Intramolecular higher order packing of parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad of GGA triplet repeat DNA

GGA triplet repeats are widely dispersed throughout eukaryotic genomes and are frequently located within biologically important regions such as gene regulatory regions and recombination hot spot sites. We determined the structure of d(GGA)4 (12-mer) under physiological conditions and founded the formation of an intramolecular parallel quadruplex for the first time. Later, a similar architecture to that of the intramolecular parallel quadruplex was found for a telomere DNA in the crystalline state. Here, we have determined the structure of d(GGA)8 (24-mer) under physiological conditions. Two intramolecular parallel quadruplexes comprising a G:G:G:G tetrad and a G(:A):G(:A):G(:A):G heptad are formed in d(GGA)8. These quadruplexes are packed in a tail-to-tail manner. This is the first demonstration of the intramolecular higher order packing of quadruplexes at atomic resolution. K+ ions, but not Na+ ones, are critically required for the formation of this unique structure. The elucidated structure suggests the mechanisms underlying the biological events related to the GGA triplet repeat. Furthermore, in the light of the structure, the mode of the higher order packing of the telomere DNA is discussed.     

Experimental demonstration of T:(G:G:G:G):T hexad and T:A:A:T tetrad alignments within a DNA quadruplex stem

A template-based approach was used to design unprecedented architectural motifs into a known DNA framework. The structure formed by the sequence d(GCGGTTGGAT) in 0.1 M Na(+) solution has been determined using molecular dynamics simulations constrained by distance and dihedral restraints derived from NMR experiments. The molecular topology has been previously observed for the sequence d(GCGGTGGAT) (Webba da Silva, M. (2003) Biochemistry 42, 14356-65). Insertion of a single thymine into the double chain reversal formed by the segment GGTGG results in the unprecedented experimental demonstration of a T:(G:G:G:G):T hexad. The bi-stranded hexad results from the pairing alignment of two G(T-G) triads. Each triad results from recognition of the sheared edge of a guanine by the Watson-Crick edge of a thymine of the segment GGTTGG. The alignment is stabilized by base-stacking of the thymine to the sugar pucker of the preceding thymine. The latter is involved in formation of the T:A:A:T tetrad alignment by forming a hydrogen bond with the free amino proton of a Watson-Crick aligned A:A mispair. We have thus established that residues in double chain reversal loops linking juxtaposed tetrads of a quadruplex stem may facilitate formation of yet unknown hydrogen bond alignments. By employing a systematic approach analysis of sequence motifs appearing in double chain reversals, bridging tetrad layers should allow for the prediction of topologies and architectural motifs appearing in biologically relevant genomic regions.     

Structure of a G.T.A triplet in an intramolecular DNA triplex.

A 32-base DNA oligonucleotide has been studied by one- and two-dimensional 1H NMR spectroscopy and is shown to form a stable, pyr.pur.pyr, intramolecular triple helical structure, with a four C loop and a TATA loop connecting the Watson-Crick- and Hoogsteen-paired strands, respectively. This triplex contains five T.A.T base triplets, two C+.G.C base triplets, and an unusual G.T.A base triplet which disrupts the pyr.pur.pyr motif. The G.T.A triplet consists of a Watson-Crick T.A base pair, with the T situated in the "purine strand" and the A situated in the "pyrimidine strand" and a G situated in the Hoogsteen-base-paired "pyrimidine strand" hydrogen bonded to the T. The base-pairing structure of the G.T.A triplet has been investigated and has been found to involve a single hydrogen bond from the guanine amino group to the O4 carbonyl of the thymine, leaving the guanine imino proton free. The specific amino proton involved in the hydrogen bond is the H2(2) proton. This orients the guanine such that its sugar is near the thymine methyl group. The guanine sugar adopts an N-type (C3'-endo) sugar pucker in this triplet. The stability of the G.T.A triplet within pyr.pur.pyr triplexes is discussed.     

A dimeric RNA quadruplex architecture comprised of two G:G(:A):G:G(:A) hexads,G:G:G:G tetrads and UUUU loops

Using CD and NMR, we determined the structure of an RNA oligomer, r(GGAGGUUUUGGAGG) (R14), comprising two GGAGG segments joined by a UUUU segment. A modified quadruplex structure was observed for r(GGAGGUUUUGGAGG) in solution even in the absence of K(+). An unusually stable dimeric RNA quadruplex architecture formed from two strands of r(GGAGGUUUUGGAGG) at low K(+) concentration is reported here. In each strand of r(GGAGGUUUUGGAGG), two sets of successive turns in the GGAGG segments and turns at both ends of the UUUU loops drive four G-G steps to align in a parallel manner, a core with two stacked G-tetrads being formed. Two adenine bases bind to two edges of one G:G:G:G tetrad through the sheared G:A mismatch augmenting the tetrad into a G:G(:A):G:G(:A) hexad. Thus, one molecule of r(GGAGGUUUUGGAGG) folds into a modified quadruplex comprising a G:G:G:G tetrad, a UUUU double-chain reversal loop and a G:G(:A):G:G(:A) hexad. Two such molecules further associate by stacking through the dimeric hexad-hexad interface with a rotational symmetry. The ribose rings of most nucleotides take S (close to C2'-endo) puckering, which is unusual for an RNA. K(+) can increase the stability of this quadruplex structure; the number of bound K(+) was estimated from the results of the titration experiment. Besides G:G and G:A mismatches, a network of hydrogen bonds including O4'-NH(2) and C-H..O hydrogen bonds, and the extensive base stacking contribute to the high thermodynamic stability of R14. Our results could provide the stereochemical and thermodynamic basis for elucidating the biological role of the GGAGG-containing RNA segments abundantly existing in various RNAs. Relevance to quadruplex-mediated mRNA-FMRP binding and HIV-1 genome RNA dimerization is discussed.     

Formation of a parallel-stranded DNA homoduplex by d(GGA) repeat oligonucleotides.

The GGA9-H molecules consisting of a double helical stretch followed by a single-stranded 3'-terminal overhang of nine GGA sequence repeats exhibited a gel mobility-shifted band in a concentration-dependent manner, suggestive of the intermolecular complex formation. The position of the shifted band in a gel was almost identical to that of the Y-shaped dimer marker of the same molecular weight that had the two double-helices at one side. This suggests that GGA9-H dimerizes in a parallel orientation without the formation of four-stranded hairpin structure. Since the GGA9-H homoduplex was stably formed at pH 4, 7 and 9, the formation does not require protonation or deprotonation of the N1 position of adenines. Neither does it require the N7 group of guanines responsible for Hoogsteen base pairing from the methylation interference and modification studies. Modification of the N7 group of guanines with dimethyl sulfate (DMS) did not inhibit the association and also the N7 group in the homoduplex was not protected from DMS. On the other hand, the GAA9-H having the G to A base substitution did not show such an association with either GGA9-H or GAA9-H. These results suggest that the homoduplex formation may be due to G.G base pairing through non-Hoogsteen hydrogen bonds.     

(CGA)(4): parallel, anti-parallel, right-handed and left-handed homoduplexes of a trinucleotide repeat DNA.

Conformational properties of microsatellite DNA regions are the probable reason of their expansions in genomes which lead to serious genetic diseases in some cases. Using CD spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we study in this paper conformational properties of (CGA)(4) and compare them with those of (CAG)(4) - a related repeat, connected with Huntington's disease. We show that (CGA)(4) can adopt several distinct conformations in solution. Around neutral pH it forms a parallel-stranded homoduplex containing C(+).C, G.G, and A.A base pairs. Under the same conditions (CAG)(4) forms a hairpin. At slightly alkaline pH values and low ionic strength, (CGA)(4) also folded into a hairpin which transformed into a bimolecular anti-parallel homoduplex at increasing salt concentrations. The duplex easily isomerized into left-handed Z-DNA, implying that the mismatched adenines between G.C pairs facilitate rather than hinder the B-Z transition. No similar changes took place with (CAG)(4). Thus, the conformational repertoire of (CGA)(4) includes parallel, anti-parallel, right-handed, and left-handed homoduplexes. In contrast, (CAG)(4) invariably adopts only a single conformation, namely the very stable hairpin.     

The effect of sodium, potassium and ammonium ions on the conformation of the dimeric quadruplex formed by the Oxytricha nova telomere repeat oligonucleotide d(G(4)T(4)G(4)).

The DNA sequence d(G(4)T(4)G(4)) [Oxy-1.5] consists of 1.5 units of the repeat in telomeres of Oxytricha nova and has been shown by NMR and X-ray crystallographic analysis to form a dimeric quadruplex structure with four guanine-quartets. However, the structure reported in the X-ray study has a fundamentally different conformation and folding topology compared to the solution structure. In order to elucidate the possible role of different counterions in this discrepancy and to investigate the conformational effects and dynamics of ion binding to G-quadruplex DNA, we compare results from further experiments using a variety of counterions, namely K(+), Na(+)and NH(4)(+). A detailed structure determination of Oxy-1.5 in solution in the presence of K(+)shows the same folding topology as previously reported with the same molecule in the presence of Na(+). Both conformations are symmetric dimeric quadruplexes with T(4)loops which span the diagonal of the end quartets. The stack of quartets shows only small differences in the presence of K(+)versus Na(+)counterions, but the T(4)loops adopt notably distinguishable conformations. Dynamic NMR analysis of the spectra of Oxy-1.5 in mixed Na(+)/K(+)solution reveals that there are at least three K(+)binding sites. Additional experiments in the presence of NH(4)(+)reveal the same topology and loop conformation as in the K(+)form and allow the direct localization of three central ions in the stack of quartets and further show that there are no specific NH(4)(+)binding sites in the T(4)loop. The location of bound NH(4)(+)with respect to the expected coordination sites for Na(+)binding provides a rationale for the difference observed for the structure of the T(4)loop in the Na(+)form, with respect to that observed for the K(+)and NH(4)(+)forms.     

Effect of gamma-hydroxypropano deoxyguanosine, the major acrolein-derived adduct, on monomolecular quadruplex structure of telomeric repeat d(TTAGGG)(4)

The three oligodeoxyribonucleotides (ODNs) a-c, having the telomeric repeat d(TTAGGG)(4) sequence and incorporating gamma-hydroxypropano deoxyguanosine at different positions, were synthesized. Gel electrophoresis and CD analyses indicated that the ODNs assume monomolecular quadruplex structures in Na+ and in K+ buffers. The T(m) values, obtained by CD melting experiments, showed that the presence of the acrolein-dG adduct into the ODN b decreases the thermal stability of the monomolecular quadruplex structure in Na+ solution, whereas for a and c no significant effect could be detected in the same experimental conditions. On the contrary, all ODNs a-d show the same behaviour in K+ buffer. These findings are briefly discussed.     

Drug recognition and stabilisation of the parallel-stranded DNA quadruplex d(TTAGGGT)4 containing the human telomeric repeat

The NMR structure of the parallel-stranded DNA quadruplex d(TTAGGGT)(4), containing the human telomeric repeat, has been determined in solution in complex with a fluorinated pentacyclic quino[4,3,2-kl]acridinium cation (RHPS4). RHPS4 has been identified as a potent inhibitor of telomerase at submicromolar levels (IC(50) value of 0.33(+/-0.13)microM), exhibiting a wide differential between telomerase inhibition and acute cellular toxicity. All of the data point to RHPS4 exerting its chemotherapeutic potency through interaction with, and stabilisation of, four-stranded G-quadruplex structures. RHPS4 forms a dynamic interaction with d(TTAGGGT)(4), as evident from 1H and 19F linewidths, with fast exchange between binding sites induced at 318 K. Perturbations to DNA chemical shifts and 24 intermolecular nuclear Overhauser effects (NOEs) identify the 5'-ApG and 5'-GpT steps as the principle intercalation sites; a structural model has been refined using NOE-restrained molecular dynamics. The central G-tetrad core remains intact, with drug molecules stacking at the ends of the G-quadruplex. The partial positive charge on position 13-N of the acridine ring appears to act as a "pseudo" potassium ion and is positioned above the centre of the G-tetrad in the region of high negative charge density. In both ApG and GpT intercalation sites, the drug is seen to converge to the same orientation in which the pi-system of the drug overlaps primarily with two bases of each G-tetrad. The drug is held in place by stacking interactions with the G-tetrads; however, there is some evidence for a more dynamic, weakly stabilised A-tetrad that stacks partially on top of the drug at the 5'-end of the sequence. Together, the interactions of RHPS4 increase the t(m) of the quadruplex by approximately 20 degrees C. There is no evidence for drug intercalation within the G-quadruplex; however, the structural model strongly supports end-stacking interactions with the terminal G-tetrads.     

Method for direct discrimination of intra- and intermolecular hydrogen bonds, and characterization of the G(:A):G(:A):G(:A):G heptad, with scalar couplings across hydrogen bonds

Discrimination of intra- and intermolecular hydrogen bonds in a symmetric multimer has not been accomplished yet, although such discrimination would provide a crucial basis for construction of the multimeric architecture of nucleic acids by NMR. We have developed a direct and unambiguous method for such discrimination involving the use of scalar couplings across hydrogen bonds. The method has been validated with a symmetric dimer of d(GGGCTTTTGGGC), for which the structure including both intra- and intermolecular hydrogen bonds was already reported. This has demonstrated that our method can clearly discriminate these two kinds of hydrogen bonds. Then, the method was applied to a symmetric dimer of d(GGAGGAGGAGGA) and has provided decisive information on its multimeric architecture. Additionally, the values for scalar couplings across hydrogen bonds for G:G and G:A base pairs in the G(:A):G(:A):G(:A):G heptad formed by d(GGAGGAGGAGGA) were determined for the first time. This determination has provided an insight into the nature of the heptad.     

G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates

Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K(d) values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 +/- 6 and 77 +/- 6 Pm, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/RHAU in targeting in vivo RNA quadruplex structures.     

Characterization and DNA interaction of the Pt(II)(pq)(bdt) complex: A theoretical and experimental research

A mononuclear complex Pt(pq)(bdt) (1) (where pq = 2-(2′pyridyl)quinoxaline and bdt = benzene-1,2-dithiolate) has been prepared and characterized by NMR spectroscopy, ES mass spectroscopy and elemental analysis. Furthermore, its molecular and electronic structure has been fully elucidated by means of the density functional theory (DFT) and time-dependent density functional theory (TDDFT). The former reveals an extensive distortion of the planarity of complex 1, while the latter an intense mixed metal ligand to ligand charge transfer (MM′LLCT) transition in the visible region of the spectrum. Interactions of complex 1, the free ligands pq and bdt with double stranded calf thymus DNA before and after illumination were studied by UV-spectrophotometric (melting curves) and circular dichroism (CD) measurements, indicating that complex 1 is able to form adducts with DNA and to distort the double helix by changing the base stacking. Under our experimental conditions, it is unclear that complex 1 can photocleave DNA. Viscosity changes of calf thymus DNA (CT-DNA) in the presence of an incremental amount of complex 1 demonstrate that in very low ratios, [1]/[DNA]  0.02, this complex binds intercalatively to the DNA, while in higher ratios a partial or a non-classical interacalation should occur due to the tetrahedral distortion of the molecule and the existence of the dithiolato-ligand.     

A series of meso-tris(N-methyl-pyridiniumyl)-(4-alkylamidophenyl) porphyrins: Synthesis,interaction with DNA and antibacterial activity

A series of meso-5,10,15-tris(N-methyl-4-pyridiniumyl)-20-(4-alkylamidophenyl) porphyrins were synthesized by derivatizing the amino group on the phenyl ring with the following hydrophobic groups: –C(O)C₇F₁₅, –C(O)CHCH₂, C(O)CH₃, –C(O)C₇H₁₅, and –C(O)C₁₅H₃₁. The cationic tris-pyridiumyl porphyrin core serves as a DNA binding motif and a photosensitizer to photomodify DNA molecules. The changes of the UV–Vis absorption spectra during the titration of these porphyrins with calf thymus DNA revealed a large bathochromic shift (up to 14 nm) and a hypochromicity (up to 55%) of the porphyrins Soret bands, usually considered as proof of porphyrin intercalation into DNA. Association constants (K) calculated according to the McGhee and von Hippel model, were in the range of 10⁶–10⁷ M⁻¹. An increase in hydrophobicity of the substituents at the 20−meso-position produced higher binding affinity. These porphyrins caused photomodification of the supercoiled plasmid DNA when a green laser beam at 532 nm was applied. Those with higher surface activity acted more efficiently as DNA photomodifiers. The porphyrin with a perfluorinated alkyl chain (–COC₇F₁₅) at the meso-20-position inhibited the growth of gram-positive bacteria (S. aureus, or S. epidermidis). Other porphyrins exhibited moderate activity against both gram-negative and gram-positive organisms.     

Poly(A)-binding protein interaction with elF4G stimulates picornavirus IRES-dependent translation.

The eukaryotic mRNA 3' poly(A) tail and the 5' cap cooperate to synergistically enhance translation. This interaction is mediated, at least in part, by elF4G, which bridges the mRNA termini by simultaneous binding the poly(A)-binding protein (PABP) and the cap-binding protein, elF4E. The poly(A) tail also stimulates translation from the internal ribosome binding sites (IRES) of a number of picornaviruses. elF4G is likely to mediate this translational stimulation through its direct interaction with the IRES. Here, we support this hypothesis by cleaving elF4G to separate the PABP-binding site from the portion that promotes internal initiation. elF4G cleavage abrogates the stimulatory effect of poly(A) tail on translation. In addition, translation in extracts in which elF4G is cleaved is resistant to inhibition by the PABP-binding protein 2 (Paip2). The elF4G cleavage-induced loss of the stimulatory effect of poly(A) on translation was mimicked by the addition of the C-terminal portion of elF4G. Thus, PABP stimulates picornavirus translation through its interaction with elF4G.     

A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.

The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin.     

Unusual dimeric Zn(II)-cytosine complexes: new models of the interaction of Zn(II) with DNA and RNA

Synthesis and crystal structure of two Zn(II) dimer complexes with 1-methylcytosine (1-MeC) are reported. In complex [Zn(2)Cl(4)(mu-1-MeC-O2,N3)(2)] (1), two 1-MeC ligands are bridging two ZnCl(2) moieties. In [Zn(2)(1-MeC-N3)(4)(mu-SO(4))(2)].2H(2)O (2), the sulfates act as bridging ligands and 1-MeC are linked via N3 to Zn(II) as terminal ligands. Both complexes represent the first examples of Zn(II)-pyrimidine dimers. The potential biological significance of 1 and 2 is discussed.     

Eukaryotic initiation factors 4A (eIF4A) and 4G (eIF4G) mutually interact in a 1:1 ratio in vivo.

mRNA translation in eukaryotic cells involves a set of proteins termed translation initiation factors (eIFs), several of which are involved in the binding of ribosomes to mRNA. These include eIF4G, a modular scaffolding protein, and eIF4A, an RNA helicase, of which two closely related forms are known in mammals, eIF4A(I) and eIF4A(II). In mammals, eIF4G possesses two independent sites for binding eIF4A, whereas in other eukaryotes (e.g. yeast) only one site appears to be present, thus raising the issue of the stoichiometry of eIF4G.eIF4A complexes in different eukaryotes. We show that in human embryonic kidney cells eIF4G is associated with eIF4A(I) or eIF4A(II) but not with both simultaneously, suggesting a stoichiometry of 1:1 rather than 1:2. To confirm this, eIF4A(I) or eIF4A(II) was expressed in a tagged form in these cells, and complexes with eIF4G were again isolated. Complexes containing tagged eIF4A(I) or eIF4A(II) contained no endogenous eIF4A, supporting the notion that eIF4G binds only one molecule of eIF4A. Each binding site in eIF4G can bind either eIF4A(I) or eIF4A(II). The data imply that the second binding site in mammalian eIF4A does not bind an additional eIF4A molecule and that initiation factor complexes in different eukaryotes contain one eIF4A per eIF4G.