| Nucleic Acids Research | Pages |
Intercalated cytosine motif and novel adenine clusters in the crystal structure of the Tetrahymena telomere
Introduction
Materials And Methods
Results
Two different cytosine tetraplexes
Two adenine clusters
Three modes of base pairing
Discussion
Comparison with previous results
The bridging adenine clusters
Acknowledgements
References
Intercalated cytosine motif and novel adenine clusters in the crystal structure of the Tetrahymena telomere
PDB accession no. 294D
ABSTRACT
INTRODUCTION
Telomere DNA located at chromosome ends with many repeating sequences plays a vital role in chromosomal stability (1,2). It is important in both the normal control of cell proliferation and the abnormal growth of cancer (3). The first telomere DNA was isolated from the ciliate Tetrahymena thermophila in the early 1970s (4). Its G-rich strand contains repeats of a short sequence, d(GGGGTT), and its complementary C-rich strand contains repeated d(AACCCC). Both of these repeating segments can exist as four-stranded molecules as well as in DNA duplex form. It has long been known that polymers containing cytosine can form three hydrogen bonds with another cytosine if they are hemiprotonated (5-8). More recent NMR experiments on d(TC5) and related sequences yielded an unusual structural motif: an intercalated tetraplex (I motif), in which the same C·C+ pairings were seen in two parallel-stranded duplexes intercalated into each other in an antiparallel fashion (9). The first crystal structure of a C-rich sequence d(C4) confirmed the novel I motif and revealed more detailed structural information (10). Subsequently, several additional crystal studies of sequences with cytosine stretches have also revealed the I motif and showed structural variation among different sequences (11-13).
In these sequences, the bases attached to the cytosine tetraplex have shown a great degree of structural variability. In the metazoan telomeric sequence d(TAACCC), a stabilized loop was formed by TAA. However, in the Tetrahymena telomeric sequence, d(AACCCC), the structure displays a novel structural motif: the adenine cluster (A cluster). The adenines located at the 5[prime]-end of each strand form two different types of A clusters, with three stacking base pairs in one and four stacking base pairs in another. Three different base pairing modes are involved. The stacked A·A base pairs in each A cluster also stack upon the two different types of cytosine tetraplexes in orthogonal directions to form alternating A cluster-C tetraplex base stacking continuously along the x- and z-axes. These features have some similarities with another recently solved structure d(AACCC) (L.Chen, L.Cai, Q.Gao and A.Rich, in preparation). There are two cytosine tetraplexes in an asymmetric unit, however, there are significant differences in their geometries.
MATERIALS AND METHODS
The oligodeoxyribonucleotide d(AACCCC) was synthesized on an Applied Biosystem DNA synthesizer. It was then purified by HPLC with a linear gradient of 5-40% acetonitrile in 0.1 M triethylammonium acetate buffer, pH 7.0. Crystals were grown at room temperature by vapor diffusion using the sitting drop method from solutions containing 2.0 mM d(AACCCC) and 100 mM sodium cacodylate buffer adjusted to various pH values and equilibrated with a reservoir of 70% ammonium sulfate. The best crystal, measuring 0.3 × 0.2 × 0.1 mm, was obtained with buffer at pH 7.5. The crystal diffracted to 2.5 Å resolution. It crystallizes in space group P22121 with cell dimensions a = 35.93, b = 52.33, c = 76.94 Å. All diffraction data were collected on a Rigaku R-AXIS II imaging plate system at 4°C and processed with the PROCESS program provided by the Molecular Structure Corporation. The data set was collected to 2.5 Å resolution, with 64 frames at a crystal-to-plate distance of 120 mm using 4° oscillations. There were 4628 independent reflections above the 1[sigma] (I) level from 20 to 2.5 Å. Seventy-five percent of the reflections were observed in the resolution shell between 2.75 and 2.5 Å. Overall completeness from 20 to 2.5 Å is 86.5%. See Table 1 for a summary of crystal data and data collection statistics.
Table 1.
| Crystal data for d(AACCCC) | |
| Space group | P22121 |
| Unit cell | a = 35.93 Å, b = 52.33 Å, c = 76.94 Å |
| Strands per unit cell | 32 |
| Strands per asymmetric unit | 8 |
| Summary of data collection statistics | |
| Resolution | 20-2.5 Å |
| Number of observations | 33 551 |
| Number of unique reflections | 4628 |
| Overall completeness | 86.5% |
| Outermost shell | 2.75-2.5 Å |
| Outermost shell completeness | 75% |
| R-merge | 6% |
| Refinement statistics | |
| Resolution | 10-2.5 Å |
| Number of reflections | 4628 |
| Number of non-hydrogen DNA atoms | 836 |
| Number of water molecules | 61 |
| RMS bond length | 0.016 Å |
| RMS bond angle | 3.7° |
| R-factor | 0.21 |
| Free R-factor | 0.29 |
Several I motif crystal structures have been solved using molecular replacement techniques (11-13). This structure was also solved by that method using XPLOR (14). The starting model used the I motifs from the crystal structure of d(AACCC), which was solved by the single isomorphous replacement and single anomalous scattering method as the crystal soaked with HgCl2 was isomorphous to the native crystal (L.Chen, L.Cai, A.Gao and A.Rich, in preparation). Rotation and translation searches with that model at various resolution ranges of the d(AACCCC) diffraction data always led to the same orientation of the molecule in the lattice. This clearly showed that the asymmetric unit contained eight independent strands of d(AACCCC), enough to form two independent cytosine tetraplexes. The position of the molecule showed that orientation of the helical axis of one tetraplex was parallel to the x-axis and the helical axis of the other parallel to the z-axis. This stacking pattern is in agreement with the native Patterson map of the molecule. After several cycles of rigid body refinement using 10-2.5 Å data, the difference map allowed us to identify the missing adenines and the extra cytosines. We then carried out simulated annealing refinement, leading to an R-factor of 25.2%. Twenty cycles of restrained individual isotropic B-factor refinement followed. Well-ordered water molecules were then located from the difference Fourier map (Fo - Fc) and added as oxygen atoms to the model only if they had a peak height of >3[sigma] in the difference density map. A total of 61 water molecules were found in this way. A final round of refinement completed the structural determination with an R-factor of 0.213 and root mean square (RMS) deviations from ideal bond lengths and angles of 0.016 Å and 3.744°, respectively. The free R-factor (15) based on a random subset of 10% of the reflections is 29%. The refinement statistics are listed in Table 1. The coordinates have been deposited in the Brookhaven Protein Data Bank (accession no. 294D).
RESULTS
Two different cytosine tetraplexes
The oligonucleotide d(AACCCC) crystallizes in the orthorhombic space group P22121. There are eight strands in the asymmetric unit, enough to form two cytosine tetraplexes. Figure
Careful inspection clearly shows that the configurations of the two cytosine tetraplexes differ in a subtle way. Tetraplex 1 (Fig.
In each tetraplex, the interaction of two parallel duplexes yields a quadruplex with two wide and two narrow grooves which, as in d(C4), are largely symmetrical about the helical axis. The narrow groove is made up of two closely packed strands in antiparallel orientation. The two backbone chains fit into each other remarkably well in a zig-zag fashion. They are so close to each other that some interchain P-P distances are even shorter than intrachain ones. In tetraplex 1, the average intrachain P-P distance is 6.33 Å. The average interchain P-P distance across the minor groove is 6.36 Å, with the shortest being 5.62 Å. The average interchain P-P distance across the minor groove for tetraplex 2 is comparable at 6.81 Å. The minor groove is so narrow that there is little room left to trap anything. Indeed, we find no water molecules inside the minor groove.
a
 b  |
Figure 1. Structure of cytosine tetraplexes. (a) Cytosine tetraplex 1 of structure d(AACCCC). (Left) A schematic diagram illustrating the overall configuration of tetraplex 1. The two strands that are parallel and form hydrogen bonds between their cytosine bases are colored black, while the other two are colored white. (Right) View into the major groove of tetraplex 1. The major groove is wide and open. The center of the molecule is composed of intercalating cytosine residues held together by C·C+ base pairs. Note that there are two adenine residues at the 5[prime]-end of each strand and that they project away from the center of the molecule. The outermost C·C+ base pairs of the tetraplex are from the 3[prime]-end of each strand. (b) Cytosine tetraplex 2 of structure d(AACCCC). (Left) A schematic diagram illustrating the overall configuration of tetraplex 2. The two strands that are parallel and form hydrogen bonds between cytosine bases are colored black, while the other two are colored white. Residues with asterisks represent symmetry-related residues (equivalently, we could have chosen the asymmetric unit in such a way that four strands in the asymmetric unit would form tetraplex 2). (Right) View into the major groove of tetraplex 2. The intercalating motif here is very similar to that of tetraplex 1. However, the outermost C·C+ base pairs of the tetraplex are from the 5[prime]-end of each strand.
In contrast, the major grooves are very wide. The average interchain P-P distances across the major grooves of tetramers 1 and 2 are 16.09 and 15.19 Å, respectively. This symmetric feature of two broad grooves is very different from that seen in the metazoan telomeric structure d(TAACCC) (12), where one broad groove is very flat and the phosphate groups in the other broad groove are rotated away from the center and bend over towards each other, stabilized by the bridging water molecules between phosphate oxygens and cytosine N4 groups. Both major grooves in d(AACCCC) are very flat. Figure
Figure 2. Two adjacent layers of C·C+ base pairs from tetraplex 2 along with two water molecules that are within 3.5 Å of the base pairs. The view is down the axis of the molecule, which is the z-axis. Unlike structures such as d(AACCC) and d(TAACCC), the broad grooves of this structure are essentially flat and the phosphates are not bent over. There is also no water molecule bridging the cytosine N4 amino group with the phosphate oxygens on the opposite side of the groove. The absence of this feature shows the variability of cytosine tetraplexes.
A novel feature of this structure is the presence of two groupings containing only adenine residues. They provide the interactions which hold the lattice together. The adenine bases, as shown in Figure
Two adenine clusters
a
b
c
d
Figure 3. Adenine clusters of d(AACCCC). (a) A schematic diagram of adenine cluster 1 illustrating the formation of A cluster 1 and its relation to the cytosine residues of the strands. There are two parallel backbone A·A base pairs, A20*-A30* and A21*-A31*. The other two A·A base pairs, A2*-A11 and A1*-A12, have antiparallel backbones. Every cytosine portion of the four strands combines with three other symmetry-related cytosines strands (not shown) to form tetraplex 1. Thus, there are four cytosine tetraplexes 1 connected by A cluster 1. (b) Skeletal view of A cluster 1 connecting four cytosine strands which belong to four different cytosine tetraplexes. It consists of four stacking A·A base pairs. It stacks on two cytosine tetraplexes 2 (not shown), at both the top and bottom, forming a continuous stacking along the z-axis. (c) A schematic diagram of A cluster 2. Note there are only three stacking base pairs. The other two bases stack on each other, tilted ~38° from the other three base pairs. Like A cluster 1, A cluster 2 connects four cytosine tetraplexes 2. Of the three A·A base pairs, A61-A72 and A41-A51 are parallel while A42-A62 is antiparallel. (d) Skeletal view of A cluster 2 connecting four cytosine strands which belong to four different cytosine tetraplexes. It has three stacking A·A base pairs shown at the top. It stacks on two cytosine tetraplexes 1 (not shown), at both the top and bottom, forming continuous stacking along the x-axis. At the lower right, two stacking bases A52 and A71 are shown.
Another adenine cluster, A cluster 2, also made up of eight adenine residues, has most of the bases perpendicular to the x-axis. As shown in the schematic diagram of Figure
a, b
 c, d  |
Figure 4. Various A·A base pairs are shown in an electron density map plotted at 2[sigma]. (a) Base pair A20*-A30* with parallel backbones. It is a symmetric A·A N7-amino group base pairing. (b) Base pair A21*-A31* with parallel backbones. It is a symmetric A·A N1-amino group base paring. (c) Base pair A61-A72 with parallel backbones. It is an asymmetric A·A N1-amino group, N7-amino group base pairing. Note that A61 is in the syn conformation. (d) Base pair A42-A62 with antiparallel backbones. It is an asymmetric A·A N1-amino group, N7-amino group base pairing.
Three modes of base pairing
Close inspection of Figure
DISCUSSION
Comparison with previous results
Compared with the previously solved C-rich crystal structures, d(AACCCC) reveals many interesting features. The four-stranded, intercalated cytosine segment is an extremely stable and predominant feature of the structure. It is interesting to note that the crystals were grown over a wide range of pH, ranging from pH 5.0 to 8.0. The formation of C·C+ base pairs depends on hemiprotonation of the cytosines (18,6-8). In poly[d(C)], the hemi-protonated structure was stable up to pH 7 (7). The fact that crystals of d(AACCCC) can grow at pH 7.5 and 8.0 indicates that the stable nature of the tetraplex and the packing forces raised the pK for hemi-protonation to an even higher value. This reinforces the possibility that the Tetrahymena telomere could adopt the intercalation motif in vivo at physiological pH, possibly in the presence of binding proteins.
Aside from the general structural similarity in I motifs, we have found many variations. One notable difference is the presence of two different conformations of cytosine tetraplexes. In one tetraplex, as in all previously reported C tetraplex crystals, the outermost base pairs are from the 5[prime]-end of each strand; in the other, however, the outermost base pairs are from the 3[prime]-end of each strand. This suggests that the two conformations are energetically comparably favorable, leaving open the possibility that telomere sequences might adopt either one of the two conformations, depending on the contributions of the non-cytosine residues.
Despite the apparent similarity of all cytosine tetraplex conformations, each individual strand varies considerably from structure to structure. The average twists between covalently linked cytosines vary from 12.4° for d(C4) to 16.6° for d(AACCCC). When the common I motif portion of the structures are superimposed, the RMS differences are quite considerable, especially among the sugar-phosphate backbones. For example, the RMS difference between tetraplex 1 in d(AACCCC) and tetraplex 1 in d(C4) is 1.26 Å, with the cytosine bases having an RMS difference of 0.45 Å while the backbones have one of 1.55 Å.
In all the structures solved, the tetraplexes show considerable differences from structure to structure and the differences are mainly due to those between the sugar-phosphate backbones. The positions of the cytosine bases are relatively stable and often almost superimposable. This might be expected, given the less flexible nature of the C·C+ base pairing associated with three strong planar hydrogen bonds. In contrast, the sugar-phosphate backbones are intrinsically more flexible, partly due to their lack of torsional restraints and partly due to strong electrostatic repulsion between phosphate groups in the narrow grooves. Where the backbones are close together, they may be stablized by C-H···O hydrogen bonds as well as van der Waals interactions (19). These variable aspects of the cytosine tetraplex might be important if telomere sequences adopt different conformations under differing biological conditions.
The bridging adenine clusters
Even though there is some variability in the cytosine tetraplex among different structures, the major variation is seen in the non-cytosine part of the structure. Unlike the other telomeric sequence solved, namely the metazoan telomere d(TAACCC) (12), where the adenine/thymine segment of the structure folds back on itself to form a stable loop, the adenines in this structure adopt an entirely different conformation. In this case, the adenines adopt three different kinds of A·A base pairs and are an essential lattice building block. There are two adenine residues per strand. In cytosine tetraplex 1, which points along the x-axis, there are four stacked pairs of adenine residues. As seen in Figure
A rather interesting three-dimensional network is formed, in which the adenine clusters play a key role in assembling the complex (Fig. Sequences containing stretches of cytosines and adenines are found in telomeres (1) and also occur in segments scattered throughout the genome. They may also exist in large RNAs such as group I and group II introns and ribosomal and spliceosomal RNAs. The recent crystal structure of the P4-P6 domain of the T.thermophila intron (20,21) revealed adenosine platforms in which two adjacent adenine residues contribute to key components of the domain tertiary structure. The crystal structure of the Tetrahymena telomeric sequence d(AACCCC) shows two different novel adenine clusters that play a key role in building the crystal lattice and stabilizing the structure. The abundance of adenosine residues in internal loops of many RNAs and the ability of A clusters observed in this structure to form stabilized tertiary structures suggests the possibility that A clusters, like the adenosine platforms observed in a group I intron fragment, could be a motif present in large RNAs to facilitate folding and be responsible for long range tertiary interactions. This crystal structure shows that the telomeric sequence can adopt a very different structural conformation from standard B-DNA. Does this structural conformation occur in vivo? We do not have the answer yet. The fact that both C-rich sequences and complementary G-rich sequences can form tetraplexes (22-24) makes it possible that the two structures could act in concert or one could promote formation of the other. Such an event could play an important role in DNA self-recognition, which is essential in many biological systems (10).
a
b
c
Figure 5. The organization of adenine clusters and cytosine tetraplexes. (a) (Previous page) Stereo view of the three-dimensinal network formed by continuous stacking along the x- and z-axes. The box shown is the unit cell of the crystal. (b) A cluster 1 stacks on two symmetry-related cytosine tetraplexes 2, at both the top and bottom, creating continuous stacking along the z-axis. (c) A cluster 2 stacks on two symmetry-related cytosine tetraplexes 1, at both the top and bottom, creating continuous stacking along the x-axis.
ACKNOWLEDGEMENTS
This research was supported by grants from the National Institutes of Health, the National Science Foundation and the Department of Energy through Los Alamos National Laboratories.
REFERENCES
This article has been cited by other articles:
This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 30 Sep 1998
Copyright©Oxford University Press, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
J.-L. Leroy
The formation pathway of i-motif tetramers
Nucleic Acids Res.,
July 1, 2009;
37(12):
4127 - 4134.
[Abstract]
[Full Text]
[PDF]
X. Shi and J. B. Chaires
Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures
Nucleic Acids Res.,
January 23, 2006;
34(2):
e14 - e14.
[Abstract]
[Full Text]
[PDF]
N. Esmaili and J. L. Leroy
i-motif solution structure and dynamics of the d(AACCCC) and d(CCCCAA) tetrahymena telomeric repeats
Nucleic Acids Res.,
January 12, 2005;
33(1):
213 - 224.
[Abstract]
[Full Text]
[PDF]
K. Reblova, N.'a Spackova, J. E. Sponer, J. Koca, and J. Sponer
Molecular dynamics simulations of RNA kissing-loop motifs reveal structural dynamics and formation of cation-binding pockets
Nucleic Acids Res.,
December 1, 2003;
31(23):
6942 - 6952.
[Abstract]
[Full Text]
[PDF]
T. Sunami, J. Kondo, T. Kobuna, I. Hirao, K. Watanabe, K.-i. Miura, and A. Takenaka
Crystal structure of d(GCGAAAGCT) containing a parallel-stranded duplex with homo base pairs and an anti-parallel duplex with Watson-Crick base pairs
Nucleic Acids Res.,
December 1, 2002;
30(23):
5253 - 5260.
[Abstract]
[Full Text]
[PDF]
This Article 
Abstract
Print PDF (430K)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (20)
Request Permissions
Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Articles by Cai, L.
Articles by Rich, A.
Search for Related Content
PubMed
PubMed Citation
Articles by Cai, L.
Articles by Rich, A.
Social Bookmarking
What's this?