1H-NMR Study of the Quadruplex [d(TGGGT)]4 Containing a Modified Thymine

Abstract

A NMR structural study of quadruplex [d(TGGGT)]₄ containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.     

A thymine tetrad in d(TGGGGT) quadruplexes stabilized with Tl+/Na+ ions

We report two new structures of the quadruplex d(TGGGGT)₄ obtained by single crystal X-ray diffraction. In one of them a thymine tetrad is found. Thus the yeast telomere sequences d(TG_1–3) might be able to form continuous quadruplex structures, involving both guanine and thymine tetrads. Our study also shows substantial differences in the arrangement of thymines when compared with previous studies. We find five different types of organization: (i) groove binding with hydrogen bonds to guanines from a neighbour quadruplex; (ii) partially ordered groove binding, without any hydrogen bond; (iii) stacked thymine triads, formed at the 3′ends of the quadruplexes; (iv) a thymine tetrad between two guanine tetrads. Thymines are stabilized in pairs by single hydrogen bonds. A central sodium ion interacts with two thymines and contributes to the tetrad structure. (v) Completely disordered thymines which do not show any clear location in the crystal. The tetrads are stabilized by either Na⁺ or Tl⁺ ions. We show that by using MAD methods, Tl⁺ can be unambiguously located and distinguished from Na⁺. We can thus determine the preference for either ion in each ionic site of the structure under the conditions used by us.     

Chromatographic separation of oligodeoxynucleotides with identical length: application to purification of oligomers containing a modified base.

A general procedure is described for separation and purification of oligodeoxynucleotides of identical length but different base composition, in particular, of oligomers containing modified bases such as 4-substituted thymines and 6-substituted guanines, using an anion-exchange column (either Mono Q or NucleoPac). The modified oligomers can be well separated from the analogous oligomers containing unmodified thymine or guanine under the basic conditions of the chromatography. The effects of oligomer length, base composition, and lipophilicity on the separation are discussed. A general rule which can be used for prediction of the order of elution of different oligomers and for estimation of tautomeric form of a modified base in the oligomer is presented.     

Uracil interference, a rapid and general method for defining protein-DNA interactions involving the 5-methyl group of thymines: the GCN4-DNA complex.

We describe a novel uracil interference method for examining protein contacts with the 5-methyl group of thymines. The protein of interest is incubated with target DNA containing randomly distributed deoxyuracil substitutions that is generated by carrying out the polymerase chain reaction in the presence of a mixture of TTP and dUTP. After separating DNA-protein complexes away from unbound DNA, the locations of deoxyuracil residues that either do or do not interfere with DNA-binding are determined by cleavage with uracil-N-glycosylase followed by piperidine. Using this uracil interference assay, we show that the methyl groups of the four core thymines, but not the two peripheral thymines, of the optimal binding site (ATG-ACTCAT) are important for high affinity binding of GCN4. Similar, but not identical, results are obtained using KMnO4 interference, another method used for studying protein-DNA interactions involving thymine residues. These observations strongly suggest that GCN4 directly contacts the 5-methyl groups of the four core thymines that lie in the major groove of the target DNA. Besides providing specific structural information about protein-DNA complexes, uracil interference should also be useful for identifying DNA-binding proteins and their target sites in eukaryotic promoter regions.     

Sequence dependence for bypass of thymine glycols in DNA by DNA polymerase I.

Single-stranded phage DNAs containing thymine glycols were prepared by oxidation with osmium tetroxide (OsO4) and were used as templates for DNA synthesis by E. coli DNA polymerase I. The induction of thymine glycol lesions in DNA, as measured by immunoassay, quantitatively accounted for an inhibition of in vitro DNA synthesis on modified templates. Analysis of termination sites for synthesis by DNA polymerase I (Klenow fragment) showed that DNA synthesis terminated at most template thymine sites in OsO4-treated DNA, indicating that incorporation occurred opposite putative thymine glycols in DNA. Nucleotides 5' and 3' to putative thymine glycol sites affect the reaction, however, since termination was not observed at thymines in the sequence 5'-CTPur-3'. Conversion of thymine glycols to urea residues in DNA by alkali treatment caused termination of DNA synthesis one nucleotide 3' to template thymine sites, including thymines in the 5'-CTPur-3' sequence, showing that the effect of surrounding sequence is on the elongation reaction by DNA polymerase rather than differential damage induction by OsO4.     

In vitro and in vivo ligation-mediated polymerase chain reaction analysis of a polypurine/polypyrimidine sequence upstream of the mouse metallothionein-I gene

The mouse metallothionein-I homopurine/homopyrimidine (MT-I R/Y) sequence is a 128-base pair element located approximately 1.2 kilobase pairs upstream of the MT-I gene. Previous in vitro studies of this sequence in purified plasmids indicated the formation of a non-B DNA structure stabilized by acidic pH and negative supercoiling. We now present a detailed in vitro and in vivo analysis of the MT-I R/Y sequence using chemical probes of DNA structure and ligation-mediated polymerase chain reaction. In vivo analysis suggests neither profound base unpairing nor protein binding within the MT-I R/Y sequence before or after metal induction of MT-I. We conclude for this element that the propensity to adopt an unusual DNA structure in vitro does not imply the occurrence of such a structure in vivo. We were able to show both in purified genomic DNA and in vivo that only isolated thymines and the 3' terminal thymine in strings of consecutive thymines are modified significantly by KMnO(4), indicating an altered thymine accessibility pattern within the R/Y sequence. This KMnO(4) reactivity pattern is more consistent and predictable within the R/Y sequence when compared with flanking sequences. We propose a simple steric interference model to explain the observed pattern of KMnO(4) modification of thymines.     

Crystal structure of an RNA quadruplex containing inosine tetrad: implications for the roles of NH2 group in purine tetrads

Polyinosinic acid has been known to adopt the four-stranded helical structure but its basic unit, inosine tetrad (I tetrad), has not been determined at the atomic level. Here we report the crystal structure of an RNA quadruplex containing an I tetrad at 1.4 A resolution. The I tetrad has one cyclic hydrogen bond N1...O6 with the bond length of 2.7 A. A water bridge is observed in the minor groove side of the base tetrad. Even though it is sandwiched by guanine tetrads (G tetrads), the I tetrad is buckled towards the 3' side of the tetrad plane, which results from the different interaction strength with K ions on two sides of the tetrad plane. Comparison with both G tetrad and adenine tetrad indicates that lack of NH2 in the C2 position makes the I tetrad prone to buckle for interactions with ligands. Two U*(G-G-G-G) base pentads are observed at the junction of the 5' termini of two quadruplexes. The uridine residue in the base pentad is engaged in two hydrogen bonding interactions (N2(G)-H...O2(U) and O2'(G)-H...O4(U)) and a water-mediated interaction (N3(G) and N3(U)) with the G tetrad. We also discuss the roles of amino group in purine tetrads and the inter-quadruplex interactions in RNA molecules. These quadruplexes may interact with each other by stacking, groove binding and intercalation.     

Specificity and Efficiency of Thymidine Incorporation in Escherichia coli Lacking Thymidine Phosphorylase

A mutant of Escherichia coli lacking the catabolic enzyme thymidine phosphorylase readily incorporates exogenous thymidine into deoxyribonucleic acid (DNA) even when provided at concentrations as low as 0.2 mug/ml. Incorporation by this prototrophic strain occurs specifically into DNA, since, with radioactively labeled thymidine, (i) more than 98% is incorporated into alkali-stable material, (ii) at least 90% is recovered as thymine after brief formic acid hydrolysis, and (iii) at least 90% is incorporated into material with the buoyant density of DNA. During growth in medium containing thymidine, the bacteria obtain approximately half of their DNA thymines from the exogenous thymidine and half from endogenous synthesis. The thymines and cytosines of DNA can be simultaneously and specifically labeled by thymidine-2-(14)C and uridine-5-(3)H, respectively. The mutant, which does not degrade thymidine, retains the ability to degrade the thymidine analogue 5-bromodeoxyuridine.     

An eight-stranded helical fragment in RNA crystal structure: implications for tetraplex interaction

Multistranded helical structures in nucleic acids play various functions in biological processes. Here we report the crystal structure of a hexamer, rU(BrdG)r(AGGU),at 1.5 A resolution containing a structural complex of an alternating antiparallel eight-stranded helical fragment that is sandwiched in two tetraplexes. The octaplex is formed by groove binding interaction and base tetrad intercalation between two tetraplexes. Two different forms of octaplexes have been proposed, which display different properties in interaction with proteins and nucleic acids. Adenines form a base tetrad in the novel N6-H em leader N3 conformation and further interact with uridines to form an adenine-uridine octad in the reverse Hoogsteen pairing scheme. The conformational flexibility of adenine tetrad indicates that it can optimize its conformation in different interactions.     

Proton magnetic resonance studies of ultraviolet-irradiated apurinic acid

In apurinic acid, a single-stranded polydeoxyribonucleotide easily obtained upon depurination of DNA, the proton resonances arising from thymine and cytosine are readily observable in aqueous solution of 25°C. Two methyl thymine resonances, centered at 1.88 ppm and separated by 0.045 ppm, are observed. We attribute the downfield methyl resonance to thymines with no pyrimidine nearest neighbors and the upfield methyl resonance to thymines having pyrimidine neighbors in the 3′ and/or 5′ positions. Upon ultraviolet irradiation, the upfield methyl and thymine H-6 resonances decrease in amplitude and two methyl resoances appear at 1.63 and 1.52 ppm, corresponding, respectively, to cytosine-thymine and thymine-thymine cyclobutane dimers. Photoreversal eliminates these two minor methyl resonances from the pmr spectrum. We conclude that apurinic acid provides a suitable model system for pmr studies of chemically modified pyrimidine bases in DNA.     

SPORE DEVELOPMENT IN THE LIVERWORT RICCARDIA PINGUIS

The ontogeny of spores of the liverwort Riccardia pinguis was studied at the light and electron microscope levels. Three stages of development were arbitrarily defined: spore mother cell (SMC); early tetrad with nonpigmented and unsculptured walls; and mature tetrad with pigmented and sculptured spore walls. The SMC is quadrilobed with a two-layered SMC wall, containing a central nucleus, many chloroplasts, spherosomes, and other organelles. During and following meiosis cell plates form from coalescing Golgi vesicles. These plates by continued coalescence eventually form a septum, completing the tetrad. This septum comprises middle lamella and primexine; within the latter the exine forms. By continued addition of vesicle contents to the septum and dorsal surfaces of the tetrad, the exine (sexine and nexine) and intine layers of the spore wall are laid down. The contents of the vesicles change successively during wall formation, corresponding to the different wall layers being formed. It is concluded that wall formation is under the exclusive control of the spore protoplast, and that the pattern of the mature exine is determined by the primexine. Rearrangement of organelles and other cellular components during sporogenesis is described.     

Conformational polymorphism in telomeric structures: Loop orientation and interloop pairing in d(G4TnG4)

Sequence repeats constituting the telomeric regions of chromosomes are known to adopt a variety of unusual structures, consisting of a G tetraplex stem and short stretches of thymines or thymines and adenines forming loops over the stem. Detailed model building and molecular mechanics studies have been carried out for these telomeric sequences to elucidate different types of loop orientations and possible conformations of thymines in the loop. The model building studies indicate that a minimum of two thymines have to be interspersed between guanine stretches to form folded-back structures with loops across adjacent strands in a G tetraplex (both over the small as well as large groove), while the minimum number of thymines required to build a loop across the diagonal strands in a G tetraplex is three. For two repeat sequences, these hairpins, resulting from different types of folding, can dimerize in three distinct ways—i.e., with loops across adjacent strands and on same side, with loops across adjacent strands and on opposite sides, and with loops across diagonal strands and on opposite sides—to form hairpin dimer structures. Energy minimization studies indicate that all possible hairpin dimers have very similar total energy values, though different structures are stabilized by different types of interactions. When the two loops are on the same side, in the hairpin dimer structures of d(G₄T_nG₄), the thymines form favorably stacked tetrads in the loop region and there is interloop hydrogen bonding involving two hydrogen bonds for each thymine–thymine pair. Our molecular mechanics calculations on various folded-back as well as parallel tetraplex structures of these telomeric sequences provide a theoretical rationale for the experimentally observed feature that the presence of intervening thymine stretches stabilizes folded-back structures, while isolated stretches of guanines adopt a parallel tetraplex structure. © 1994 John Wiley & Sons, Inc.     

5-(1-propargylamino)-2'-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent triplex A6-L-T6-L-T6at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by approximately 12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds approximately 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.     

EcoRI DNA methyltransferase-DNA interactions.

We present a novel strategy with synthetic hemimethylated DNA substrates containing uracil for thymine and inosine for guanosine replacements and EcoRI DNA methyltransferase to characterize the importance of major groove hydrophobic groups to the sequence-specific modification of DNA. The bacterial Mtase uses S-adenosyl-L-methionine to methylate the double-stranded DNA site 5'GAATTC3' at the N6 position of the central adenosine of each strand. Uracil substitution in either strand at the outer thymine (5'GAATUC3') causes 2.2- and 1.7-fold improvements in specificity (kcat/KmDNA). The fact that the specificity constant for the substrate containing uracil in both strands is identical to the value expected for noninteracting substitutions suggests that no significant methyltransferase-DNA interactions are altered beyond the site of either substitution. Similar analysis of the internal thymine (5'GAAUTC3') also shows these methyl groups to make a negative contribution to specificity, although the observed nonadditivity with the doubly modified substrate clearly shows methyltransferase-DNA interactions beyond the site of substitution to be affected in this case. To further probe the effect of analogue incorporation on methyltransferase-DNA interactions beyond the site of substitution, the relatively "silent" and additive uracil changes (5'GAATUC3') were combined with inosine for guanosine substitutions (e.g., 5'IAATTC3') known to have significant negative effects on specificity. In contrast to the additivity observed with the outer thymines, these studies show significant changes in methyltransferase-DNA interactions caused by the removal of the thymine methyls. Our results implicate a complex and flexible methyltransferase-DNA interface in which subtle structural changes in the substrate are transmitted over the entire canonical site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structures of B-DNA with incorporated 2'-deoxy-2'-fluoro-arabino-furanosyl thymines: implications of conformational preorganization for duplex stability.

The fundamental conformational states of right-handed double helical DNA, the A- and B-forms, are associated with distinct puckers of the sugar moieties. The furanose conformation itself is affected by the steric and electronic nature of the ring substituents. For example, a strongly electronegative substituent at the C2' position, such as in the 2'-deoxy-2'-fluoro ribo furanosyl analogue, will drive the conformational equilibrium towards the C3'- endo type (north). Conversely, the 2'-deoxy-2'-fluoro arabino furanosyl modification with opposite stereochemistry at C2' appears to have a preference for a C2'- endo type pucker (south). Incorporation of 2'-fluoroarabinofuranosyl thymines was previously shown to enhance the thermodynamic stability of B-DNA duplexes. We have determined the crystal structures of the B-DNA dodecamer duplexes [d(CGCGAASSCGCG)]2and [d(CGCGAASTCGCG)]2with incorporated 2'-deoxy-2'-fluoroarabinofuranosyl thymines S (south) at 1.55 A resolution. In the crystal structures, all S residues adopt an O4'- endo conformation (east), well compatible with an overall B-form duplex geometry. In addition to the increased rigidity of S nucleosides, a clathrate-like ordered water structure around the 2'-fluorines may account for the observed larger thermodynamic stability of DNA duplexes containing 2'-deoxy-2'-fluoroarabino thymidines.     

Osmium tetroxide reactivity of DNA bases in nucleotide sequencing and probing of DNA structure.

Osmium tetroxide, 2,2'-bipyridine (Os,bipy) has been widely applied as a probe of the DNA structure. To obtain information about reactivity of DNA bases toward this probe synthetic homopolynucleotides poly(dT), poly(dC), poly(dG) and poly(dA) were treated with Os,bipy and the content of modified bases measured by stripping voltammetry and absorption spectrophotometry. After 20 hours' treatment strong modification of poly(dT) and poly(dC) and weak modification of poly(dG) were observed, while no modification was detected in poly(dA). At short incubation times under conditions close to those usually used in probing the DNA structure the extent of poly(dT) modification was more than 10 times higher than that of poly(dC). Thus, in single-stranded DNA Os,bipy reacts with T much greater than C and G. Due to the fast reaction of thymines with Os,bipy (and osmium tetroxide, pyridine) these chemicals can be applied in Maxam-Gilbert nucleotide sequencing as agents specific for thymines in single-stranded DNA.     

The role of minor groove functional groups in DNA hydration

Here we describe the crystal structure of modified [d(CGCGAATTCGCG)]₂ refined to 2.04 Å. The modification, which affects only the two thymines at the central ApT step, involves isosteric removal of the 2-keto oxygen atoms and substitution of the N1 nitrogen with carbon. The crystal structure reveals the ability of this modified thymine to effectively base pair with adenine in [d(CGCGAAtTCGCG)]₂. The structure also suggests that the minor groove ‘spine of hydration’ is destabilized but essentially intact.     

Dimeric DNA quadruplex containing major groove-aligned A-T-A-T and G-C-G-C tetrads stabilized by inter-subunit Watson-Crick A-T and G-C pairs

We report on an NMR study of unlabeled and uniformly 13C,15N-labeled d(GAGCAGGT) sequence in 1 M NaCl solution, conditions under which it forms a head-to-head dimeric quadruplex containing sequentially stacked G-C-G-C, G-G-G-G and A-T-A-T tetrads. We have identified, for the first time, a slipped A-T-A-T tetrad alignment, involving recognition of Watson-Crick A-T pairs along the major groove edges of opposing adenine residues. Strikingly, both Watson-Crick G-C and A-T pairings within the direct G-C-G-C and slipped A-T-A-T tetrads, respectively, occur between rather than within hairpin subunits of the dimeric d(GAGCAGGT) quadruplex. The hairpin turns in the head-to-head dimeric quadruplex involve single adenine residues and adds to our knowledge of chain reversal involving edgewise loops in DNA quadruplexes. Our structural studies, together with those from other laboratories, definitively establish that DNA quadruplex formation is not restricted to G(n) repeat sequences, with their characteristic stacked uniform G-G-G-G tetrad architectures. Rather, the quadruplex fold is a more versatile and robust architecture, accessible to a range of mixed sequences, with the potential to facilitate G-C-G-C and A-T-A-T tetrad through major and minor groove alignment, in addition to G-G-G-G tetrad formation. The definitive experimental identification of such major groove-aligned mixed A-T-A-T and G-C-G-C tetrads within a quadruplex scaffold, has important implications for the potential alignment of duplex segments during homologous recombination.     

Cell surface of a tetrads-forming mutant of Micrococcus luteus: chemical treatment of the cells and teichuronic acids on the surface

Tetrads-forming mutant MT cells of Micrococcus luteus, both treated with chemical reagents and non-treated, were observed with a scanning electron microscope (SEM). The agglutinability of the cells with antiserum containing anti-teichuronic acid antibody was examined. The binding of protein A-gold particles to the cells, mediated with the antiserum, was also observed with SEM. A tetrad surface, not surface of each of four "unit monococci" constituting a tetrad, consisted of two or three smooth areas with borders. The difference in the surface features between M. luteus wild-type IFO 3333 (Monodane et al, Microbiol. Immunol. 33: 165-174, 1989) and the mutant MT cells is discussed.     

The short‐chain oxidoreductase Q9HYA2 from Pseudomonas aeruginosa PAO1 contains an atypical catalytic center

The characteristic oxidation or reduction reaction mechanisms of short‐chain oxidoreductase (SCOR) enzymes involve a highly conserved Asp‐Ser‐Tyr‐Lys catalytic tetrad. The SCOR enzyme Q9HYA2 from the pathogenic bacterium Pseudomonas aeruginosa was recognized to possess an atypical catalytic tetrad composed of Lys118‐Ser146‐Thr159‐Arg163. Orthologs of Q9HYA2 containing the unusual catalytic tetrad along with conserved substrate and cofactor recognition residues were identified in 27 additional species, the majority of which are bacterial pathogens. However, this atypical catalytic tetrad was not represented within the Protein Data Bank. The crystal structures of unligated and NADPH‐complexed Q9HYA2 were determined at 2.3 Å resolution. Structural alignment to a polyketide ketoreductase (KR), a typical SCOR, demonstrated that Q9HYA2's Lys118, Ser146, and Arg163 superimposed upon the KR's catalytic Asp114, Ser144, and Lys161, respectively. However, only the backbone of Q9HYA2's Thr159 overlapped KR's catalytic Tyr157. The Thr159 hydroxyl in apo Q9HYA2 is poorly positioned for participating in catalysis. In the Q9HYA2–NADPH complex, the Thr159 side chain was modeled in two alternate rotamers, one of which is positioned to interact with other members of the tetrad and the bound cofactor. A chloride ion is bound at the position normally occupied by the catalytic tyrosine hydroxyl. The putative active site of Q9HYA2 contains a chemical moiety at each catalytically important position of a typical SCOR enzyme. This is the first observation of a SCOR protein with this alternate catalytic center that includes threonine replacing the catalytic tyrosine and an ion replacing the hydroxyl moiety of the catalytic tyrosine.