Solution structure of a DNA double helix incorporating four consecutive non-Watson-Crick base-pairs

A series of DNA 21-mers containing a variety of the 4 x 4 internal loop sequence 5'-CAAG-3'/3'-ACGT-5' were studied using nuclear magnetic resonance (NMR) methodology and distance geometry (DG)/molecular dynamics (MD) approaches. Such oligomers exhibit excellent resolution in the NMR spectra and reveal many unusual NOEs (nuclear Overhauser effect) that allow for the detailed characterization of a DNA hairpin incorporating a track of four different non-Watson-Crick base-pairs in the stem. These include a wobble C.A base-pair, a sheared A.C base-pair, a sheared A.G base-pair, and a wobble G.T base-pair. Significantly different twisting angles were observed between the base-pairs in internal loop that results with excellent intra-strand and inter-strand base stacking within the four consecutive mismatches and the surrounding canonical base-pairs. This explains why it melts at 52 degrees C even though five out of ten base-pairs in the stem adopt non-Watson-Crick pairs. However, the 4 x 4 internal loop still fits into a B-DNA double helix very well without significant change in the backbone torsion angles; only zeta torsion angles between the tandem sheared base-pairs are changed to a great extent from the gauche(-) domain to the trans domain to accommodate the cross-strand base stacking in the internal loop. The observation that several consecutive non-canonical base-pairs can stably co-exist with Watson-Crick base-pairs greatly increases the limited repertoire of irregular DNA folds and reveals the possibility for unusual structural formation in the functionally important genomic regions that have potential to become single-stranded.     

NMR study of a heterochiral DNA hairpin:impact of L-enantiomery in the loop.

We carried out a structural study of the DNA heterochiral strand d (AGCTTATCAT(L)CGATAAGCT), -AT(L)C-, where T(L) (L thymine ) replaces T (natural D-thymine). -AT(L)C- is a structural analog of -ATC- that belongs to a strong topoisomerase II DNA cleavage site and which has been shown to resolve into a hairpin structure with a stem formed by eight Waston-Crick base-pairs and a single residue loop closed by an A.C sheared base-pair. Although - AT(L)C-, like its parent -ATC-, folds into a hairpin structure at low and high DNA concentrations it displays a lower stability (Tm of 56 degrees C versus 58.5 degrees C). Several NMR features in -AT(L)C- account for the disruption of the A.C pairing in the loop and a weakening of the C.G base-pair stability at the stem-loop junction. For instance, the exchange of the loop imino protons with solvent is accelerated compared with the natural oligonucleotide -ATC-. The higher flexibility of the heterochiral loop is confirmed by the results of NMR restrained molecular dynamics. In the calculated final structures of -AT(L)C-, the T10(L) residue moves the A9 and C11 residues away, thus preventing the loop closure through a C.A sheared base-pair and the achievement of a good base-base or sugar-base stacking. Actually, most of the stabilizing interactions present in -ATC- are lost in the heterochiral - AT(L)C- explaining its weaker stability.     

Structure of a T4 hairpin loop on a Z-DNA stem and comparison with A-RNA and B-DNA loops

The synthetic DNA oligomer C-G-C-G-C-G-T-T-T-T-C-G-C-G-C-G crystallizes as a Z-DNA hexamer, capped at one end by a T4 loop. The crystals are monoclinic, space group C2, with a = 57.18 A, b = 21.63 A, c = 36.40 A, beta = 95.22 degrees, and one hairpin molecule per asymmetric unit. The structure of the z-hexamer stem was determined by molecular replacement, and the T4 loop was positioned by difference map methods. The final R factor at 2.1 A resolution for hairpin plus 70 water molecules is 20% for 2 sigma data, with a root-mean-square error of 0.26 A. The (C-G)3 stem resembles the free Z-DNA hexamer with minor crystal packing effects. The T4 loop differs from that observed on a B-DNA stem in solution, or in longer loops in tRNA, in that it shows intraloop and intermolecular interactions rather than base stacking on the final base-pair of the stem. Bases T7, T8 and T9 stack with one another and with the sugar of T7. Two T10 bases from different molecules stack between the C1-G12 terminal base-pairs of a third and fourth molecule, to simulate a T.T "base-pair". Distances between thymine N and O atoms suggest that the two thymine bases are hydrogen bonded, and a keto-enol tautomer pair is favored over disordered keto-keto wobble pairs. The hairpin molecules pack in the crystal in herringbone columns in a manner that accounts well for the observed relative crystal growth rates in a, b and c directions. Hydration seems to be most extensive around the phosphate groups, with lesser hydration within the grooves.     

Stable formation of a pyrimidine-rich loop hairpin in a cruciform promoter.

We have determined the solution structure of a TCC-loop hairpin in the cruciform promoter for the bacteriophage N4 virion RNA polymerase (N4 vRNAP). This hairpin and its complementary GGA-loop hairpin are extruded at physiological superhelical density and are required for vRNAP recognition. Contrary to its complementary GGA-loop, the three pyrimidines in the TCC-loop are all unpaired. However, with the help of two juxtaposed stem Watson-Crick G.C base-pairs, each nucleotide in the loop employs a special method to stabilize the hairpin structure. The resulting structures display extensive loop base-stacking rearrangement yet minor backbone distortion, which is largely accomplished through some loop zeta and alpha torsional angle changes. Consistent with the structural studies, UV melting of the GAAGCTCCGCTTC hairpin revealed a higher melting temperature (66 degrees C) than that of the GAACGTCCCGTTC hairpin (58 degrees C) with reversed stem G.C base-pairs, indicating significant contribution from the extra three loop-stem H-bonds. Thermodynamic parameters DeltaG degrees 25of the GAAGCTCCGCTTC hairpin and its complementary GAAGCGGAGCTTC hairpin are -4.1 and -4. 3 kcal/mol respectively, indicating approximately equal contribution of each hairpin to the cruciform formation of the N4 virion RNA polymerase promoter. No significant loop dynamics in the microsecond to millisecond NMR time-scale was observed, and the abundant well-defined exchangeable and non-exchangeable proton NOEs allowed us to efficiently determine a well-converged family for the final structures of the TCC-loop hairpin.     

Cooperative binding of tetrameric p53 to DNA

We analysed by analytical ultracentrifugation and fluorescence anisotropy the binding of p53 truncation mutants to sequence-specific DNA. The synthetic 30 base-pair DNA oligomers contained the 20 base-pair recognition elements for p53, consisting of four sites of five base-pairs per p53 monomer. We found that the binding at low ionic strengths was obscured by artifacts of non-specific binding and so made measurements at higher ionic strengths. Analytical ultracentrifugation of the construct p53CT (residues 94-360, containing the DNA-binding core and tetramerization domains) gave a dissociation constant of approximately 3 microM for its dimer-tetramer equilibrium, similar to that of full-length protein. Analytical ultracentrifugation and fluorescence anisotropy showed that p53CT formed a complex with the DNA constructs with 2:1 stoichiometry (dimer:DNA). The binding of p53CT (1-100 nm range) to DNA was highly cooperative, with a Hill coefficient of 1.8 (dimer:DNA). The dimeric L344A mutant of p53CT has impaired tetramerization. It bound to full-length DNA p53 recognition sequence, but with sixfold less affinity than wild-type protein. It did not form a detectable complex with a 30-mer DNA construct containing two specific five base-pair sites and two random sites, emphasizing the high co-operativity of the binding. The fundamental active unit of p53 appears to be the tetramer, which is induced by DNA binding, although it is a dimer at low concentrations.     

Base sequence and helix structure variation in B and A DNA

The observed propeller twist in base-pairs of crystalline double-helical DNA oligomers improves the stacking overlap along each individual helix strand. But, as proposed by Calladine, it also leads to clash or steric hindrance between purines at adjacent base-pairs on opposite strands of the helix. This clash can be relieved by: (1) decreasing the local helix twist angle between base-pairs; (2) opening up the roll angle between base-pairs on the side on which the clash occurs; (3) separating purines by sliding base-pairs along their long axes so that the purines are partially pulled out of the stack (leading to equal but opposite alterations in main-chain torsion angle delta at the two ends of the base-pair); and (4) flattening the propeller twist of the offending base-pairs. Simple sum functions, sigma 1 through sigma 4, are defined, by which the expected local variation in helix twist, base roll angle, torsion angle delta and propeller twist may be calculated from base sequence. All four functions are quite successful in predicting the behavior of B DNA. Only the helix twist and base roll functions are applicable to A DNA, and the helix twist function begins to fail for an A helical RNA/DNA hybrid. Within these limits, the sequence-derived sum functions match the observed helix parameter variation quite closely, with correlation coefficients greater than 0.900 in nearly all cases. Implications of this sequence-derived helix parameter variation for repressor-operator interactions are considered.     

Sequence-specific recognition of DNA. Nuclear magnetic resonance assignments and structural comparison of wild-type and mutant lambda OR3 operator DNA

The resonances of all the base protons and most of the sugar protons in both strands of the 17 base-pair OR3 operator of the phage lambda, and of the vC3 single base-pair mutant, have been assigned using two-dimensional nuclear magnetic resonance methods. The chemical shift and nuclear Overhauser effect data for these two DNA sequences reveal no structural perturbation at sites distal to the mutation, neither are there significant changes in structure immediately surrounding the altered base-pair in the mutant sequence. These results are consistent with the model proposed by Ohlendorf et al. (1982), based on crystallographic data on the cro protein, for the OR3-cro protein interaction. The data from these solution studies are examined and discussed in the light of this model. This work demonstrates that nuclear magnetic resonance chemical shifts and nuclear Overhauser effect intensities provide a method for comparing the solution structures of DNA molecules. From the resolution available in the spectra of the 17 base-pair operators studied, it is clear that DNA duplexes of up to 30 or more base-pairs can be studied using phase-sensitive methods.     

Genetic conversion of G.C base-pairs to A.U base-pairs in a transfer RNA

The cloverleaf stem segments of the suppressor gene of bacteriophage T4 tRNA(Gln) contain ten G.C and ten A.U base-pairs. To gain a better appreciation of the G.C base-pair requirement, we isolated multiple mutants of this suppressor gene in which base-pairs of G.C were replaced by A.U. One active suppressor gene contained only A.U base-pairs on the anticodon stem, indicating that G.C base-pairs in this region of tRNA(Gln) are not essential for function. In contrast, replacement was not possible at two base-pairs on the D stem and at one base-pair on the T stem.     

Structure and dimerization of HIV-1 kissing loop aptamers

Dimerization of two homologous strands of genomic RNA is an essential feature of the retroviral replication cycle. In HIV-1, genomic RNA dimerization is facilitated by a conserved stem-loop structure located near the 5' end of the viral RNA called the dimerization initiation site (DIS). The DIS loop is comprised of nine nucleotides, six of which define an autocomplementary sequence flanked by three conserved purine residues. Base- pairing between the loop sequences of two copies of genomic RNA is necessary for efficient dimerization. We previously used in vitro evolution to investigate a possible structural basis for the marked sequence conservation of the DIS loop. In this study, chemical structure probing, measurements of the apparent dissociation constants, and computer structure analysis of dimerization-competent aptamers were used to analyze the dimers' structure and binding. The selected aptamers were variants of the naturally occurring A and B subtypes. The data suggest that a sheared base-pair closing the loop of the DIS is important for dimerization in both subtypes. On the other hand, the open or closed state of the last base-pair in the stem differed in the two subtypes. This base-pair appeared closed in the subtype A DIS dimer and open in subtype B. Finally, evidence for a cross-talk between nucleotides 2, 5, and 6 was found in some, but not all, loop contexts, indicating some structural plasticity depending on loop sequence. Discriminating between the general rules governing dimer formation and the particular characteristics of individual DIS aptamers helps to explain the affinity and specificity of loop-loop interactions and could provide the basis for development of drugs targeted against the dimerization step during retroviral replication.     

Unique tertiary and neighbor interactions determine conservation patterns of Cis Watson-Crick A/G base-pairs

X-ray, phylogenetic and quantum chemical analysis of molecular interactions and conservation patterns of cis Watson-Crick (W.C.) A/G base-pairs in 16S rRNA, 23S rRNA and other molecules was carried out. In these base-pairs, the A and G nucleotides interact with their W.C. edges with glycosidic bonds oriented cis relative to each other. The base-pair is stabilised by two hydrogen bonds, the C1'-C1' distance is enlarged and the G(N2) amino group is left unpaired. Quantum chemical calculations show that, in the absence of other interactions, the unpaired amino group is substantially non-planar due to its partial sp(3) pyramidalization, while the whole base-pair is internally propeller twisted and very flexible. The unique molecular properties of the cis W.C. A/G base-pairs make them distinct from other base-pairs. They occur mostly at the ends of canonical helices, where they serve as interfaces between the helix and other motifs. The cis W.C. A/G base-pairs play crucial roles in natural RNA structures with salient sequence conservation patterns. The key contribution to conservation is provided by the unpaired G(N2) amino group that is involved in a wide range of tertiary and neighbor contacts in the crystal structures. Many of them are oriented out of the plane of the guanine base and utilize the partial sp(3) pyramidalization of the G(N2). There is a lack of A/G to G/A covariation, which, except for the G(N2) position, would be entirely isosteric. On the contrary, there is a rather frequent occurrence of G/A to G/U covariation, as the G/U wobble base-pair has an unpaired amino group in the same position as the cis W.C. G/A base-pair. The cis W.C. A/G base-pairs are not conserved when there is no tertiary or neighbor interaction. Obtaining the proper picture of the interactions and phylogenetic patterns of the cis W.C. A/G base-pairs requires a detailed analysis of the relation between the molecular structures and the energetics of interactions at a level of single H-bonds and contacts.     

The conformational variability of an adenosine.inosine base-pair in a synthetic DNA dodecamer.

A crystal structure analysis of the synthetic deoxydodecamer d(CGCAAATTIGCG) which contains two adenosine.inosine (A.I) mispairs has revealed that, in this sequence, the A.I base-pairs adopt a A(anti).I(syn) configuration. The refinement converged at R = 0.158 for 2004 reflections with F greater than or equal to 2 sigma(F) in the range 7.0-2.5A for a model consisting of the DNA duplex and 71 water molecules. A notable feature of the structure is the presence of an almost complete spine of hydration spanning the minor groove of the whole of the (AAATTI)2 core region of the duplex. pH-dependent ultraviolet melting studies have suggested that the base-pair observed in the crystal structure is, in fact, a protonated AH+ (anti).I(syn) species and that the A.I base-pairs in the sequence studied display the same conformational variability as A.G mispairs in the sequence d(CGCAAATTGGCG). The AH+(anti).I(syn) base-pair predominates below pH 6.5 and an A(anti).I(anti) mispair is the major species present between pH 6.5 and 8.0. The protonated base-pairs are held together by two hydrogen bonds one between N6(A) and O6(I) and the other between N1(A) and N7(I). This second hydrogen bond is a direct result of the protonation of the N1 of adenosine. The ultraviolet melting studies indicate that the A(anti).I(anti) base-pair is more stable than the A(anti).G(anti) base-pair but that the AH+(anti).I(syn) base pair is less stable than its AH+(anti).G(syn) analogue. Possible reasons for this observation are discussed.     

Characterization of base-pair opening in deoxynucleotide duplexes using catalyzed exchange of the imino proton

Using nuclear magnetic resonance line broadening, longitudinal relaxation and magnetization transfer from water, we have measured the imino proton exchange times in the duplex form of the 10-mer d-CGCGATCGCG and in seven other deoxy-duplexes, as a function of the concentration of exchange catalysts, principally ammonia. All exchange times are catalyst dependent. Base-pair lifetimes are obtained by extrapolation to infinite concentration of ammonia. Lifetimes of internal base-pairs are in the range of milliseconds at 35 degrees C and ten times more at 0 degrees C. Lifetimes of neighboring pairs are different, hence base-pairs open one at a time. Lifetimes of d(G.C) are about three times longer than those of d(A.T). The nature of neighbors usually has little effect, but lifetime anomalies that may be related to sequence and/or structure have been observed. In contrast, there is no anomaly in the A.T base-pair lifetimes of d-CGCGA[TA]5TCGCG, a model duplex of poly[d(A-T)].poly[d(A-T)]. The d(A.T) lifetimes are comparable to those of r(A.U) that we reported previously. End effects on base-pair lifetimes are limited to two base-pairs. The low efficiency of exchange catalysts is ascribed to the small dissociation constant of the deoxy base-pairs, and helps to explain why exchange catalysis had been overlooked in the past. This resulted in a hundredfold overestimation of base-pair lifetimes. Cytosine amino proteins have been studied in the duplex of d-CGm5CGCG. Exchange from the closed base-pair is indicated. Hence, the use of an amino exchange rate to evaluate the base-pair dissociation constant would result in erroneous, overestimated values. Catalyzed imino proton exchange is at this time the safest and most powerful, if not the only probe of base-pair kinetics. We propose that the single base-pair opening event characterized here may be the only mode of base-pair disruption, at temperatures well below the melting transition.     

Resolving fast and slow motions in the internal loop containing stem-loop 1 of HIV-1 that are modulated by Mg2+ binding: role in the kissing-duplex structural transition

Stem loop 1 (SL1) is a highly conserved hairpin in the 5′-leader of the human immunodeficiency virus type I that forms a metastable kissing dimer that is converted during viral maturation into a stable duplex with the aid of the nucleocapsid (NC) protein. SL1 contains a highly conserved internal loop that promotes the kissing–duplex transition by a mechanism that remains poorly understood. Using NMR, we characterized internal motions induced by the internal loop in an SL1 monomer that may promote the kissing–duplex transition. This includes micro-to-millisecond secondary structural transitions that cause partial melting of three base-pairs above the internal loop making them key nucleation sites for exchanging strands and nanosecond rigid-body stem motions that can help bring strands into spatial register. We show that while Mg²⁺ binds to the internal loop and arrests these internal motions, it preserves and/or activates local mobility at internal loop residues G272 and G273 which are implicated in NC binding. By stabilizing SL1 without compromising the accessibility of G272 and G273 for NC binding, Mg²⁺ may increase the dependence of the kissing–duplex transition on NC binding thus preventing spontaneous transitions from taking place and ensuring that viral RNA and protein maturation occur in concert.     

Characterization of the Structure and Melting Behavior of the Loop I fragment of ColE1 RNA I

Abstract

We have synthesized two RNA fragments: a 42-mer corresponding to the full loop I sequence of the loop I region of ColE1 antisense RNA (RNA I), plus three additional Gs at the 5′-end, and a 31-mer which has 11 5′-end nucleotides (G(-2)-U9) deleted. The secondary structure of the 42-mer, deduced from one- and two-dimensional NMR spectra, consists of a stem of 11 base-pairs which contains a U-U base-pair and a bulged C base, a 7 nucleotide loop, and a single-stranded 5′ end of 12 nucleotides. The UV-melting study of the 42-mer further revealed a multi-step melting behavior with transition temperatures 32°C and 71°C clearly discernible. In conjunction with NMR melting study the major transition at 71°C is assigned to the overall melting of the stem region and the 32°C transition is assigned to the opening of the loop region. The deduced secondary structure agrees with that proposed for the intact RNA I and provides structural bases for understanding the specificity of RNase E.     

Proton exchange and base-pair lifetimes in a deoxy-duplex containing a purine-pyrimidine step and in the duplex of inverse sequence

Using proton relaxation and magnetization transfer from water we have measured the imino proton exchange kinetics in two dodecadeoxynucleotide duplexes. One is formed by the self-complementary sequence 5'-d(C-C-T-T-T-C-G-A-A-A-G-G), the other by the inverse sequence. The imino proton exchange rates are found to depend on the concentration of ammonia or imidazole, acting as basic catalysts of proton exchange. Extrapolation of exchange times to infinite catalyst concentration yields the base-pair lifetimes, for instance 40 milliseconds for the central G.C base-pair of the 5'-d(C-C-T-T-T-C-G-A-A-A-G-G) duplex and four milliseconds for its A.T neighbour, at 15 degrees C. These results differ markedly from those reported by other laboratories for similar deoxy compounds. An explanation of the discrepancy has been proposed recently. Differences between base-pair lifetimes indicate that opening is not co-operative. From the catalyst efficiency relative to exchange from isolated nucleosides, we estimate the dissociation constant of each base-pair, e.g. 0.3 x 10(-6) and 1.5 x 10(-5) at 15 degrees C, for the same G.C and A.T base-pairs. The lifetime and dissociation constant of corresponding base-pairs of the two duplexes are similar, except for the central G.C base-pair. This correlates with differences in the solution structures reported by others. We have completed the assignments of the imino protons and of the six cytosine amino protons of the 5'-d(G-G-A-A-A-G-C-T-T-T-C-C) 12-mer. A new base-pair numbering scheme is proposed.     

Selection of cleavage site by mammalian tRNA 3' processing endoribonuclease

Mammalian tRNA 3' processing endoribonuclease (3' tRNase) removes 3' trailers from pre-tRNAs by cleaving the RNA immediately downstream of the discriminator nucleotide. Although 3' tRNase can recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA, in some cases cleavage occurs at multiple sites near the discriminator. We investigated what features of pre-tRNA determine the cleavage site using various pre-tRNAArg variants and purified pig enzyme. Because the T stem-loop and the acceptor stem plus a 3' trailer are sufficient for recognition by 3' tRNase, we constructed variants that had additions and/or deletions of base-pairs in the T stem and/or the acceptor stem. Pre-tRNAs lacking one and two acceptor stem base-pairs were cleaved one and two nucleotides and two and three nucleotides, respectively, downstream of the discriminator. On the other hand, pre-tRNA variants containing extra acceptor stem base-pairs were cleaved only after the discriminator. The cleavage site was shifted to one and two nucleotides downstream of the discriminator by deleting one base-pair from the T stem, but was not changed by additional base-pairs in the T stem. Pre-tRNA variants that contained an eight base-pair acceptor stem plus a six base-pair T stem, an eight base-pair acceptor stem plus a four base-pair T stem, or a six base-pair acceptor stem plus a six base-pair T stem were all cleaved after the original nucleotide. In general, pre-tRNA variants containing a total of more than 11 bp in the acceptor stem and the T stem were cleaved only after the discriminator, and pre-tRNA variants with a total of N bp (N is less than 12) were cleaved 12-N and 13-N nt downstream of the discriminator. Cleavage efficiency of the variants decreased depending on the degree of structural changes from the authentic pre-tRNA. This suggests that the numbers of base-pairs of both the acceptor stem and the T stem are important for recognition and cleavage by 3' tRNase.     

Analysis of local helix geometry in three B-DNA decamers and eight dodecamers

Local variations in B-DNA helix structure are compared among three decamers and eight dodecamers, which contain examples of all ten base-pair step types. All pairwise combinations of helix parameters are compared by linear regression analysis, in a search for internal relationships as well as correlations with base sequence. The primary conclusions are: (1) Three-center hydrogen bonds between base-pairs occur frequently in the major groove at C-C, C-A, A-A and A-C steps, but are less convincing at C-C and C-T steps in the minor groove. The requirements for large base-pair propeller are (1) that the base-pair should be A.T rather than G.C, and (2) that it be involved in a major groove three-center hydrogen bond with the following base-pair. Either condition alone is insufficient. Hence, a large propeller is expected at the leading base-pair of A-A and A-C steps, but not at A-T, T-A, C-A or C-C steps. (2) A systematic and quantitative linkage exists between helix variables twist, rise, cup and roll, of such strength that the rise between base-pairs can hardly be described as an independent variable at all. Two typical patterns of behavior are observed at steps from one base-pair to the next: high twist profile (HTP), characterized by high twist, low rise, positive cup and negative roll, and low twist profile (LTP), marked by low twist, high rise; negative cup and positive roll. Examples of HTP are steps G-C, G-A and Y-C-A-R, where Y is pyrimidine and R is purine. Examples of LTP steps are C-G, G-G, A-G and C-A steps other than Y-C-A-R. (3) The minor groove is especially narrow across the two base-pairs of the following steps: A-T, T-A, A-A and G-A. (4) In general, base step geometry cannot be correlated solely with the bases that define the step in question; the two flanking steps also must be taken into account. Hence, local helix structure must be studied in the context, not of two base-pairs: A-B, but of four: x-A-B-y. Calladine's rules, although too simple in detail, were correct in defining the length of sequence over which a given perturbation is expressed. Whereas ten different two-base steps are possible, allowing for the identity of complementary sequences, there are 136 different four-base steps. Only 33 of these 136 four-base steps are represented in the decamer and dodecamer structures solved to date, and hence it is premature to try to set up detailed structural algorithms. (5) The sugar-phosphate backbone chains of B-DNA place strong limits on sequence-induced structural variation, damping down most variables within four or five base-pairs, and preventing purine-purine anti-anti mismatches from causing bulges in the double helix. Hence, although short-range sequence-induced deformations (or deformability) are observed, long-range deformations propagated down the helix are not to be expected.     

Solution structure of a DNA duplex containing 8-hydroxy-2'-deoxyguanosine opposite deoxyguanosine

Deoxyguanosine residues are hydroxylated by reactive oxygen species at the C-8 position to form 8-hydroxy-2'-deoxyguanosine (8-OG), one of the most important mutagenic lesions in DNA. Though the spontaneous G:C to C:G transversions are rare events, the pathways leading to this mutation are not established. An 8-OG:G mispair, if not corrected by DNA repair enzymes, could lead to G:C to C:G transversions. NMR spectroscopy and restrained molecular dynamics calculations are used to refine the solution structure of the base mismatch formed by the 8-OG:G pair on a self complementary DNA dodecamer duplex d(CGCGAATT(8-O)GGCG)(2). The results reveal that the 8-OG base is inserted into the helix and forms Hoogsteen base-pairing with the G on the opposite strand. The 8-OG:G base-pairs are seen to be stabilized by two hydrogen bonding interactions, one between the H7 of the 8-OG and the O6 of the G, and a three-center hydrogen bonding between the O8 of the 8-OG and the imino and amino protons of the G. The 8-OG:G base-pairs are very well stacked between the Watson-Crick base-paired flanking bases. Both strands of the DNA duplex adopt right-handed conformations. All of the unmodified bases, including the G at the lesion site, adopt anti glycosidic torsion angles and form Watson-Crick base-pairs. At the lesion site, the 8-OG residues adopt syn conformations. The structural studies demonstrate that 8-OG(syn):G(anti) forms a stable pair in the interior of the duplex, providing a basis for the in vivo incorporation of G opposite 8-OG. Calculated helical parameters and backbone torsional angles, and the observed 31P chemical shifts, indicate that the structure of the duplex is perturbed near lesion sites, with the local unwinding of the double helix. The melting temperature of the 8-OG:G containing duplex is only 2.6 deg. C less than the t(m) of the unmodified duplex.     

Elucidating a key intermediate in homologous DNA strand exchange: structural characterization of the RecA-triple-stranded DNA complex using fluorescence resonance energy transfer

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA-tsDNA complex), has been reported. We present a systematic characterization of the helical geometries of the three DNA strands of the RecA-tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. FRET donor and acceptor dyes were used to label different DNA strands, and the interfluorophore distances were inferred from energy transfer efficiencies measured as a function of the base-pair separation between the two dyes. The energy transfer efficiencies were first measured on a control RecA-dsDNA complex, and the calculated helical parameters (h approximately 5 A, Omega(h) approximately 20 degrees ) were consistent with structural conclusions derived from electron microscopy (EM) and other classic biochemical methods. Measurements of the helical parameters for the RecA-tsDNA complex revealed that all three DNA strands adopt extended and unwound conformations similar to those of RecA-bound dsDNA. The structural data are consistent with the hypothesis that this complex is a late, post-strand-exchange intermediate with the outgoing strand shifted by about three base-pairs with respect to its registry with the incoming and complementary strands. Furthermore, the bases of the incoming and complementary strands are displaced away from the helix axis toward the minor groove of the heteroduplex, and the bases of the outgoing strand lie in the major groove of the heteroduplex. We present a model for the strand exchange intermediate in which homologous contacts preceding strand exchange arise in the minor groove of the substrate dsDNA.     

Thermodynamics of RNA hairpins containing single internal mismatches.

Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions. Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities. The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site. UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins. Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability. In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities. The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed.