A palindromic repeat sequence adopts a stable fold back structure under supercoiling

A synthetic deoxyoligonucleotide containing five palindromic repeats of GGATCC self assembles to form a parallel four-stranded structure held together by G-tetrads that shows slower mobility than duplex DNA. This structure is hypersensitive to S1 nuclease and resistant to DMS modification. The same oligonucleotide when cloned in a plasmid forms a different structure under supercoiling that persists stably even in the cleaved out insert. On polyacrylamide gel electrophoresis, the cleaved out insert moves to a position midway between the duplex and parallel four-stranded forms of the oligonucleotide. Upon S1 nuclease treatment, the cleaved out insert shows a discreet band of 18 base pairs, suggesting an unfolded region in the middle. All the guanines in the cleaved out insert are sensitive to DMS modification and produce a positive peak at 285 nM in the circular dichroism spectrum, a signature of fold back tetraplex structures. We propose a fold back quadruplex structure for the insert under supercoiling with only A.T.A.T and G.C.G.C tetrads. This is the first suggestive evidence of a general tetraplex motif without G quartets as that proposed for generalized recombination.     

DNA sequence recognition by the antitumor drug ditercalinium

The antitumor drug ditercalinium is a rare example of a noncovalent DNA-binding ligand that forms bisintercalation complexes via the major groove of the double helix. Previous structural studies have revealed that the two connected pyridocarbazolium chromophores intercalate into DNA with the positively charged bis(ethylpiperidinium) linking chain oriented to the wide groove side of the helix. Although the interaction of ditercalinium with short oligonucleotides containing 4-6 contiguous GC base pairs has been examined in detail by biophysical and theoretical approaches, the sequence preference for ditercalinium binding to long DNA fragments that offer a wide variety of binding sites has been investigated only superficially. Here we have investigated both sequence preferences and possible molecular determinants of selectivity in the binding of ditercalinium to DNA, primarily using methods based upon DNase I footprinting. A range of multisite DNA substrates, including several natural restriction fragments and different PCR-generated fragments containing unconventional bases (2,6-diaminopurine, inosine, uridine, 5-fluoro- and 5-methylcytosine, 7-deazaguanine, 7-deazaadenine, and N(7)-cyanoboranoguanine), have been employed to show that ditercalinium selectively recognizes certain GC-rich sequences in DNA and to identify some of the factors which affect its DNA-binding sequence selectivity. Specifically, the footprinting data have revealed that the 2-amino group on the purines or the 5-methyl group on the pyrimidines is not essential for the formation of ditercalinium-DNA complexes whereas the major groove-oriented N(7) of guanine does appear as a key element in the molecular recognition process. The loss of N(7) at guanines but not adenines is sufficient to practically abolish sequence-selective binding of ditercalinium to DNA. Thus, as expected for a major groove binding drug, the N(7) of guanine is normally required for effective complex formation with GC base pairs, but interestingly the substitution of the N(7) with a relatively bulky cyanoborane group does not markedly affect the sequence recognition process. Therefore, the hydrogen bond accepting capability at N(7) of guanines is not sufficient to explain the GC-selective drug-DNA association, and the implications of these findings are considered.     

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.     

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes stabilized by the formation of G.A base pairs.

Alternating d(GA)n DNA sequences form antiparallel stranded homoduplexes which are stabilized by the formation of G.A pairs. Three base pairings are known to occur between adenine and guanine: AH+ (anti).G(syn), A(anti).G(anti) and A(syn).G(anti). Protonation of the adenine residues is not involved in the stabilization of this structure, since it is observed at any pH value from 8.3 to 4.5; at pH < or = 4.0 antiparallel stranded d(GA.GA) DNA is destabilized. The results reported in this paper strongly suggest that antiparallel stranded d(GA.GA) homoduplexes are stabilized by the formation of alternating A(anti).G(anti) and G(anti).A(syn) pairs. In this structure, all guanine residues are in the anti conformation with their N7 position freely accessible to DMS methylation. On the other hand, adenines in one strand adopt the anti conformation, with their N7 position also free for reaction, while those of the opposite strand are in the syn conformation, with their N7 position hydrogen bonded to the guanine N1 group of the opposite strand. A regular right-handed helix can be generated using alternating G(anti).A(syn) and A(anti).G(anti) pairs.     

Towards a better understanding of the unusual conformations of the alternating guanine-adenine repeat strands of DNA

Alternating guanine-adenine strands of DNA are known to self-associate into a parallel-stranded homoduplex at neutral pH, fold into an ordered single-stranded structure at acid pH, and adopt yet another ordered single-stranded conformer in aqueous ethanol. The unusual conformers melt cooperatively and exhibit distinct circular dichroism spectra suggestive of a substantial conformational order, but their molecular structures are not known yet. Here, we have probed the molecular structures using guanine and adenine analogs lacking the N7 atom, and thus unable of Hoogsteen pairing, or those restrained in the less-frequent syn glycosidic orientation. The studies showed that the syn glycosidic orientation of dA residues promoted the neutral homoduplex, whereas the syn orientation of dG was incompatible with the homoduplex. In addition, Hoogsteen pairing of dA seemed to be a crucial property of the homoduplex whereas dG did not pair in this way. The situation was the same in both single-stranded conformers with the dG residues. On the other hand, the presence of N7 was important with dA but its syn geometry was not favorable. The present data can be used as restraints to model the unusual molecular structures of the alternating guanine-adenine strands of DNA.     

An alternating sheared AA pair and elements of stability for a single sheared purine-purine pair flanked by sheared GA pairs in RNA

A previous NMR structure of the duplex 5'GGU GGA GGCU/PCCG AAG CCG5' revealed an unusually stable RNA internal loop with three consecutive sheared GA pairs. Here, we report NMR studies of two duplexes, 5'GGU GGA GGCU/PCCA AAG CCG5' (replacing the UG pair with a UA closing pair) and 5'GGU GAA GGCU/PCCG AAG CCG5' (replacing the middle GA pair with an AA pair). An unusually stable loop with three consecutive sheared GA pairs forms in the duplex 5'GGU GGA GGCU/PCCA AAG CCG5'. The structure contrasts with that reported for this loop in the crystal structure of the large ribosomal subunit of Deinococcus radiodurans [Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) Cell 107, 679-688]. The middle AA pair in the duplex 5'GGU GAA GGCU/PCCG AAG CCG5' rapidly exchanges orientations, resulting in alternative base stacking and pseudosymmetry with exclusively sheared pairs. The U GAA G/G AAG C internal loop is 2.1 kcal/mol less stable than the U GGA G/G AAG C internal loop at 37 degrees C. Structural, energetic, and dynamic consequences upon functional group substitutions within related 3 x 3 and 3 x 6 internal loops are also reported.     

Kinetic evidence for inefficient and error-prone bypass across bulky N2-guanine DNA adducts by human DNA polymerase iota

DNA polymerase (pol) iota has been proposed to be involved in translesion synthesis past minor groove DNA adducts via Hoogsteen base pairing. The N2 position of G, located in minor groove side of duplex DNA, is a major site for DNA modification by various carcinogens. Oligonucleotides with varying adduct size at G N2 were analyzed for bypass ability and fidelity with human pol iota. Pol iota effectively bypassed N2-methyl (Me)G and N2-ethyl(Et)G, partially bypassed N2-isobutyl(Ib)G and N2-benzylG, and was blocked at N2-CH2(2-naphthyl)G (N2-NaphG), N2-CH2(9-anthracenyl)G (N2-AnthG), and N2-CH2(6-benzo[a]pyrenyl)G. Steady-state kinetic analysis showed decreases of kcat/Km for dCTP insertion opposite N2-G adducts according to size, with a maximal decrease opposite N2-AnthG (61-fold). dTTP misinsertion frequency opposite template G was increased 3-11-fold opposite adducts (highest with N2-NaphG), indicating the additive effect of bulk (or possibly hydrophobicity) on T misincorporation. N2-IbG, N2-NaphG, and N2-AnthG also decreased the pre-steady-state kinetic burst rate compared with unmodified G. High kinetic thio effects (S(p)-2'-deoxycytidine 5'-O-(1-thiotriphosphate)) opposite N2-EtG and N2-AnthG (but not G) suggest that the chemistry step is largely interfered with by adducts. Severe inhibition of polymerization opposite N2,N2-diMeG compared with N2-EtG by pol eta but not by pol iota is consistent with Hoogsteen base pairing by pol iota. Thus, polymerization by pol iota is severely inhibited by a bulky group at G N2 despite an advantageous mode of Hoogsteen base pairing; pol iota may play a limited role in translesion synthesis on bulky N2-G adducts in cells.     

Intramolecular DNA triplexes in supercoiled plasmids. I. Effect of loop size on formation and stability

The capacity of four oligopurine.oligopyrimidine (pur.pyr) sequences with different lengths of interruptions in the center [GAA)4(N)n(GAA)4G) (n = 3, 5, 7, and 9) to adopt intramolecular DNA triplexes was evaluated in recombinant plasmids. The hyperreactive patterns of the pur.pyr inserts to specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) at the base pair level demonstrate that intramolecular triplexes with identical 12-base triads in the stem but with different loop sizes (4, 6, 8, and 10 bases) can form in supercoiled plasmids. Furthermore, the extent of OsO4 modification was measured as a function of temperature and of average negative supercoil density. In addition, the transition free energy of B-DNA to triplexes at pH 4.5 was determined by two-dimensional electrophoresis. These comparative studies show that longer loops require more supercoil energy for triplex formation and are less thermostable than triplexes with shorter loops. Also, it may be that not only the loop size but the base composition of the loop region affects the structural transition and triplex stability. Thus, these results significantly broaden the range of natural pur.pyr sequences that may adopt triplexes.     

The structure of yeast tRNA(Asp). A model for tRNA interacting with messenger RNA

The anticodon of yeast tRNA(Asp), GUC, presents the peculiarity to be self-complementary, with a slight mismatch at the uridine position. In the orthorhombic crystal lattice, tRNA(Asp) molecules are associated by anticodon-anticodon interactions through a two-fold symmetry axis. The anticodon triplets of symmetrically related molecules are base paired and stacked in a normal helical conformation. A stacking interaction between the anticodon loops of two two-fold related tRNA molecules also exists in the orthorhombic form of yeast tRNA(Phe). In that case however the GAA anticodon cannot be base paired. Two characteristic differences can be correlated with the anticodon-anticodon association: the distribution of temperature factors as determined from the X-ray crystallographic refinements and the interaction between T and D loops. In tRNA(Asp) T and D loops present higher temperature factors than the anticodon loop, in marked contrast to the situation in tRNA(Phe). This variation is a consequence of the anticodon-anticodon base pairing which rigidifies the anticodon loop and stem. A transfer of flexibility to the corner of the tRNA molecule disrupts the G19-C56 tertiary interactions. Chemical mapping of the N3 position of cytosine 56 and analysis of self-splitting patterns of tRNA(Asp) substantiate such a correlation.     

pH-dependent conformational flexibility within the ribosomal peptidyl transferase center.

A universally conserved adenosine, A2451, within the ribosomal peptidyl transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the peptidyl transferase center, the exact nature and physiological relevance of which is not known.     

Nickel-guanine interactions in DNA: crystal structure of nickel-d[CGTGTACACG]2

The aim of this study was to clarify whether Ni2+ ions could bind to guanine bases in a standard B-DNA duplex and eventually induce a B-->Z transition. We have determined by X-ray crystallography at 3.1 A resolution the structure of the alternating deoxynucleotide d(CGTGTACACG), which contains both internal and terminal guanines. The duplex is in the B form. It is shown that nickel ions bind selectively to the N7 atom of guanine 10, which is in an extra-helical position, and guanine 2, which is in the terminal position of the duplex. It does not bind to guanine 4, which lies within a standard B-DNA tract. This simple but unambiguous result proves that nickel ions select between different guanines via steric accessibility. Guanine-Ni2+-guanine bridges among symmetry-related duplexes have also been found. These bridges may explain why Ni2+ ions may act either as a precipitant or a renaturing agent for DNA under certain conditions. The biochemical interaction of nickel with DNA can thus be related to its capacity to specifically bind to B-DNA regions with exposed guanines. Also, from the structural point of view, we have found a terminal cytosine, which forms a C.G:C reverse-Hoogsteen triple structure with a base pair of a neighbor duplex. This type of triplet is seldom found and is here described for the first time for a DNA structure.     

Efficacy of a Parainfluenza Virus 5 (PIV5)-Based H7N9 Vaccine in Mice and Guinea Pigs: Antibody Titer towards HA Was Not a Good Indicator for Protection

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.     

Differential helix stabilities and sites pre-organized for tertiary interactions revealed by monitoring local nucleotide flexibility in the bI5 group I intron RNA

The local environment at adenosine residues in the bI5 group I intron RNA was monitored as a function of Mg(2+) using both the traditional method of dimethyl sulfate (DMS) N1 methylation and a new approach, selective acylation of 2'-amine substituted nucleotides. These probes yield complementary structural information because N1 methylation reports accessibility at the base pairing face, whereas 2'-amine acylation scores overall residue flexibility. 2'-Amine acylation robustly detects RNA secondary structure and is sensitive to higher order interactions not monitored by DMS. Disruption of RNA structure due to the 2'-amine substitution is rare and can be compensated by stabilizing folding conditions. Peripheral helices that do not interact with other parts of the RNA are more stable than both base paired helices and tertiary interactions in the conserved catalytic core. The equilibrium state of the bI5 intron RNA, prior to assembly with its protein cofactor, thus features a relatively loosely packed core anchored by more stable external stem-loop structures. Adenosine residues in J4/5 and P9.0 form structures in which the nucleotide is constrained but the N1 position is accessible, consistent with pre-organization to form long-range interactions with the 5' and 3' splice sites.     

Poly(8-aminoguanylic acid): formation of ordered self-structures and interaction with poly(cytidylic acid).

Poly(8-aminoguanylic acid) has in neutral solution a novel ordered structure of high stability. The 8-amino group permits formation of three hydrogen bonds between two residues along the "top", or long axis, of the purines. The usual hydrogen bonding protons and Watson-Crick pairing sites are not involved in the association. The bonding scheme has a twofold rotation axis and is hemiprotonated at N(7). Poly(8NH2G) is converted by alkaline titration (pK = 9.7) to a quite different ordered structure, which is the favored form over the range approximately pH 10-11. The bonding scheme appears to be composed of a planar, tetrameric array of guanine residues, in which the 8-amino group does not participate in interbase hydrogen bonding. Poly (8NH2G) does not interact with poly(C) in neutral solution because of the high stability of the hemiprotonated G-G self-structure. Titration to the alkaline plateau, however, permits ready formation of a two-stranded Watson-Crick helix. In contrast to the monomer 8NH2GMP, poly(8NH2G) does not form a triple helix with poly(C) under any conditions. The properties of the ordered structures are interpreted in terms of a strong tendency of the 8-amino group to form a third interbase hydrogen bond, when this possibility is not prevented by high pH.     

Structural models for the interaction of Cd(II) with DNA: trans-[Cd(9-RGH-N7)2(H2O)4]2+

Reactions of Cd(NO(3))(2) with the model nucleobases 9-alkylguanine in water at neutral pH, give the compounds trans-[Cd(9-RGH-N7)(2)(H(2)O)(4)](NO(3))(2)(R=Me, Et), with the 9-alkylguanine ligands bound to the metal cation at the N(7) position. The X-ray structures of both compounds are reported. The six-coordinate Cd(II) complexes consist of a highly regular octahedral geometry in which the two 9-alkylguanine ligands are in a trans position to each other and approximately collinear with the metal cation. In addition, the networks of both compounds show interesting features. Thus, intramolecular H-bonds between O(6) and a coordinated water molecule are present, and self-association of guanines via H-bonding of N(3)-H...N(2) take place, leading to a 1D supramolecular polymeric ribbon. Density functional theory calculations have been applied to both compounds in order to study the stability of N(7) metalated guanine-guanine associations by comparing experimental and theoretical results. The potential relevance with regard to possible Cd(II)-DNA cross-links is briefly discussed.     

Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement

The binding of PNA (peptide nucleic acid) T₂CT₂CT₄-LysNH₂ to the double-stranded DNA target 5′ -A₂GA₂GA₄ was studied by KMnO₄ and dimethylsulfate (DMS) probing. It is found that upon sequence-specific strand displacement binding of the PNA to the dsDNA target concomitant protection of the N-7 of guanines within the target takes place. It is furthermore shown that the binding of this PNA is more efficient at pH 5.5 that at pH 6.5 and very inefficient at pH 7.5. These results clearly indicate that C⁺G Hoogsteen base pairing is present and important for binding and that the strand displacement complex therefore involves a PNA·DNA-PNA triplex.     

Suppression of Glutamic Acid Codons by Mutant Glycine Transfer Ribonucleic Acid

In previous mutational studies with mutant trpA46 (Gly [GGA] --> Glu [GAA] at position 211 of the tryptophan synthetase alpha chain) of Escherichia coli, no missense suppressors were detected. Such suppressors have now been obtained by single mutations in gly Vins, the structural gene for a GGA/G-reading, mutationally altered form of gly V transfer ribonucleic acid (tRNA) (tRNA(Gly) which reads GGU/C). A trpA46 strain containing the gly Vins alteration was mutagenized with hydroxylamine, and suppressor mutations were detected in the prototrophs obtained. Eighteen independent suppressors were examined and shown to have alterations which map in the gly V region. Chromatography of the glycyl-tRNAs of one suppressed mutant on a benzoylated diethylaminoethyl-cellulose column revealed an alteration in the tRNA(ins) (Gly) peak. The trpA46 suppressor mutation thus appears to involve a change of tRNA(ins) (Gly) from a GGA/G (Gly) reader to a GAA (Glu) reader. Since this suppressor presumably retains the "wobble" pairing of gly Vins tRNA, it was used to select the conversion of GAU (Asp211) to GAG (Glu211) in the alpha chain. supD (serine-inserting amber suppressor) was then used to obtain the conversion of GAG (Glu211) to UAG211. Missense revertants of trpA (UAG211) are being isolated as a means of introducing new codons which can be used in the selection of additional missense suppressors.     

Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease.

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.