Solution Structures of the Huntington's Disease DNA Triplets, (CAG)n

Summary

Highly polymorphic DNA triplet repeats, (CAG)_n, are located inside the first exon of the Huntington's disease gene. Inordinate expansion of this repeat is correlated with the onset and progression of the disease. NMR spectroscopy, gel electrophoresis, digestion by single-strand specific PI enzyme, and in vitro replication assay have been used to investigate the structural basis of (CAG)_n expansion. Nondenaturing gel electrophoresis and ID ¹H NMR studies of (CAG)₅ and (CAG)₆ reveal the presence of hairpins and mismatched duplexes as the major and minor populations respectively. However, at high DNA concentrations (i.e., 1.0–2.0 mM that is typically required for 2D NMR experiments) both (CAG)₅ and (CAG)₆ exist predominantly in mismatched duplex forms. Mismatched duplex structures of (CAG)₅and (CAG)₆ are useful, because they adequately model the stem of the biologically relevant hairpins formed by (CAG).,. We, therefore, performed detailed NMR spectroscopic studies on the duplexes of (CAG)₅ and (CAG)₆. We also studied a model duplex, (CGCAGCG)₂ that contains the underlined building block of the duplex. This duplex shows the following structural characteristics: (i) all the nucleotides are in (C2′-endo, anti) conformations, (ii) mismatched A?A base pairs are flanked by two Watson-Crick G?C base pairs and (iii) A?A base pairs are stably stacked (and intra-helical) and are formed by a single N6-H—N1 hydrogen bond. The nature of A?A pairing is confirmed by temperature-dependent HMQC and HMQC-NOESY experiments on the [(CA*G)₅]₂ duplex where the adenines are ¹⁵N-labeled at N6. Temperature-and pH-dependent imino proton spectra, nondenaturing electrophoresis, and PI digestion data demonstrate that under a wide range of solution conditions longer (CAG)_n repeats (n>10) exist exclusively in hairpin conformation with two single-stranded loops. Finally, an in vitro replication assay with (CAG)₈₂₁ inserts in the Ml3 single-stranded DNA templates shows a replication bypass for the (CAG)₂₁ insert but not for the (CAG)₈ insert in the template. This demonstrates that for a sufficiently long insert (n=21 in this case), a hairpin is formed by the (CAG)., even in presence of its complementary strand. This observation implies that the formation of hairpin by the (CAG)_n may cause slippage during replication and thus may explain the observed length polymorphism.     

Modelling studies on neurodegenerative disease-causing triplet repeat sequences d(GGC/GCC)n and d(CAG/CTG)n

Model building and molecular mechanics studies have been carried out to examine the potential structures for d(GGC/GCC)₅ and d(CAG/CTG)₅ that might relate to their biological function and association with triplet repeat expansion diseases. Model building studies suggested that hairpin and quadruplex structures could be formed with these repeat sequences. Molecular mechanics studies have demonstrated that the hairpin and hairpin dimer structures of triplet repeat sequences formed by looping out of the two strands are as favourable as the corresponding B-DNA type hetero duplex structures. Further, at high salt condition, Greek key type quadruplex structures are energetically comparable with hairpin dimer and B-DNA type duplex structures. All tetrads in the quadruplex structures are well stacked and provide favourable stacking energy values. Interestingly, in the energy minimized hairpin dimer and Greek key type quadruplex structures, all the bases even in the non-G tetrads are cyclically hydrogen bonded, even though the A, C and T-tetrads were not hydrogen bonded in the starting structures.     

A molecular dynamics study of the effect of G.T mispairs on the conformation of DNA in solution

The effect of G.T mispair incorporation into a double-helical environment was examined by molecular dynamics simulation. The 60-ps simulations performed on the two hexanucleotide duplexes d (G3C3)2 and d(G3TC2)2 included 10 Na+ counterions and first hydration shell waters. The resulting backbone torsional angle trajectories were analyzed to select time spans representative of conformational domains. The average backbone angles and helical parameters of the last time span for both duplexes are reported. During the simulation the hexamers retained B-type DNA structures that differed from typical A- or B-DNA forms. The overall helical structures for the two duplexes are vary similar. The presence of G.T mispairs did not alter the overall helical structure of the oligonucleotide duplex. Large propeller twist and buckle angles were obtained for both duplexes. The purine/pyrimidine crossover step showed a large decrease in propeller twist in the normal duplex but not in the mismatch duplex. Upon the formation of wobble mispairs in the mismatched duplex, the guanines moved into the minor groove and the thymines moved into the major groove. This helped prevent purine/purine clash and created a deformation in the relative orientation of the glycosidic bonds. It also exposed the free O4 of the thymines in the major groove and N2 of the guanines in the minor groove to interactions with solvent and counterions. These factors seemed to contribute to the apparently higher rigidity of the mismatched duplex during the simulation.     

Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A.A, C.C, G.G, and T.T mismatches

Thermodynamic measurements are reported for 51 DNA duplexes with A.A, C.C, G.G, and T.T single mismatches in all possible Watson-Crick contexts. These measurements were used to test the applicability of the nearest-neighbor model and to calculate the 16 unique nearest-neighbor parameters for the 4 single like with like base mismatches next to a Watson-Crick pair. The observed trend in stabilities of mismatches at 37 degrees C is G.G > T.T approximately A.A > C.C. The observed stability trend for the closing Watson-Crick pair on the 5' side of the mismatch is G.C >/= C.G >/= A.T >/= T.A. The mismatch contribution to duplex stability ranges from -2.22 kcal/mol for GGC.GGC to +2.66 kcal/mol for ACT.ACT. The mismatch nearest-neighbor parameters predict the measured thermodynamics with average deviations of DeltaG degrees 37 = 3.3%, DeltaH degrees = 7. 4%, DeltaS degrees = 8.1%, and TM = 1.1 degrees C. The imino proton region of 1-D NMR spectra shows that G.G and T.T mismatches form hydrogen-bonded structures that vary depending on the Watson-Crick context. The data reported here combined with our previous work provide for the first time a complete set of thermodynamic parameters for molecular recognition of DNA by DNA with or without single internal mismatches. The results are useful for primer design and understanding the mechanism of triplet repeat diseases.     

Exploration of pairing constraints identifies a 9 base-pair core within box C/D snoRNA-rRNA duplexes

2'-O-ribose methylation of eukaryotic ribosomal RNAs is guided by RNA duplexes consisting of rRNA and box C/D small nucleolar (sno)RNA sequences, the methylated sites invariably mapping five positions apart from the D box. Here we have analyzed the RNA duplex pairing constraints by investigating the features of 415 duplexes from the fungus, plant and animal kingdoms, and the evolution of those duplexes within the 124 sets they group into. The D-box upstream 1st and >or=15th positions consist of Watson-Crick base-pairs, G:U base-pairs and mismatched bases with ratios close to random assortments; these positions display single base differences in >60% of the RNA duplex sets. The D-box upstream 2nd to 11th positions have >90% Watson-Crick base-pairs; they display single base mutations with a U-shaped distribution of lower values of 0% and 1.6% at the methylated site 5th and 4th positions, and double compensatory mutations leading to new Watson-Crick base-pairs with an inverted U-shaped distribution of higher values at the 8th to 11th positions. Half of the single mutations at the 3rd to 11th positions resulted in G:U base-pairing, mainly through A-->G mutations in the rRNA strands and C-->T mutations in the snoRNA strands. Double compensatory mutations at the 3rd to 11th positions are extremely frequent, representing 36% of all mutations; they frequently arose from an A-->G mutation in the rRNA strands followed by a T-->C mutation in the snoRNA strands. Differences in the mutational pathways through which the rRNA and snoRNA strand evolved must be related to differences in the rRNA and snoRNA copy number and gene organization. Altogether these data identify the D-box upstream 3rd to 11th positions as box C/D snoRNA-rRNA duplex cores. The impact of the pairing constraints on the evolution of the 9 base-pair RNA duplex cores is discussed.     

Sheared-type G(anti).C(syn) base-pair: a unique d(GXC) loop closure motif

Stable DNA loop structures closed by a novel G.C base-pair have been determined for the single-residue d(GXC) loops (X=A, T, G or C) in low-salt solution by high-resolution nuclear magnetic resonance (NMR) techniques. The closing G.C base-pair in these loops is not of the canonical Watson-Crick type, but adopts instead a unique sheared-type (trans Watson-Crick/sugar-edge) pairing, like those occurring in the sheared mismatched G.A or A.C base-pair, to draw the two opposite strands together. The cytidine residue in the closing base-pair is transformed into the rare syn domain to form two H-bonds with the guanine base and to prevent the steric clash between the G 2NH(2) and the C H-5 protons. Besides, the sugar pucker of the syn cytidine is still located in the regular C2'-endo domain, unlike the C3'-endo domain adopted for the pyrimidines of the out-of-alternation left-handed Z-DNA structure. The facile formation of the compact d(GXC) loops closed by a unique sheared-type G(anti).C(syn) base-pair demonstrates the great potential of the single-stranded d(GXC) triplet repeats to fold into stable hairpins.     

Structural polymorphism exhibited by a quasipalindrome present in the locus control region (LCR) of the human beta-globin gene cluster

Structural polymorphism of DNA is a widely accepted property. A simple addition to this perception has been our recent finding, where a single nucleotide polymorphism (SNP) site present in a quasipalindromic sequence of β-globin LCR exhibited a hairpin-duplex equilibrium. Our current studies explore that secondary structures adopted by individual complementary strands compete with formation of a perfect duplex. Using gel-electrophoresis, ultraviolet (UV)-thermal denaturation, circular dichroism (CD) techniques, we have demonstrated the structural transitions within a perfect duplex containing 11 bp quasipalindromic stretch (TGGGG(G/C)CCCCA), to hairpins and bulge duplex forms. The extended version of the 11 bp duplex, flanked by 5 bp on both sides also demonstrated conformational equilibrium between duplex and hairpin species. Gel-electrophoresis confirms that the duplex coexists with hairpin and bulge duplex/cruciform species. Further, in CD spectra of duplexes, presence of two overlapping positive peaks at 265 and 285 nm suggest the features of A- as well as B-type DNA conformation and show oligomer concentration dependence, manifested in A → B transition. This indicates the possibility of an architectural switching at quasipalindromic region between linear duplex to a cruciform structure. Such DNA structural variations are likely to be found in the mechanics of molecular recognition and manipulation by proteins.     

DNA-substrate sequence specificity of human G:T mismatch repair activity.

G:T mispairs in DNA originate spontaneously via deamination of 5-methylcytosine. Such mispairs are restored to normal G:C pairs by both E. coli K strains and human cells. In this study we have analyzed the repair by human cell extracts of G:T mismatches in various DNA contexts. We performed two sets of experiments. In the first, repair was sequence specific in that G:T mispairs at CpG sites at four different CpG sites were repaired, but a G:T mismatch at a GpG site was not. Cytosine hemimethylation did not block repair of a substrate containing a CpG/GpT mismatch. In the second set of experiments, substrates with a G:T mismatch at a fixed position were constructed with an A, T, G, or C 5' to the mismatched G, and alterations in the complementary strand to allow otherwise perfect Watson-Crick pairing. All were incised just 5' to the mismatched T and competed for repair incision with a G:T substrate in which a C was 5' to the mismatched G. Thus human G:T mismatch activity shows sequence specificity, incising G:T mismatched pairs at some DNA sites, but not at others. At an incisable site, however, incision is little influenced by the base 5' to the mismatched G.     

Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus.

The mechanism of disease-associated trinucleotide repeat length variation may involve slippage of the triplet-containing strand at the replication fork, generating a slipped-strand DNA structure. We recently reported formation in vitro of slipped-strand DNA (S-DNA) structures when DNAs containing triplet repeat blocks of myotonic dystrophy or fragile X diseases were melted and allowed to reanneal to form duplexes. Here additional evidence is presented that is consistent with the existence of S-DNA structures. We demonstrate that S-DNA structures can form between two complementary strands containing equal numbers of repeats. In addition, we show that both the propensity for S-DNA formation and the structural complexity of S-DNAs formed increase with increasing repeat length. S-DNA structures were also analyzed by electron microscopy, confirming that the two strands are slipped out of register with respect to each other and confirming the structural polymorphism expected within long tracts of trinucleotide repeats. For (CTG)50.(CAG)50 two distinct populations of slipped structures have been identified: those involving </=10 repeats per slippage, which appear as bent/kinked DNA molecules, and those involving >10 repeats, which have multiple loops or hairpins indicative of complex alternative DNA secondary structures.     

The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH

UV absorption and CD spectroscopy, along with polyacrylamide gel electrophoresis, were used to study conformational properties of DNA fragments containing the trinucleotide repeat (GCC)_n (n = 4, 8 or 16), whose expansion is correlated with the fragile X chromosome syndrome. We have found that the conformational spectrum of the (GCC)_n strand is wider than has been shown so far. (GCC)_n strands adopt the hairpin described in the literature under a wide range of salt concentrations, but only at alkaline (>7.5) pH values. However, at neutral and slightly acid pH (GCC)₄ and (GCC)₈ strands homodimerize. Our data suggest that the homodimer is a bimolecular tetraplex formed by two parallel-oriented hairpins held together by hemi-protonated intermolecular C·C⁺ pairs. The (GCC)₁₆ strand forms the same tetraplex intramolecularly. We further show that below pH 5 (GCC)_n strands generate intercalated cytosine tetraplexes, whose molecularity depends on DNA strand length. They are tetramolecular with (GCC)₄, bimolecular with (GCC)₈ and monomolecular with (GCC)₁₆. i-Tetraplex formation is a complex and slow process. The neutral tetraplex, on the other hand, arises with fast kinetics under physiological conditions. Thus it is a conformational alternative of the (GCC)_n strand duplex with a complementary (GGC)_n strand.     

Structural features and hydration of a dodecamer duplex containing two C.A mispairs.

X-ray diffraction techniques have been used to characterise the crystal and molecular structure of the deoxyoligomer d(C-G-C-A-A-A-T-T-C-G-C-G) at 2.5A resolution. The final R factor is 0.19 with the location of 78 solvent molecules. The oligomer crystallises in a B-DNA type conformation with two strands coiled about each other to produce a duplex. This double helix consists of four A.T and six G.C Watson-Crick base pairs and two C.A mispairs. The mismatched base pairs adopt a "wobble" type structure with the cytosine displaced laterally into the major groove, the adenine into the minor groove. We have proposed that the two close contacts observed in the C.A pairing represent two hydrogen bonds one of which results from protonation of adenine. The mispairs are accommodated in the double helix with small adjustments in the conformation of the sugar-phosphate backbone. Details of the backbone conformation, base stacking interactions, thermal parameters and the hydration are now presented and compared with those of the native oligomer d(C-G-C-G-A-A-T-T-C-G-C-G) and with variations of this sequence containing G.T and G.A mispairs.     

Bimolecular triplex formation between 5'-d-(AG)nT4(CT)n and 5'-d-(TC)n as functions of helix length and buffer

It was observed that a group of unusually stable DNA hairpins (Hn: 5'-d-(AG)nT4(CT)n, n = 2-4) were directed to homopyrimidine sequences (Pn: 5'-d-(TC)n) by py x pu x py-type triplex formation, resulting in high binding affinity and specificity. The spectroscopic results (UV and CD) showed that the short bimolecular triplex Hn:Pn could be formed in acidic conditions (pH 4.5-6.0) as helix length n > 2, and further extending to neutral pH as n = 4. This hairpin strategy for recognition of a pyrimidine strand has a substantial binding advantage over either the conventional linear analog or simple Watson-Crick complement. Triplex stability of Hn with Pn is not only pH-dependent, as expected for triplexes involving C+. GC triads, but also sensitive to the buffer. The triplex H4:P4 was formed in the phosphate buffers of pH 6.0-7.0 but already dissociated above pH 6.5 in the buffer of cacodylate, MOPSO or PIPES. By contrast, the nature of a buffer had no major influence on stability of a hairpin duplex. Here we provide a simple triplex system, and the data presented here may be useful in defining the experimental conditions necessary to stabilize triplex DNA.     

The crystal structure of the octamer [r(guauaca)dC]2 with six Watson-Crick base-pairs and two 3' overhang residues

The crystal structure of an alternating RNA octamer, r(guauaca)dC (RNA bases are in lower case while the only DNA base is in upper case), with two 3' overhang residues one of them a terminal deoxycytosine and the other a ribose adenine, has been determined at 2.2 A resolution. The refined structure has an Rwork 18.6% and Rfree 26.8%. There are two independent duplexes (molecules I and II) in the asymmetric unit cell, a = 24.95, b = 45.25 and c = 73.67 A, with space group P2(1)2(1)2(1). Instead of forming a blunt end duplex with two a+.c mispairs and six Watson-Crick base-pairs, the strands in the duplex slide towards the 3' direction forming a two-base overhang (radC) and a six Watson-Crick base-paired duplex. The duplexes are bent (molecule I, 20 degrees; molecule II, 25 degrees) and stack head-to-head to form a right-handed superhelix. The overhang residues are looped out and the penultimate adenines of the two residues at the top end (A15) are anti and at the bottom (A7) end are syn. The syn adenine bases form minor groove A*(G.C) base triples with C8-H...N2 hydrogen bonds. The anti adenine in molecule II also forms a triple and a different C2-H...N3 hydrogen bond, while the other anti adenine in molecule I does not, it stacks on the looped out overhang base dC. The 3' terminal deoxycytosines form two stacked hemiprotonated trans d(C.C)+ base-pairs and the pseudo dyad related molecules form four consecutive deoxyribose and ribose zipper hydrogen bonds in the minor groove.     

Methylation of slipped duplexes, snapbacks and cruciforms by human DNA(cytosine-5)methyltransferase.

When human DNA(cytosine-5)methyltransferase was used to methylate a series of snapback oligodeoxy-nucleotides of differing stem lengths, each containing a centrally located CG dinucleotide recognition site, the enzyme required a minimum of 22 base pairs in the stem for maximum activity. Extrahelical cytosines in slipped duplexes that were 30 base pairs in length acted as effective methyl acceptors and were more rapidly methylated than cytosines that were Watson-Crick paired. Duplexes containing hairpins of CCG repeats in cruciform structures in which the enzyme recognition sequence was disrupted by a C.C mispair were also more rapidly methylated than control Watson-Crick-paired duplexes. Since enzymes have higher affinities for their transition states than for their substrates, the results with extrahelical and mispaired cytosines suggest that these structures can be viewed as analogs of the transition state intermediates produced during catalysis by methyltransferases.     

79 Thermodynamic and kinetic studies of trinucleotide repeat (TNR) DNA

The expansion of trinucleotide repeat (TNR) DNA has been linked to several neurodegenerative diseases (McMurray, 2010). The number of repeats is usually a characteristic indication of the severity of TNR-related diseases, with longer repeats giving higher propensity to expand and earlier onset of symptoms (López, Cleary, & Pearson, 2010). It is generally accepted that formation of noncanonical secondary structures, such as stem-loop hairpins or slipouts, contributes to the expansion mechanisms during aberrant DNA replication or repair processes (Mirkin, 2007). The stability of these hairpins is considered an important factor (Paiva & Sheardy, 2005). In this work, we used differential scanning calorimetry (DSC) and UV–Vis spectroscopy to study the thermodynamic and kinetic stability of a series of (CTG)_n and (CAG)_n TNR stem-loop hairpins and their corresponding (CTG)_n/(CAG)_n duplexes (n?=?6–14). We found that hairpins with n?=?even and n?=?even?+?1 (odd) repeats possess very similar thermodynamic stability. But, when converting to the canonical duplex form, odd-repeat hairpins are more stabilized compared to those of their even-repeat counterparts. Within both even- and odd-repeat series, hairpins with longer repeats are thermodynamically more stabilized compared to the shorter ones. Kinetic experiments of the stem-loop hairpin to duplex conversion revealed a longer lifetime for the even-repeat hairpins, while the odd-repeat hairpins convert to duplexes 10-fold faster. Also, hairpins with increased number of repeats are more resistant to the conversion when considered within the even- or odd-repeat series individually. Taken together, although it is thermodynamically more favored that hairpins containing longer repeats convert to canonical duplex form; On the contrary, these longer hairpins are kinetically trapped during the conversion and therefore can persist the noncanonical structures, which allows TNR expansion.     

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Helix-coil transition of parallel-stranded DNA. Thermodynamics of hairpin and linear duplex oligonucleotides

The stabilities have been determined of different DNA double helices constructed with the two constituent strands in a parallel orientation. These molecules incorporate polarity-inverting loop structures (hairpins) or nucleotide sequences (duplexes) which impose the desired polarity on the two strands constituting the sugar-phosphate backbone. The hairpins consisted of d(A.T)n stems (n = 8 or 10) and either a 5'-p-5' linkage in a d(C)4 loop (ps-C8 and ps-C10) or a 3'-p-3' linkage in a d(G)4 loop (ps-G10). The linear duplexes had 21-nt (ps-C2.C3) and 25-nt (ps-D1.D2, ps-D3.D4) mixed A,T sequences and normal chemical linkages throughout. Reference molecules with normal antiparallel helical orientations (hairpins aps-C8, aps-C10, and aps-G10 and duplexes aps-C3.C7, aps-D1.D3, and aps-D2.D4) were also synthesized and studied. Hydrogen bonding in ps-DNA is via reverse Watson-Crick base pairs, and the various constructs display spectroscopic, chemical, biochemical, and electrophoretic properties distinct from those of their aps counterparts. For example, both the ps and aps molecules show a pronounced UV absorption hyperchromicity upon melting, but the spectral distribution is not the same. Thus, the difference spectra (ps-aps) in the native state are characterized by a positive peak at 252 nm, an isosbestic point at 267 nm, and a negative peak at 282 nm. Temperature-dependent absorbances were recorded at selected wavelengths and in the form of complete spectra to derive the thermodynamic parameters for the helix-coil transitions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and thermodynamic analysis of the conformational states of self-complementary hexanucleotides 5′-d(GCATGC) and 5′-d(GCTAGC) in Aqueous Solution

The conformational states of hexanucleotides 5′-d(GCATGC) and 5′-d(GCTAGC) capable of forming hairpins in aqueous solution were studied by 1D and 2D ¹H NMR and molecular dynamics. The equilibrium thermodynamic parameters were determined for the formation of duplexes and hairpins, and the spatial structures were computed for the GCATGC and GCTAGC conformers. The mobility of the hexamer constituents was evaluated by nanosecond molecular dynamics simulation. The possible causes of the observed difference in the thermodynamic stability of the duplex and the hairpin are discussed.     

Structures of the mismatched duplex d(GGGTGCCC) and one of its Watson-Crick analogues d(GGGCGCCC)

The mismatched duplex d(GGGTGCCC) (I) and its two Watson-Crick analogues (dGGGCGCCC) (II) and d(GGGTACCC) (III) were synthesized. The X-ray crystal structures of (I) and (II) were determined at resolutions of 2.5 and 1.7 A (1 A = 0.1 nm) and refined to R factors of 15 and 16%, respectively. (I) and (II) crystallize as A-DNA doublehelical octamers in space groups P61 and P4(3)2(1)2, respectively, and are stable at room temperature. The central two G.T mispairs of (I) adopt the wobble geometry as observed in other G.T mismatches. The two structures differ significantly in their local conformational features at the central helical regions as well as in some global ones. In particular, T-G adopts a large helical twist (44 degrees) whereas C-G adopts a small one (24 degrees). This difference can be rationalized on the basis of simple geometrical considerations. Base-pair stacking energies which were calculated for the two duplexes indicate that (I) is destabilized with respect to (II). Helix-coil transition measurements were performed for each of the three oligomers by means of ultraviolet absorbance spectrophotometry. The results indicate that the stability of the duplexes and the co-operativity of the transition are in the following order: (I) less than (III) less than (II). Such studies may help in understanding why certain regions of DNA are more likely to undergo spontaneous mutations than others.     

Theoretical study of the geometrical arrangement of GT and GA wobble pairs in two short duplexes, Proton NMR chemical shifts and interaction energy calculations

NMR shielding constants are calculated for the base protons of duplexes formed by the dodecamer d(CGTGAATTCGCG) and the decamer d(CCAAGATTGG). A good agreement with experimental data is obtained for B-DNA helices in which the wobble GT and GA pairs are in the plane of the corresponding GC pairs of the parent duplexes formed by d(CGCGAATTCGCG) and d(CCAAGCTTGG), if the glycosyl bonds of T and G or A and G are symmetrical with respect to the dyad axis of the Watson-Crick GC pair. Interaction energy calculations show that this type of geometrical arrangement, which implies a distortion of the ribonphosphate backbone of both strands of the duplexes are more stable than those in which only one strand has its conformation modified by the presence of the wobble pair. For the duplex containing the GA pair, NMR chemical shifts as well as interaction energy computations favour the Watson-Crick hydrogen bonding scheme. The variation of the different contributions (intrastrand, interstrand, pair-pair) to the interaction energy between the bases of the duplexes, with the geometrical arrangement of the wobble pairs, is reported.