Molecular modeling studies of a parallel stranded quadruplexes containing a 8-bromoadenosine

Truncated sequences of human telomeric DNA can readily assemble to form parallel stranded quadruplexes containing A- and G-tetrads. The formation of an A-tetrad is highly context-dependent and the relationship between the formation of an A-tetrad and the glycosidic torsion angle of the adenosine residues implicated has not been completely clarified so far. In order to give a further insight in this issue we synthesized the modified oligomers d(ABrGGGT) and d(TABrGGGT), two different truncations of the human telomeric sequence containing a 8-bromoadenosine residue, named ABr. NMR data show that both the modified oligomers are able to perfectly fold into highly symmetric quadruplexes with all strands parallel to each other. Molecular modeling studies were performed on both [d(ABrGGGT)]4 and [d(TABrGGGT)]4, indicating that a bulky substituent, such as a bromine atom at the C8 position of adenines, can force the glycosidic bond to adopt a syn conformation, stabilizing the resulting quadruplexes.     

MOLECULAR MODELING STUDIES OF A PARALLEL STRANDED QUADRUPLEXES CONTAINING A 8-BROMOADENOSINE

Truncated sequences of human telomeric DNA can readily assemble to form parallel stranded quadruplexes containing A- and G-tetrads. The formation of an A-tetrad is highly context-dependent and the relationship between the formation of an A-tetrad and the glycosidic torsion angle of the adenosine residues implicated has not been completely clarified so far. In order to give a further insight in this issue we synthesized the modified oligomers d(ABrGGGT) and d(TABrGGGT), two different truncations of the human telomeric sequence containing a 8-bromoadenosine residue, named ABr. NMR data show that both the modified oligomers are able to perfectly fold into highly symmetric quadruplexes with all strands parallel to each other. Molecular modeling studies were performed on both [d(ABrGGGT)]4 and [d(TABrGGGT)]4, indicating that a bulky substituent, such as a bromine atom at the C8 position of adenines, can force the glycosidic bond to adopt a syn conformation, stabilizing the resulting quadruplexes.     

NMR observation of T-tetrads in a parallel stranded DNA quadruplex formed by Saccharomyces cerevisiae telomere repeats.

We report here the NMR structure of the DNA sequence d-TGGTGGC containing two repeats of Saccharomyces cerevisiae telomere DNA which is unique in that it has a single thymine in the repeat sequence and the number of Gs can vary from one to three. The structure is a novel quadruplex incor-porating T-tetrads formed by symmetrical pairing of four Ts via O4-H3 H-bonds in a plane. This is in contrast to the previous results on other telomeric sequences which contained more than one T in the repeat sequences and they were seen mostly in the flexible regions of the structures. We observed that the T4-tetrad was nicely accommodated in the center of the G-quadruplex, but it caused a small underwinding of the right handed helix. The T tetrad stacked well on the adjacent G3-tetrad, but poorly on the G5 tetrad. Likewise, T1 also formed a stable T-tetrad at the 5' end of the quadruplex. To our knowledge, this is the first report of T-tetrad formation in DNA structures. These observations are of significance from the points of view of both structural diversity and specific recognitions.     

Structure of a two-G-tetrad intramolecular G-quadruplex formed by a variant human telomeric sequence in K+ solution: insights into the interconversion of human telomeric G-quadruplex structures

Human telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. The telomeric sequence shows intrinsic structure polymorphism. Here we report a novel intramolecular G-quadruplex structure formed by a variant human telomeric sequence in K⁺ solution. This sequence forms a basket-type intramolecular G-quadruplex with only two G-tetrads but multiple-layer capping structures formed by loop residues. While it is shown that this structure can only be detected in the specifically truncated telomeric sequences without any 5′-flanking residues, our results suggest that this two-G-tetrad conformation is likely to be an intermediate form of the interconversion of different telomeric G-quadruplex conformations.     

Protection of DNA ends by telomeric 3' G-tail sequences

The extreme ends of eukaryotic chromosomes contain 3' extensions in the form of single-stranded G-rich repeats, referred to as telomeric 3' G-tails or overhangs. Increasing evidence has suggested that telomeric 3' G-tails can adopt a G-quadruplex conformation both in vitro and in vivo. However, the role of G-quadruplexes on the structure and function of telomeric 3' G-tails remains unclear. In the current study, we showed that the human telomeric 3' G-tail sequence protected the duplex DNA ends in cis from being recognized as double strand breaks. This protection is dependent on the G-quadruplex conformation of the 3' G-tail sequence. These results suggest that the ability of telomeric 3' G-tails to adopt the endprotecting G-quadruplex conformation may be one of the reasons for the existence of the evolutionarily conserved G-stretch motifs in telomeric DNA sequences.     

Structural polymorphism of intramolecular quadruplex of human telomeric DNA: effect of cations, quadruplex-binding drugs and flanking sequences

G-quadruplex structures formed in the telomeric DNA are thought to play a role in the telomere function. Drugs that stabilize the G-quadruplexes were shown to have anticancer effects. The structures formed by the basic telomeric quadruplex-forming unit G(3)(TTAG(3))(3) were the subject of multiple studies. Here, we employ (125)I-radioprobing, a method based on analysis of the distribution of DNA breaks after decay of (125)I incorporated into one of the nucleotides, to determine the fold of the telomeric DNA in the presence of TMPyP4 and telomestatin, G-quadruplex-binding ligands and putative anticancer drugs. We show that d[G(3)(TTAG(3))(3)(125)I-CT] adopts basket conformation in the presence of NaCl and that addition of either of the drugs does not change this conformation of the quadruplex. In KCl, the d[G(3)(TTAG(3))(3)(125)I-CT] is most likely present as a mixture of two or more conformations, but addition of the drugs stabilize the basket conformation. We also show that d[G(3)(TTAG(3))(3)(125)I-CT] with a 5'-flanking sequence folds into (3+1) type 2 conformation in KCl, while in NaCl it adopts a novel (3+1) basket conformation with a diagonal central loop. The results demonstrate the structural flexibility of the human telomeric DNA; and show how cations, quadruplex-binding drugs and flanking sequences can affect the conformation of the telomeric quadruplex.     

The cohering telomeres of Oxytricha.

We have studied the process by which purified Oxytricha macronuclear DNA associates with itself to form large aggregates. The various macronuclear DNA molecules all have the same terminal or telomeric DNA sequences that are shown below. 5' C4A4C4A4C4--mean length----G4T4G4T4G4T4G4T4G4 G4T4G4T4G4T4G4T4G4-----2.4 kb------C4A4C4A4C4. When incubated at high concentrations, these telomeric sequences cohere with one another to form an unusual structure--one that is quite different from any DNA structure so far described. The evidence for this is the following: 1) These sequences cohere albeit slowly, in the presence of relatively high concentrations of Na+, and no other cation tested. This contrasts with the rapid coherence of complementary single-chain terminals of normal DNA (sticky ends) which occurs in the presence of any cation tested. 2) If the cohered form is transferred into buffers containing a special cation, K+, it becomes much more resistant to dissociation by heating. We estimate that K+ increases the thermal stability by 25 degrees or more. The only precedent known (to us) for a cation-specific stabilization is that seen in the quadruplex structure formed by poly I. The thermal stability of double helical macronuclear DNA depends on the cation concentration, but not the cation type. Limited treatment with specific nucleases show that the 3' and 5'-ended strands are essential for the formation of the cohering structure. Once in the cohered form, the telomeric sequences are protected from the action of nucleases. Coherence is inhibited by specific, but not by non-specific, synthetic oligomers, and by short telomeric fragments with or without their terminal single chains. We conclude that the coherence occurs by the formation of a novel condensed structure that involves the terminal nucleotides in three or four chains.     

Substitution of adenine for guanine in the quadruplex-forming human telomere DNA sequence G(3)(T(2)AG(3))(3)

We have studied the formation and structural properties of quadruplexes of the human telomeric DNA sequence G(3)(T(2)AG(3))(3) and related sequences in which each guanine base was replaced by an adenine base. None of these single base substitutions hindered the formation of antiparallel quadruplexes, as shown by circular dichroism, gel electrophoresis, and UV thermal stability measurements in NaCl solutions. Effect of substitution did differ, however, depending on the position of the substituted base. The A-for-G substitution in the middle quartet of the antiparallel basket scaffold led to the most distorted and least stable structures and these sequences preferred to form bimolecular quadruplexes. Unlike G(3)(T(2)AG(3))(3), no structural transitions were observed for the A-containing analogs of G(3)(T(2)AG(3))(3) when sodium ions were replaced by potassium ions. The basic quadruplex topology remained the same for all sequences studied in both salts. As in vivo misincorporation of A for a G in the telomeric sequence is possible and potassium is a physiological salt, these findings may have biological relevance.     

Characterization of the Structural Conformation Adopted by (TTAGGG)(n) Telomeric DNA Repeats of Different Length in Closed Circular DNA

Abstract Telomeric DNA sequences are known to adopt unusual DNA structures upon protonation when contained into negatively supercoiled DNA. In this paper, the structural properties of (T(2)AG(3))(n) telomeric sequences of different length is analyzed in detail. Transition to the protonated form is observed at very low pH for (T(2)AG(3))(n<8) sequences. Formation of the protonated form is facilitated by negative supercoiling. The patterns of chemical modification obtained with different chemical reagents indicate that protonation induces denaturation of the (T(2)AG(3))(n) telomeric sequences. Upon denaturation, the "C-rich" strand becomes structured forming, most likely, hairpin-like conformations stabilized by the formation of C(+)·C pairs and, probably, of A(+)·A pairs. The "G-rich" strand of the (T(2)AG(3))(8) sequence shows also signs of becoming structured giving rise to various structural conformers which might include triple- and tetra-stranded conformations. However, in the case of shorter sequences, the "G-rich" strand remains basically single-stranded.     

Deciphering the mechanism of thermodynamic accommodation of telomeric oligonucleotide sequences by the Schizosaccharomyces pombe protection of telomeres 1 (Pot1pN) protein

Linear chromosomes terminate in specialized nucleoprotein structures called telomeres, which are required for genomic stability and cellular proliferation. Telomeres end in an unusual 3' single-strand overhang that requires a special capping mechanism to prevent inappropriate recognition by the DNA damage machinery. In Schizosaccharomyces pombe, this protective function is mediated by the Pot1 protein, which binds specifically and with high affinity to telomeric ssDNA. We have characterized the thermodynamics and accommodation of both cognate and noncognate telomeric single-stranded DNA (ssDNA) sequences by Pot1pN, an autonomous ssDNA-binding domain (residues 1-187) found in full-length S. pombe Pot1. Direct calorimetric measurements of cognate telomeric ssDNA binding to Pot1pN show favorable enthalpy, unfavorable entropy, and a negative heat-capacity change. Thermodynamic analysis of the binding of noncognate telomeric ssDNA to Pot1pN resulted in unexpected changes in free energy, enthalpy, and entropy. Chemical-shift perturbation and structural analysis of these bound noncognate sequences show that these thermodynamic changes result from the structural rearrangement of both Pot1pN and the bound oligonucleotide. These data suggest that the ssDNA-binding interface is highly dynamic and, in addition to the conformation observed in the crystal structure of the Pot1pN/d(GGTTAC) complex, capable of adopting alternative thermodynamically equivalent conformations.     

Isolation of telomeric DNA from the filamentous fungus Podospora anserina and construction of a self-replicating linear plasmid showing high transformation frequency.

It has been previously shown that linear plasmids bearing Tetrahymena telomeric sequences are able to replicate autonomously in the filamentous fungus Podospora anserina (1). However, autonomous replication occurs in only 50-70% of the transformants, suggesting a defect in the recognition of the Tetrahymena telomeric template by the putative P. anserina telomerase so that only a fraction of entering DNA is stabilized into linear extrachromosomal molecules. We have cloned DNA sequences added to the Tetrahymena (T2G4)n ends of the linear plasmid. Nucleotide sequencing showed that these sequences are exclusively composed of T2AG3 repeat units. Hybridization experiments of Bal31 treated DNA showed that T2AG3 repeats are confined within 200 bp in chromosomal P. anserina telomeres. A new plasmid has been constructed so that after linearization, the terminal sequences contain T2AG3 repeats. This linear molecule transforms P. anserina with a high frequency (up to 1.75 x 10(4) transformants/micrograms), autonomous replication occurs in 100% of the transformants and the plasmid copy number is about 2-3 per nucleus. These results underscore the importance of the telomeric repeat nucleotide sequence for efficient recognition as functional telomeric DNA in vivo and provide the first step toward the development of an artificial chromosome cloning system for filamentous fungi.     

Ciliate telomerase RNA structural features.

Telomerase RNA is an integral part of telomerase, the ribonucleoprotein enzyme that catalyzes the synthesis of telomeric DNA. The RNA moiety contains a templating domain that directs the synthesis of a species-specific telomeric repeat and may also be important for enzyme structure and/or catalysis. Phylogenetic comparisons of telomerase RNA sequences from various Tetrahymena spp. and hypotrich ciliates have revealed two conserved secondary structure models that share many features. We have cloned and sequenced the telomerase RNA genes from an additional six Tetrahymena spp. (T. vorax, T. borealis, T. australis, T. silvana, T. capricornis and T. paravorax). Inclusion of these sequences, most notably that from T. paravorax, in a phylogenetic comparative analysis allowed us to more narrowly define structural elements that may be necessary for a minimal telomerase RNA. A primary sequence element, positioned 5' of the template and conserved between all previously known ciliate telomerase RNAs, has been reduced from 5'-(C)UGUCA-3' to the 4 nt sequence 5'-GUCA-3'. Conserved secondary structural features and the impact they have on the general organization of ciliate telomerase RNAs is discussed.     

Diffusion-ordered nuclear magnetic resonance spectroscopy for analysis of DNA secondary structural elements

Structure determination of secondary DNA structural elements, such as G-quadruplexes, gains an increasing importance as fundamental physiological roles are being associated with the formation of such structures in vivo. A truncated native DNA sequence generally requires further optimization to obtain a candidate with desired nuclear magnetic resonance (NMR) properties for structural analysis in solution. The optimum sequence is expected to form one dominant, stable molecular entity in solution with well-resolved NMR peaks. However, DNA sequences are prone to form structures composed of one, two, three, or four strands depending on sequence and solution conditions. The thorough characterization of the molecularity (stoichiometry and molecular weight) and appropriate solution conditions for sequences with different modifications traditionally applies analytical techniques that generally do not represent the solution conditions for NMR structure determination. Here we present the application of diffusion-ordered NMR spectroscopy as a useful analytical tool for the optimization and analysis of DNA secondary structural elements, specifically, the DNA G-quadruplex structures, including those formed in the human telomeric sequence and in the promoter regions of bcl-2 and c-myc genes.     

Structural study of four-stranded quadruplex structures containing 2'-deoxy-8-(propyn-1-yl)adenosine

In this paper, we report the NMR structural study of two quadruplex structures formed by truncations of the human telomeric sequence and containing a modified base, namely d(AprGGGT) and d(TAprGGGT), where Apr indicates 2'-deoxy-8-(propyn-1-yl)adenosines. Both oligonucleotides have been found to form 4-fold symmetric G-quadruplex structures with all strands parallel and equivalent to each other and characterized by higher thermal stabilities than the natural counterparts. The presence of the propynyl groups affects the conformations of the 5' edge of both quadruplexes in such a way to prevent the formation of one of the two possible H-bond patterns observed for a canonical A-tetrad. The increased thermal stabilities of the modified quadruplexes seem to be mostly due to a prevalent syn glycosidic conformation assumed by the Apr residues.     

Interaction of human telomeric DNA with N-methyl mesoporphyrin IX

The remarkable selectivity of N-methyl mesoporphyrin IX (NMM) for G-quadruplexes (GQs) is long known, however its ability to stabilize and bind GQs has not been investigated in detail. Through the use of circular dichroism, UV-visible spectroscopy and fluorescence resonance energy transfer (FRET) melting assay we have shown that NMM stabilizes human telomeric DNA dAG(3)(TTAG(3))(3) (Tel22) and is selective for its parallel conformation to which it binds in 1:1 stoichiometry with a binding constant of ≈ 1.0 × 10(5)M(-1). NMM does not interact with an antiparallel conformation of Tel22 in sodium buffer and is the second example in the literature, after TOxaPy, of a ligand with an excellent selectivity for a specific GQ structure. NMM's stabilizing ability toward predominantly parallel GQ conformation is universal: it stabilizes a variety of biologically relevant G-rich sequences including telomeres and oncogene promoters. The N-methyl group is integral for selectivity and stabilization, as the unmethylated analogue, mesoporphyrin IX, does not stabilize GQ DNA in FRET melting assays. Finally, NMM induces the isomerization of Tel22 into a structure with increased parallel component in K(+) but not in Na(+) buffer. The ability of NMM to cause structural rearrangement and efficient stabilization of Tel22 may bear biological significance.     

S1 nuclease sensitivity of a double-stranded telomeric DNA sequence.

We examined structural properties of poly d(C4A2).d(T2G4), the telomeric DNA sequence of the ciliated protozoan Tetrahymena. Under conditions of high negative supercoiling, poly d(C4A2).d(T2G4) inserted in a circular plasmid vector was preferentially sensitive to digestion with S1 nuclease. Only the C4A2 strand was sensitive to first-strand S1 cutting, with a markedly skewed pattern of hypersensitive sites in tracts of either 46 or 7 tandem repeats. Linear poly d(C4A2).(T2G4) showed no preferential S1 sensitivity, no circular dichroism spectra indicative of a Z-DNA conformation, no unusual Tm, and no unusual migration in polyacrylamide gel electrophoresis. The S1 nuclease sensitivity properties are consistent with a model proposed previously for supercoiled poly d(CT).d(AG) (Pulleyblank et al., Cell 42:271-280, 1985), consisting of a double-stranded, protonated, right-handed underwound helix. We propose that this structure is shared by related telomeric sequences and may play a role in their biological recognition.     

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.     

DNA recognition and binding by the Euplotes telomere protein.

The 51-kDa telomere protein from Euplotes crassus binds to the extreme terminus of macronuclear telomeres, generating a very salt-stable telomeric DNA-protein complex. The protein recognizes both the sequence and the structure of the telomeric DNA. To explore how the telomere protein recognizes and binds telomeric DNA, we have examined the DNA-binding specificity of the purified protein using oligonucleotides that mimic natural and mutant versions of Euplotes telomeres. The protein binds very specifically to the 3' terminus of single-stranded oligonucleotides with the sequence (T4G4) > or = 3 T4G2; even slight modifications to this sequence reduce binding dramatically. The protein does not bind oligonucleotides corresponding to the complementary C4A4 strand of the telomere or to double-stranded C4A4.T4G4-containing sequences. Digestion of the telomere protein with trypsin generates an N-terminal protease-resistant fragment of approximately 35 kDa. This 35-kDa peptide appears to comprise the DNA-binding domain of the telomere protein as it retains most of the DNA-binding characteristics of the native 51-kDa protein. For example, the 35-kDa peptide remains bound to telomeric DNA in 2 M KCl. Additionally, the peptide binds well to single-stranded oligonucleotides that have the same sequence as the T4G4 strand of native telomeres but binds very poorly to mutant telomeric DNA sequences and double-stranded telomeric DNA. Removal of the C-terminal 15 kDa from the telomere protein does diminish the ability of the protein to bind only to the terminus of a telomeric DNA molecule.     

Binding properties of human telomeric quadruplex multimers: a new route for drug design

Human telomeric G-quadruplex structures are known to be promising targets for an anticancer therapy. In the past decade, several research groups have been focused on the design of new ligands trying to optimize the interactions between these small molecules and the G-quadruplex motif. In most of these studies, the target structures were the single quadruplex units formed by short human DNA telomeric sequences (typically 21-26 nt). However, the 3′-terminal single-stranded human telomeric DNA is actually 100-200 bases long and can form higher-order structures by clustering several consecutive quadruplex units (multimers). Despite the increasing number of structural information on longer DNA telomeric sequences, very few data are available on the binding properties of these sequences compared with the shorter DNA telomeric sequences.In this paper we use a combination of spectroscopic (CD, UV and fluorescence) and calorimetric techniques (ITC) to compare the binding properties of the (TTAGGG)₈TT structure formed by two adjacent quadruplex units with the binding properties of the (AG₃TT)₄ single quadruplex structure. The three side-chained triazatruxene derivative azatrux and TMPyP4 cationic porphyrin were used as quadruplex ligands. We found that, depending on the drug, the number of binding sites per quadruplex unit available in the multimer structure was smaller or greater than the one expected on the basis of the results obtained from individual quadruplex binding studies. This work suggests that the quadruplex units along a multimer structure do not behave as completely independent. The presence of adjacent quadruplexes results in a diverse binding ability not predictable from single quadruplex binding studies. The existence of quadruplex-quadruplex interfaces in the full length telomeric overhang may provide an advantageous factor in drug design to enhance both affinity and selectivity for DNA telomeric quadruplexes.     

Hairpin and parallel quartet structures for telomeric sequences.

The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.