An eclectic DNA structure adopted by human telomeric sequence under superhelical stress and low pH.

We have found, with the aid of 2-D gel electrophoresis, that double-stranded human telomeric repeat, (T2AG3)12.(C3TA2)12, being cloned within a plasmid, forms a protonated superhelically-induced structure. Experiments on chemical and enzymatic probing also indicate that the human telomeric repeats adopt an unusual structure. We have proposed an eclectic model for this structure in which four different elements coexist: a non-orthodox intramolecular triplex stabilized by the canonical protonated C.G*C+ base-triads and highly enriched by noncanonical base-triads; the intramolecular quadruplex formed by a portion of the G-rich strand; the single-stranded region encompassing a portion of the G-rich strand and, probably, the (C,A)-hairpin formed by a portion of the C-rich strand.     

Stability of intramolecular DNA quadruplexes: comparison with DNA duplexes

We have determined the stability of intramolecular quadruplexes that are formed by a variety of G-rich sequences, using oligonucleotides containing appropriately placed fluorophores and quenchers. The stability of these quadruplexes is compared with that of the DNA duplexes that are formed on addition of complementary C-rich oligonucleotides. We find that the linkers joining the G-tracts are not essential for folding and can be replaced with nonnucleosidic moieties, though their sequence composition profoundly affects quadruplex stability. Although the human telomere repeat sequence d[G(3)(TTAG(3))(3)] folds into a quadruplex structure, this forms a duplex in the presence of the complementary C-rich strand at physiological conditions. The Tetrahymena sequence d[G(4)(T(2)G(4))(3)], the sequence d[G(3)(T(2)G(3))(3)], and sequences related to regions of the c-myc promoter d(G(4)AG(4)T)(2) and d(G(4)AG(3)T)(2) preferentially adopt the quadruplex form in potassium-containing buffers, even in the presence of a 50-fold excess of their complementary C-rich strands, though the duplex predominates in the presence of sodium. The HIV integrase inhibitor d[G(3)(TG(3))(3)] forms an extremely stable quadruplex which is not affected by addition of a 50-fold excess of the complementary C-rich strand in both potassium- and sodium-containing buffers. Replacing the TTA loops of the human telomeric repeat with AAA causes a large decrease in quadruplex stability, though a sequence with AAA in the first loop and TTT in the second and third loops is slightly more stable.     

GG sequence of DNA and the human telomeric sequence react with cis-diammine-diaquaplatinum at comparable rates

G-quadruplex structures of telomeric sequences are of growing interest because they inhibit telomerase, an enzyme involved in the maintenance of telomere length of cancer cells. As we have shown previously, the antiparallel structure of G-quadruplexes can be cross-linked in vitro by the anti-tumour drug cisplatin. The question arises whether platination of quadruplex structures of human telomeric sequences by cisplatin could be relevant from a biological point of view. Therefore, we have compared the kinetics of reactions of the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), with the human telomeric quadruplex structure, a duplex DNA and a single-stranded DNA containing one specific platination GG site. The ratio between the platination rate constants was obtained using two intramolecular competition experiments: either a construct with a junction between duplex DNA containing a unique GG platination site and the quadruplex structure of the human telomeric sequence AG(3)(T(2)AG(3))(3), or a construct with a junction between duplex DNA and a single strand containing each a unique GG platination site. Those competition experiments allowed us to conclude that the platination of the quadruplex is favoured over that of the GG duplex by a factor of about two whereas the GG duplex is platinated three times faster than the GG single strand.     

Duplex and quadruplex DNA binding and photocleavage by trioxatriangulenium ion

The stable trioxatriangulenium ion (TOTA) has previously been shown to bind to and photooxidize duplex DNA, leading to cleavage at G residues, particularly 5'-GG-3' repeats. Telomeric DNA consists of G-rich sequences that may exist in either duplex or G-quadruplex forms. We have employed electrospray ionization mass spectrometry (ESI-MS) to investigate the interactions between TOTA and duplex DNA or G-quadruplex DNA. A variety of duplex decamer oligodeoxynucleotides form complexes with TOTA that can be detected by ESI-MS, and the stoichiometry and fragmentation patterns observed are commensurate with an intercalative binding mode. TOTA also forms complexes with four-stranded and hairpin-dimer G-quadruplex oligodeoxynucleotides that can be detected by ESI-MS. Both the stoichiometry and the fragmentation patterns observed by ESI-MS are different than those observed for G-tetrad end-stacking binding ligands. We have carried out (1)H NMR titrations of a four-stranded G-quadruplex in the presence of TOTA. Addition of up to 1 equiv of TOTA is accompanied by pronounced upfield shifts of the G-tetrad imino proton resonances in the NMR, which is similar to the effect observed for G-tetrad end-stacking ligands. At higher ratios of added TOTA, there is evidence for additional binding modes. Duplex DNA containing either human telomeric repeats (T(2)AG(3))(4) or the Tetrahymena telomeric repeats (T(2)G(4))(4) are readily photooxidized by TOTA, the major sites of oxidation being the central guanine residues in each telomeric repeat. These telomeric repeats were incorporated into duplex/quadruplex chimeras in which the repeats adopt a G-quadruplex structure. Analysis by denaturing polyacrylamide gel electrophoresis reveals significantly less TOTA photocleavage of these quadruplex telomeric repeats when compared to the duplex repeats.     

Formation of novel hairpin structures by telomeric C-strand oligonucleotides.

Telomeres are specialized structures at the ends of chromosomes that are required for long term chromosome stability and replication of the chromosomal terminus. Telomeric DNA consists of simple repetitive sequences with one strand G-rich relative to the other, C-rich, strand. Evolutionary conservation of this feature of telomeric repeat sequences suggests that they have specific structural characteristics involved in telomere function. Absorbance thermal denaturation, chemical modification and non-denaturing gel electrophoretic analyses showed that telomeric C-strand oligonucleotides form stable non-Watson-Crick hairpin structures containing C.C+ base pairs. Formation of such hairpins may facilitate previously reported G-strand exclusive interactions.     

Interactions of the human telomeric DNA with terbium-amino acid complexes

The human telomeric DNA can form four-stranded structures: the G-rich strand adopts a G-quadruplex conformation stabilized by G-quartets and the C-rich strand may fold into an I-motif based on intercalated C.C(+) base pairs. There is intense interests in the design and synthesis of compounds which can target telomeric DNA and inhibit the telomerase activity. Here we report the thermodynamic studies of the two newly synthesized terbium-amino acid complexes bound to the human telomeric G-quadruplex and I-motif DNA which were studied by means of UV-Visible, DNA meltings, fluorescence and circular dichroism. These two complexes can bind to the human telomeric DNA and have shown different features on DNA stability, binding stoichiometry, and sequence-dependent fluorescence enhancement. To our knowledge, this is the first report to show terbium-amino acid complexes can interact with the human telomeric DNA.     

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.     

In vitro properties of the conserved mammalian protein hnRNP D suggest a role in telomere maintenance

Mammalian chromosomes terminate with a 3' tail which consists of reiterations of the G-rich repeat, d(TTAGGG). The telomeric tail is the primer for replication by telomerase, and it may also invade telomeric duplex DNA to form terminal lariat structures, or T loops. Here we show that the ubiquitous and highly conserved mammalian protein hnRNP D interacts specifically with the G-rich strand of the telomeric repeat. A single gene encodes multiple isoforms of hnRNP D. All isoforms bind comparably to the G-rich strand, and certain isoforms can also bind tightly and specifically to the C-rich telomeric strand. G-rich telomeric sequences readily form structures stabilized by G-G pairing, which can interfere with telomere replication by telomerase. We show that hnRNP D binding to the G-rich strand destabilizes intrastrand G-G pairing and that hnRNP D interacts specifically with telomerase in human cell extracts. This biochemical analysis suggest that hnRNP D could function in vivo to destabilize structures formed by telomeric G-rich tails and facilitate their extension by telomerase.     

The role of the AT pairs in the acid denaturation of DNA.

It has been determined previously that the protonation of the GC pairs induces a DNA conformation change which leads to a "metastable" structure. The role of the AT pairs, however, is no well known because the protonation does not modify their spectral properties. By means of an indirect method based on the binding of proflavine, it has been determined that the AT pairs are protonated before the acid-induced denaturation and that they seem to be unable to assume a conformation change when protonated. These results would indicate that the protonated AT pairs may be responsible for the induction of the acid denaturation and not the GC pairs as it was thought previously.     

The solution structure of d(G(4)T(4)G(3))(2): a bimolecular G-quadruplex with a novel fold

The G-rich 11-mer oligonucleotide d(G(4)T(4)G(3)) forms a bimolecular G-quadruplex in the presence of sodium ions with a topology that is distinct from the folds of the closely related and well-characterized sequences d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)). The solution structure of d(G(4)T(4)G(3))(2) has been determined using a combination of NMR spectroscopy and restrained molecular dynamics calculations. d(G(4)T(4)G(3))(2) forms an asymmetric dimeric fold-back structure consisting of three stacked G-quartets. The two T(4) loops that span diagonally across the outer faces of the G-quartets assume different conformations. The glycosidic torsion angle conformations of the guanine bases are 5'-syn-anti-syn-anti-(T(4) loop)-anti-syn-anti in one strand and 5'-syn-anti-syn-anti-(T(4) loop)-syn-anti-syn in the other strand. The guanine bases of the two outer G-quartets exhibit a clockwise donor-acceptor hydrogen-bonding directionality, while those of the middle G-quartet exhibit the anti-clockwise directionality. The topology of this G-quadruplex, like other bimolecular fold-back structures with diagonal loops, places each strand of the G-quartet region next to a neighboring parallel and an anti-parallel strand. The two guanine residues not involved in G-quartet formation, G4 and G12 (i.e. the fourth guanine base of one strand and the first guanine base of the other strand), adopt distinct conformations. G4 is stacked on top of an adjacent G-quartet, and this base-stacking continues along with the bases of the loop residues T5 and T6. G12 is orientated away from the core of G-quartets; stacked on the T7 base and apparently involved in hydrogen-bonding interactions with the phosphodiester group of this same residue. The cation-dependent folding of the d(G(4)T(4)G(3))(2) quadruplex structure is distinct from that observed for similar sequences. While both d(G(4)T(4)G(4)) and d(G(3)T(4)G(3)) form bimolecular, diagonally looped G-quadruplex structures in the presence of Na(+), K(+) and NH(4)(+), we have observed this folding to be favored for d(G(4)T(4)G(3)) in the presence of Na(+), but not in the presence of K(+) or NH(4)(+). The structure of d(G(4)T(4)G(3))(2) exhibits a "slipped-loop" element that is similar to what has been proposed for structural intermediates in the folding pathway of some G-quadruplexes, and therefore provides support for the feasibility of these proposed transient structures in G-quadruplex formation.     

Monovalent cation induced structural transitions in telomeric DNAs: G-DNA folding intermediates

Telomeric DNA consists of G- and C-rich strands that are always polarized such that the G-rich strand extends past the 3' end of the duplex to form a 12-16-base overhang. These overhanging strands can self-associate in vitro to form intramolecular structures that have several unusual physical properties and at least one common feature, the presence of non-Watson-Crick G.G base pairs. The term "G-DNA" was coined for this class of structures (Cech, 1988). On the basis of gel electrophoresis, imino proton NMR, and circular dichroism (CD) results, we find that changing the counterions from sodium to potassium (in 20 mM phosphate buffers) specifically induces conformational transitions in the G-rich telomeric DNA from Tetrahymena, d(T2G4)4 (TET4), which results in a change from the intramolecular species to an apparent multistranded structure, accompanied by an increase in the melting temperature of the base pairs of greater than 25 degrees, as monitored by loss of the imino proton NMR signals. NMR semiselective spin-lattice relaxation rate measurements and HPLC size-exclusion chromatography studies show that in 20 mM potassium phosphate (pH 7) buffer (KP) TET4 is approximately twice the length of the form obtained in 20 mM sodium phosphate (pH 7) buffer (NaP) and that mixtures of Na+ and K+ produce mixtures of the two forms whose populations depend on the ratio of the cations. Since K+ and NH4+ are known to stabilize a parallel-stranded quadruplex structure of poly[r(I)4], we infer that the multistranded structure is a quadruplex. Our results indicate that specific differences in ionic interactions can result in a switch in telomeric DNAs between intramolecular hairpin-like or quadruplex-containing species and intermolecular quadruplex structures, all of which involve G.G base pairing interactions. We propose a model in which duplex or hairpin forms of G-DNA are folding intermediates in the formation of either 1-, 2-, or 4-stranded quadruplex structures. In this model monovalent cations stabilize the duplex and quadruplex forms via two distinct mechanisms, counterion condensation and octahedral coordination to the carbonyl groups in stacked planar guanine "quartet" base assemblies. Substituting one of the guanosine residues in each of the repeats of the Tetrahymena sequence to give the human telomeric DNA, d(T2AG3)4, results in less effective K(+)-dependent stabilization. Thus, the ion-dependent stabilization is attenuated by altering the sequence. Upon addition of the Watson-Crick (WC) complementary strand, only the Na(+)-stabilized structure dissociates quickly to form a WC double helix.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human telomeric DNA: G-quadruplex,i-motif and Watson-Crick double helix

Human telomeric DNA composed of (TTAGGG/CCCTAA)n repeats may form a classical Watson-Crick double helix. Each individual strand is also prone to quadruplex formation: the G-rich strand may adopt a G-quadruplex conformation involving G-quartets whereas the C-rich strand may fold into an i-motif based on intercalated C*C+ base pairs. Using an equimolar mixture of the telomeric oligonucleotides d[AGGG(TTAGGG)3] and d[(CCCTAA)3CCCT], we defined which structures existed and which would be the predominant species under a variety of experimental conditions. Under near-physiological conditions of pH, temperature and salt concentration, telomeric DNA was predominantly in a double-helix form. However, at lower pH values or higher temperatures, the G-quadruplex and/or the i-motif efficiently competed with the duplex. We also present kinetic and thermodynamic data for duplex association and for G-quadruplex/i-motif unfolding.     

Replication Timing of Human Telomeric DNA and Other Repetitive Sequences Analyzed by Fluorescence in Situ Hybridization and Flow Cytometry

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.     

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.     

The interaction of cisplatin with a human telomeric DNA sequence containing seventeen tandem repeats

The anti-tumour drug, cisplatin, preferentially forms adducts at G-rich DNA sequences. Telomeres are found at the ends of chromosomes and, in humans, contain the repeated DNA sequence (GGGTTA)_n that is expected to be targeted by cisplatin. Using a plasmid clone with 17 tandem telomeric repeats, (GGGTTA)₁₇, the DNA sequence specificity of cisplatin was investigated utilising the linear amplification procedure that pin-pointed the precise sites of cisplatin adduct formation. This procedure used a fluorescently labelled primer and capillary electrophoresis with laser-induced fluorescence detection to determine the DNA sequence specificity of cisplatin. This technique provided a very accurate analysis of cisplatin-DNA adduct formation in a long telomeric repeat DNA sequence. The DNA sequence specificity of cisplatin in a long telomeric tandem repeat has not been previously reported. The results indicated that the 3′-end of the G-rich strand of the telomeric repeat was preferentially damaged by cisplatin and this suggests that the telomeric DNA repeat has an unusual conformation.     

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.     

Different loop arrangements of intramolecular human telomeric (3+1) G-quadruplexes in K+ solution

Intramolecular G-quadruplexes formed by the human telomeric G-rich strand are promising anticancer targets. Here we show that four-repeat human telomeric DNA sequences can adopt two different intramolecular G-quadruplex folds in K⁺ solution. The two structures contain the (3+1) G-tetrad core, in which three G-tracts are oriented in one direction and the fourth in the opposite direction, with one double-chain-reversal and two edgewise loops, but involve different loop arrangements. This result indicates the robustness of the (3+1) core G-quadruplex topology, thereby suggesting it as an important platform for structure-based drug design. Our data also support the view that multiple human telomeric G-quadruplex conformations coexist in K⁺ solution. Furthermore, even small changes to flanking sequences can perturb the equilibrium between different coexisting G-quadruplex forms.     

Intramolecular Telomeric G-Quadruplexes Dramatically Inhibit DNA Synthesis by Replicative and Translesion Polymerases,Revealing their Potential to Lead to Genetic Change

Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.