too. The formed structure is supposed to be an obstacle for an RNA polymerase to proceed, which could explain the attenuation of gene expression at the transcription level (10). We have revealed that RNA with the related sequence also can form a similar intramolecular parallel quadruplex (36). This RNA structure is very stable, too, the melting temperature being 86 °C (36). Therefore, it is expected that the unique structure in the transcribed mRNA becomes an obstacle for the translation machinery to associate with and/or proceed along mRNA in the course of translation, which may lead to the attenuation of gene expression at the post-transcriptional level (10).
The structure elucidated in this study can also provide a clue as to the higher order packing of the telomere DNA. A quadruplex of telomere DNA only contains the tetrad, not the heptad (Fig. 5C). Therefore, the arrangement of the two quadruplexes in a head-to-tail manner, in which the G26:G32:G38: G44 tetrad of the second quadruplex is stacked on the G4:G10: G16:G22 tetrad of the first quadruplex, with a T23-T24-A25 segment serving as a linker, seems to be likely for d[AGGG-(TTAGGG)7], as proposed by Parkinson et al. (20). Because of the lack of the heptad, the tail-to-tail arrangement of the two quadruplexes, in which the G26:G32:G38:G44 tetrad is stacked on the G2:G8:G14:G20 tetrad, with folding back of the T23-T24-A25 segment, seems less likely, although it is not impossible.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
¶ Supported by Grants-in-aid for Scientific Research (numbers 12470487 and 15030214) and the Protein 3000 Project of the Ministry of Education, Culture, Sports, Science and Technology of Japan. To whom correspondence should be addressed. Tel.: 81-45-339-4264; Fax: 81-45-339-4264; E-mail: masakata{at}ynu.ac.jp.
1 The abbreviations used are: NOESY, nuclear Overhauser effect spectroscopy;
NOE, nuclear Overhauser effect; TOCSY, total correlation spectroscopy; DQF,
double quantum filtered; HSQC, heteronuclear single-quantum coherence
spectroscopy; r.m.s.d., root mean square deviation.
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REFERENCES |
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