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PubMed2005

i-motif solution structure and dynamics of the d(AACCCC) and d(CCCCAA) tetrahymena telomeric repeats.

Using NMR methods, we have resolved the i-motif structures formed by d(AACCCC) and by d(CCCCAA), two versions of the DNA sequence repeated in the telomeric regions of the C-rich strand of tetrahymena chromosomes. Both oligonucleotides form fully symmetrical i-motif tetramers built by intercalation of two hemiprotonated duplexes containing four C*C+ pairs. The structures are extremely stable. In the tetramer of d(AACCCC), the outermost C*C+ pairs are formed by the cytidines of the 5' ends of the cytidine tracts. A2 forms an A2*A2 (H6trans-N7) pair stacked to C3*C3+ and cross-strand stacked to A1. At 0 degrees C, the lifetimes of the hemiprotonated pairs range from 1 ms for the outermost pair to approximately 1 h for the innermost pairs. The tetramer of d(CCCCAA) adopts two distinct intercalation topologies in slow conformational exchange. One, whose outermost C*C+ pairs are built by the cytidines of the 5' end and the other by those of the 3' end. In both topologies, the adenosine bases are fairly well stacked to the adjacent C*C+ pairs. They are not paired but form symmetrical pseudo-pairs with their H6cis amino proton and N1 nitrogen pointing towards each other.

Adenine↗
PubMed2005

Formation and properties of hairpin and tetraplex structures of guanine-rich regulatory sequences of muscle-specific genes.

Clustered guanine residues in DNA readily generate hairpin or a variety of tetrahelical structures. The myogenic determination protein MyoD was reported to bind to a tetrahelical structure of guanine-rich enhancer sequence of muscle creatine kinase (MCK) more tightly than to its target E-box motif [K. Walsh and A. Gualberto (1992) J. Biol. Chem., 267, 13714-13718], suggesting that tetraplex structures of regulatory sequences of muscle-specific genes could contribute to transcriptional regulation. In the current study we show that promoter or enhancer sequences of various muscle-specific genes display a disproportionately high incidence of guanine clusters. The sequences derived from the guanine-rich promoter or enhancer regions of three muscle-specific genes, human sarcomeric mitochondrial creatine kinase (sMtCK), mouse MCK and alpha7 integrin formed diverse secondary structures. The sMtCK sequence folded into a hairpin structure; the alpha7 integrin oligonucleotide generated a unimolecular tetraplex; and sequences from all three genes associated to generate bimolecular tetraplexes. Furthermore, two neighboring non-contiguous guanine-rich tracts in the alpha7 integrin promoter region also paired to form a tetraplex structure. We also show that homodimeric MyoD bound bimolecular tetraplex structures of muscle-specific regulatory sequences more efficiently than its target E-box motif. These results are consistent with a role of tetrahelical structures of DNA in the regulation of muscle-specific gene expression.

Antigens, CD↗
PubMed2005

Saccharomyces cerevisiae Mre11 is a high-affinity G4 DNA-binding protein and a G-rich DNA-specific endonuclease: implications for replication of telomeric DNA.

In Saccharomyces cerevisiae, Mre11p/Rad50p/Xrs2p (MRX) complex plays a vital role in several nuclear processes including cellular response to DNA damage, telomere length maintenance, cell cycle checkpoint control and meiotic recombination. Telomeres are comprised of tandem repeats of G-rich DNA and are incorporated into non-nucleosomal chromatin. Although the structure of the yeast telomeric DNA is poorly understood, it has been suggested that the G-rich sequences can fold into G4 DNA, which has been shown to inhibit DNA synthesis by telomerase. However, little is known about the factors and mechanistic aspects of the generation of appropriate termini for DNA synthesis by telomerase. Here, we show that S.cerevisiae Mre11 protein (ScMre11p) possesses substantially higher binding affinity for G4 DNA, over single- or double-stranded DNA, and binding was inhibited by poly(dG) or porphyrin. Binding of ScMre11p to G4 DNA was most robust, compared with G2' DNA and the resulting protein-DNA complexes were strikingly very resistant to dissociation by NaCl. Remarkably, binding of ScMre11p to G4 DNA and G-rich single-stranded DNA was accompanied by the endonucleolytic cleavage at sites flanking the array of G residues and G-quartets in Mn2+-dependent manner. Collectively, these results suggest that ScMre11p is likely to play a major role in generating appropriate substrates for DNA synthesis by telomerase and telomere-binding proteins. We discuss the implications of these findings with regard to telomere length maintenance by telomerase-dependent and independent mechanisms.

DNA↗
PubMed2005

Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats.

Secondary structures of the G-rich strand of human telomere DNA fragments G3(TTAG3)n, n = 1-16, have been studied by means of circular dichroism spectroscopy and PAGE, in solutions of physiological potassium cation concentrations. It has been found that folding of these fragments into tetraplexes as well as tetraplex thermostabilities and enthalpy values depend on the number of TTAG3 repeats. The suggested topologies include, e.g. antiparallel and parallel bimolecular tetraplexes, an intramolecular antiparallel tetraplex, a tetraplex consisting of three parallel chains and one antiparallel chain, a poorly stable parallel intramolecular tetraplex, and both parallel and antiparallel tetramolecular tetraplexes. G3(TTAG3)3 folds into a single, stable and very compact intramolecular antiparallel tetraplex. With an increasing repeat number, the fragment tetraplexes surprisingly are ever less thermostable and their migration and enthalpy decrease indicate increasing irregularities or domain splitting in their arrangements. Reduced stability and different topology of lengthy telomeric tails could contribute to the stepwise telomere shortening process.

Circular Dichroism↗
PubMed2006

The PNA-DNA hybrid I-motif: implications for sugar-sugar contacts in i-motif tetramerization.

We have created a hybrid i-motif composed of two DNA and two peptide nucleic acid (PNA) strands from an equimolar mixture of a C-rich DNA and analogous PNA sequence. Nano-electrospray ionization mass spectrometry confirmed the formation of a tetrameric species, composed of PNA-DNA heteroduplexes. Thermal denaturation and CD experiments revealed that the structure was held together by C-H+-C base pairs. High resolution NMR spectroscopy confirmed that PNA and DNA form a unique complex comprising five C-H+-C base pairs per heteroduplex. The imino protons are protected from D2O exchange suggesting intercalation of the heteroduplexes as seen in DNA4 i-motifs. FRET established the relative DNA and PNA strand polarities in the hybrid. The DNA strands were arranged antiparallel with respect to one another. The same topology was observed for PNA strands. Fluorescence quenching revealed that both PNA-DNA parallel heteroduplexes are intercalated, such that both DNA strands occupy one of the narrow grooves. H1'-H1' NOEs show that both heteroduplexes are fully intercalated and that both DNA strands are disposed towards a narrow groove, invoking sugar-sugar interactions as seen in DNA4 i-motifs. The hybrid i-motif shows enhanced thermal stability, intermediate pH dependence and forms at relatively low concentrations making it an ideal nanoscale structural element for pH-based molecular switches. It also serves as a good model system to assess the contribution of sugar-sugar contacts in i-motif tetramerization.

Base Pairing↗
PubMed2006

Gene function correlates with potential for G4 DNA formation in the human genome.

G-rich genomic regions can form G4 DNA upon transcription or replication. We have quantified the potential for G4 DNA formation (G4P) of the 16 654 genes in the human RefSeq database, and then correlated gene function with G4P. We have found that very low and very high G4P correlates with specific functional classes of genes. Notably, tumor suppressor genes have very low G4P and proto-oncogenes have very high G4P. G4P of these genes is evenly distributed between exons and introns, and it does not reflect enrichment for CpG islands or local chromosomal environment. These results show that genomic structure undergoes selection based on gene function. Selection based on G4P could promote genomic stability (or instability) of specific classes of genes; or reflect mechanisms for global regulation of gene expression.

CpG Islands↗
PubMed2006

Use of thermolytic protective groups to prevent G-tetrad formation in CpG ODN type D: structural studies and immunomodulatory activity in primates.

CpG oligodeoxynucleotides (ODN) show promise as immunoprotective agents and vaccine adjuvants. CpG ODN type D were shown to improve clinical outcome in rhesus macaques challenged with Leishmania major. These ODN have a self-complementary core sequence and a 3' end poly(G) track that favors G-tetrad formation leading to multimerization. Although multimerization appears necessary for localization to early endosomes and signaling via Toll-like receptor 9 (TLR-9), it can result in product polymorphisms, aggregation and precipitation, thereby hampering their clinical applications. This study shows that functionalizing the poly(G) track of D ODN with thermolytic 2-(N-formyl-N-methyl)aminoethyl (fma) phosphate/thiophosphate protecting groups (pro-D ODN) reduces G-tetrad formation in solution, while allowing tetrad formation inside the cell where the potassium concentration is higher. Temperature-dependent cleavage of the fma groups over time further promoted formation of stable G-tetrads. Peripheral blood cells internalized pro-D ODN efficiently, inducing high levels of IFNalpha, IL-6, IFNgamma and IP-10 and triggering dendritic cell maturation. Administration of pro-D35 to macaques challenged with L.major significantly increased the number of antigen-specific IFNgamma-secreting PBMC and reduced the severity of the skin lesions demonstrating immunoprotective activity of pro-D ODN in vivo. This technology fosters the development of more efficient immunotherapeutic oligonucleotide formulations for the treatment of allergies, cancer and infectious diseases.

Adjuvants, Immunologic↗
PubMed2001

Structural transition of d(G4T4G4) from antiparallel to parallel G-quartet induced by divalent cations.

We obtained thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg2+, Ca2+, Mn2+, Co2+, or Zn2+). In addition, we also found that a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that the divalent cations are a good tool for regulating the G-quartet structures and their stability.

Base Sequence↗
PubMed2005

Structural and functional characterizations of the G-quartet and i-motif elements in retinoblastoma susceptibility genes (Rb).

The thermal stability of a G-quartet formed by incorporation of 8-methylguanine (m8G) in the DNA sequence at the 5' terminus of the Rb gene was examined by CD melting experiments. Substitutions of m8G at positions that show syn conformations in antiparallel G-quartets stabilize the structure. Formation of the G-quartet structure in the Rb gene acts as a barrier in DNA synthesis, as determined using a rapid and simple polymerase arrest assay. It was found that the C-rich complementary strand could form an i-motif structure at lower pH values.

DNA↗
PubMed2005

Reactivity of 2'-deoxyuridin-1'-yl radical in various DNA structures.

DNA C1' radicals formed by hydrogen atom abstraction from the C1' position of the sugar backbone lead to the production of 2'-deoxyribonolactone as a DNA damage lesion. Although DNA C1' radicals may be involved in a variety of DNA damage processes, there is very little information on the molecular basis of DNA structure dependence in the reactions of C1' radicals. Here, we describe a detailed study of the reactivity of 2'-deoxyuridin-1'-yl radicals in single- and double-stranded B-form DNA and G-quartets in the formation of 2'-deoxyribonolactone, using the unnatural precursor 1'-pivaloyl-2'-deoxyuridine (1).

Chromatography, High Pressure Liquid↗
PubMed2005

Tetraplex structure of fission yeast telomeric DNA and its unfolding by the interaction with telomeric DNA binding protein Pot1.

We compared the structure of fission yeast telomeric DNA, 4G4: d(GGGGTTAC)4, and nontelomeric DNA, 4T4: d(TTTTTTAC)4, and examined their interaction with telomeric DNA binding domain of telomeric DNA binding protein Pot1 (Pot1DBD). 4T4 did not form any higher-order structure, but 4G4 formed intramolecular folded tetraplex structure in the presence of Na+. Although Pot1DBD did not induce any significant structural change of 4T4, the intramolecular folded tetraplex structure of 4G4 was unfolded by the interaction with Pot1DBD. Pot1 may facilitate telomere elongation of telomerase by disrupting the tetraplex structure of the telomeric DNA.

DNA↗
PubMed2005

Octaplex formation of d(GCGAAAGC) with the double A-quartet.

We reported that the sequence gcGA[X]1Agc adopts a specific structure to form several multiplexes. In the case of X=G, an octaplex formation occurs, in which the stacked double G-quartet stabilizes the architecture through potassium cation mediations. In the case of a mutant X=A, too, it has been found that the oligomers form an octaplex. The eight central A residues form a stacked double A-quartet, which is a first example of adenine associations. Several water molecules occupy the centre to stabilize the quartets, so that the octaplex seems to be swollen as compared with that of X=G. In any multiplex formations, the building block is a base-intercalated duplexes.

Adenine↗
PubMed2006

Linker chain effect of ferrocenylnaphthalene diimide derivatives on a tetraplex DNA binding.

Spectrophotometric binding studies of a series of the naphthalene diimide derivatives, 1-7, carrying different chains with a human telomere oligonucleotide, d(TTAGG)(4) was carried out in 0.1 M AcOK-AcOH buffer (pH 5.6) and 0.1 M KCl. Under this condition, this DNA could exist as the mixture of two-type tetraplex structures and these derivatives could bind to this DNA with strong affinity of 10(6) M(-1). The effect of the linker chain is not so large in those binding affinity, but the ligand 5 having piperazine skeleton in the linker chain had relative higher affinity for this tetraplex DNA than other derivatives. Large hypochromic effect of these derivatives upon binding to the tetraplex DNA suggested that the binding mode of these derivatives might contribute the stacking interaction between the naphthalene diimide and guanine tetraplex planes.

DNA↗
PubMed2006

Development of molecular logic gates using the structural switch of telomere DNAs.

Telomere DNAs consisting of double-stranded G-rich and C-rich sequences are particularly promising as scaffolds for molecular devices because they form high-ordered structures and have a highly polymorphic nature depending on surrounding factors. Based on the structural polymorphism of telomere DNAs, excellent molecular devices such as molecular motors and switches have been reported. Here we found that the dynamic structural conversion of telomere DNAs can be controlled by both monovalent cations (M(+)) and pH (H(+)). Based on this conversion, we propose a new concept of molecular logic gates in response to the surrounding conditions (M(+) and H(+)) with fluorescence intensity changes as the output signal.

Cations, Monovalent↗
PubMed2006

Rec A-independent homologous recombination induced by a putative fold-back tetraplex DNA.

We have recently reported that a GC-rich palindromic repeat sequence presumably adopts a stable fold-back tetraplex DNA structure under supercoiling. To establish the biological significance of this structure, we inserted this sequence between two direct repeat sequences, separated by 200 bp, in a plasmid. We then investigated the effect of this sequence on homologous recombination events. Here we report that the putative fold-back DNA tetraplex structure induces homologous recombination between direct repeat sequences. Interestingly, this recombination event is independent of recA, a major driving force for homologous recombination. We think that the fold-back structure forces the repeat sequences to come into close proximity and therefore leads to strand exchange. Although triplex-induced recombination has been well documented, our results for the first time directly establish the potential of a tetraplex structure to induce recA-independent homologous recombination in vivo. This finding might have a significant implication for site-directed gene deletion in the context of the correction of genetic defects.

Base Sequence↗
PubMed2006

[DNA structure from A to Z--biological implications of structural diversity of DNA].

Deoxyribonucleic acid (DNA) is a biopolymer of nucleotides, usually adopting a double-stranded helical form in cells, with complementary base pairing holding the two strands together. The most stable is B-DNA conformation, although numerous other double helical structures can occur under specific conditions (A-DNA, Z-DNA, P-DNA). The existence of multiple-stranded (triplex, tetraplex) forms in vivo and their biological function in cells are subject of intensive studies.

Base Composition↗