Structure of a triple helical DNA with a triplex-duplex junction

Extended purine sequences on a DNA strand can lead to the formation of triplex DNA in which the third strand runs parallel to the purine strand. Triplex DNA structures have been proposed to play a role in gene expression and recombination and also have potential application as antisense inhibitors of gene expression. Triplex structures have been studied in solution by NMR, but have hitherto resisted attempts at crystallization. Here, we report a novel design of DNA sequences, which allows the first crystallographic study of DNA segment containing triplexes and its junction with a duplex. In the 1.8 A resolution structure, the sugar-phosphate backbone of the third strand is parallel to the purine-rich strand. The bases of the third strand associate with the Watson and Crick duplex via Hoogsteen-type interactions, resulting in three consecutive C(+).GC, BU.ABU (BU = 5-bromouracil), and C(+).GC triplets. The overall conformation of the DNA triplex has some similarity to the B-form, but is distinct from both A- and B-forms. There are large changes in the phosphate backbone torsion angles (particularly gamma) of the purine strand, probably due to the electrostatic interactions between the phosphate groups and the protonated cytosine. These changes narrow the minor groove width of the purine-Hoogsteen strands and may represent sequence-specific structural variations of the DNA triplex.     

Molecular mechanics studies of parallel and antiparallel phosphate-methylated DNA

Methylation of phosphate groups in oligo-dT strands leads to a parallel duplex with T · T base pairs. Molecular mechanics calculations on parallel d(TTTTTT)₂ show it to be a symmetric right-handed helix with B-DNA conformational characteristics. Phosphate methylation stabilizes the duplex by ca. 41 kcal/mol, due to removal of the interstrand phosphate electrostatic repulsions. The chirality introduced with phosphate methylation is important for the molecular geometry, since R_P methylation predominantly influences the conformation around the ζ bond (P? O_3′), while S_P methylation mostly changes the α conformation (P? O_5′). This is also true in antiparallel helices with methylated phosphates, as is shown by molecular mechanics calculations on d(GCGCGC)₂. These results may be of relevance to protein–DNA interactions, where phosphate charges are also shielded. As the pro-S_P oxygen is most available in a right-handed helix, we suggest changes around the α bond to occur upon protein complexation, leading to a widening of the major groove in the d(GCGCGC)₂ duplex (from 12 to 13 Å) and reduced minor groove (from 6 to 5 Å).     

Molecular dynamics simulation study of DNA triplex formed by mixed sequences in solution

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC).d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usualcut-of of the long range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other ligands.     

Molecular Dynamics Simulation Study of DNA Triplex Formed by Mixed Sequences in Solution

Abstract

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC)?d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usual “cut-off” of the long—range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence—dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other legends.     

Probing structural factors stabilizing antisense oligonucleotide duplexes: NMR studies of a DNA.DNA duplex containing a formacetal linkage.

The duplex formed by annealing the formacetal backbone modified dodecamer d-(CGCGTTOCH2OTTGCGC) to its complementary strand, d(GCGCAAAACGCG) (duplex I), has been studied by NMR techniques and analyzed with reference to its unmodified counterpart (duplex II). Comparison of parameters such as 2D cross-peak intensities, coupling constants, and spectral patterns indicates that structural perturbations caused by the incorporation of the formacetal linkage are minimal and localized to the central T4.A4 block. Duplex I adopts a B-type helical conformation with regular Watson-Crick base pairing and normal minor groove width. The methylene group is accommodated along the phosphate backbone in a conformation similar to that of the PO2 group found in the B-form DNA family. The central T6-T7 base pairs of duplex I melt simultaneously with the duplex, indicating a cooperative transition to single strands. Although the formacetal linkage affects global melting, as evidenced by a 3 degree C reduction in Tm for duplex I with respect to duplex II, the present study indicates that this is not the result of localized premelting at the formacetal site of duplex I but rather reflects the subtle interplay of several structural and energy factors which need to be further explored.     

The contribution of phosphate-phosphate repulsions to the free energy of DNA bending

DNA bending is important for the packaging of genetic material, regulation of gene expression and interaction of nucleic acids with proteins. Consequently, it is of considerable interest to quantify the energetic factors that must be overcome to induce bending of DNA, such as base stacking and phosphate–phosphate repulsions. In the present work, the electrostatic contribution of phosphate–phosphate repulsions to the free energy of bending DNA is examined for 71 bp linear and bent-form model structures. The bent DNA model was based on the crystallographic structure of a full turn of DNA in a nucleosome core particle. A Green's function approach based on a linear-scaling smooth conductor-like screening model was applied to ascertain the contribution of individual phosphate–phosphate repulsions and overall electrostatic stabilization in aqueous solution. The effect of charge neutralization by site-bound ions was considered using Monte Carlo simulation to characterize the distribution of ion occupations and contribution of phosphate repulsions to the free energy of bending as a function of counterion load. The calculations predict that the phosphate–phosphate repulsions account for ~30% of the total free energy required to bend DNA from canonical linear B-form into the conformation found in the nucleosome core particle.     

Prediction of the structure of the Y+.R-.R(+)-type DNA triple helix by molecular modelling.

Molecular mechanics has been used to predict the structure of the Y+.R-.R(+)-type DNA triple helix, in which a second polypurine strand binds antiparallel to the homopurine strand of a homopurine/homopyrimidine stretch of duplex DNA. From calculations on the sequence d(C)10.d(G)10.d(G)10, two likely structures emerge. One has the glycosidic torsions of the third strand bases in the anti-conformation and Hoogsteen hydrogen-bonds to the purine strand of the duplex, the other has the third strand purines in the syn orientation and uses a reverse-Hoogsteen hydrogen-bonding pattern. Despite the large structural differences between these two types of triplex, calculations performed in vacuo with a distance-dependent dielectric constant to mimic the shielding effect of solvent show them to be energetically very similar, with the latter (syn) slightly preferred. However, if explicit solvent molecules are included in the calculation, the anti conformation is found to be much preferred. This difference in the results seems to stem from an underestimation of short-range electrostatic interactions in the in vacuo simulations. When TAA or TAT base triples are substituted for the sixth CGG triple in the sequence, it is found that, for the solvated model, the third strand base of the TAA triple prefers the syn orientation while that in the TAT triple retains a preference, though reduced, for the anti conformation.     

Binding of netropsin to a DNA triple helix.

The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.     

Concomitant reconstitution of TraI-catalyzed DNA transesterase and DNA helicase activity in vitro

TraI protein of plasmid R1 possesses two activities, a DNA transesterase and a highly processive 5'-3' DNA helicase, which are essential for bacterial conjugation. Regulation of the functional domains of the enzyme is poorly understood. TraI cleaves supercoiled oriT DNA with site and strand specificity in vitro but fails to initiate unwinding from this site (nic). The helicase requires an extended region of adjacent single-stranded DNA to enter the duplex, yet interaction of purified TraI with oriT DNA alone or as an integral part of the IncF relaxosome does not melt sufficient duplex to load the helicase. This study aims to gain insights into the controlled initiation of both TraI-catalyzed activities. Linear double-stranded DNA substrates with a central region of sequence heterogeneity were used to trap defined lengths of R1 oriT sequence in unwound conformation. Concomitant reconstitution of TraI DNA transesterase and helicase activities was observed. Efficient helicase activity was measured on substrates containing 60 bases of open duplex but not on substrates containing < or =30 bases in open conformation. The additional presence of auxiliary DNA-binding proteins TraY and Escherichia coli integration host factor did not stimulate TraI activities on these substrates. This model system offers a novel approach to investigate factors controlling helicase loading and the directionality of DNA unwinding from nic.     

Homology-dependent changes in adenosine 5'-triphosphate hydrolysis during recA protein promoted DNA strand exchange: evidence for long paranemic complexes

As a first step in DNA strand exchange, recA protein forms a filamentous complex on single-stranded DNA (ssDNA), which contains stoichiometric (one recA monomer per four nucleotides) amounts of recA protein. recA protein monomers within this complex hydrolyze ATP with a turnover number of 25 min-1. Upon introduction of linear homologous duplex DNA to initiate strand exchange, this rate of ATP hydrolysis drops by 33%. The decrease in rate is complete in less than 2 min, and the rate of ATP hydrolysis then remains constant during and subsequent to the strand exchange reaction. This drop is completely dependent upon homology in the duplex DNA. In addition, the magnitude of the drop is linearly dependent upon the length of the homologous region in the linear duplex DNA. Linear DNA substrates in which pairing is topologically restricted to a paranemic joint also follow this relationship. Taken together, these properties imply that all of the available homology in the incoming duplex DNA is detected very early in the DNA strand exchange reaction, with the linear duplex DNA paired paranemically with the homologous ssDNA in the complex throughout its length. The results indicate that paranemic joints can extend over thousands of base pairs. We note elsewhere [Pugh, B. F., & Cox, M. M. (1987b) J. Biol. Chem. 262, 1337-1343] that this duplex acquires resistance to digestion by DNase with a much slower time course (30 min), which parallels the progress of strand exchange. Together these results imply that the duplex DNA is paired with the ssDNA but remains outside the nucleoprotein filament. Finally, the results also support the notion that ATP hydrolysis occurs throughout the recA nucleoprotein filament.     

Stable binding of recA protein to duplex DNA. Unraveling a paradox

recA protein binding to duplex DNA is a complicated, multistep process. The final product of this process is a stably bound complex of recA protein and extensively unwound double-stranded DNA. recA monomers within the complex hydrolyze ATP with an apparent kcat of approximately 19-22 min-1. Once the final binding state is achieved, binding and ATP hydrolysis by this complex becomes pH independent. The weak binding of recA protein to duplex DNA reported in previous studies does not, therefore, reflect an intrinsically unfavorable binding equilibrium. Instead, this apparent weak binding reflects a slow step in the association pathway. The rate-limiting step in this process involves the initiation rather than the propagation of DNA binding and unwinding. This step exhibits no dependence on recA protein concentration at pH 7.5. Extension or propagation of the recA filament is fast relative to the overall process. Initiation of binding is pH dependent and represents a prominent kinetic barrier at pH 7.5. ATP hydrolysis occurs only after the duplex DNA is unwound. The binding density of recA protein on double-stranded DNA is approximately one monomer/4 base pairs. A model for this process is presented. These results provide an explanation for several paradoxical observations about recA protein-promoted DNA strand exchange. In particular, they demonstrate that there is no thermodynamic requirement for dissociation of recA protein from the heteroduplex DNA product of strand exchange.     

Thermodynamics of polymolecular duplexes between phosphate-methylated DNA and natural DNA

Phosphate-methylated (P.M.) DNA possesses a very high affinity for complementary natural DNA, as a result of the absence of interstrand electrostatic repulsions. In this study, a model system phosphate-methylated d[Cn] with natural d(Gk) (n less than k) is chosen for an investigation of the thermodynamic properties that determine duplex stability. The enthalpy change of a melting transition is shown to be considerably larger than is observed for corresponding natural DNA duplexes. It is found that delta Hn0 of GG/CC nearest neighbor pairwise interaction equals -15.6 kcal/mol, compared to -11.0 kcal/mol for the natural analog. The entropy change is strongly dependent on the length of the natural DNA strand and the number of phosphate-methylated DNA oligomers hybridized. The results are explained by means of a model in which a cooperative effect for subsequent hybridizations of phosphate-methylated DNA oligomers is assumed, thus giving additional stability.     

DNA strand exchange and selective DNA annealing promoted by the human immunodeficiency virus type 1 nucleocapsid protein.

Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.     

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) shows significant differences to that found in solution.

The crystal structure of the RNA/DNA hybrid r(GAAGAGAAGC). d(GCTTCTCTTC) has been solved and refined at 2.5 A resolution. The refinement procedure converged at R = 0.181 for all reflections in the range 20.0-2.5 A. In the crystal, the RNA/DNA hybrid duplex has an A' conformation with all but one of the nucleotide sugar moieties adopting a C3'- endo (N) conformation. Both strands in the double helix adopt a global conformation close to the A-form and the width of the minor groove is typical of that found in the crystal structures of other A-form duplexes. However, differences are observed between the RNA and DNA strands that make up the hybrid at the local level. In the central portion of the duplex, the RNA strand has backbone alpha, beta and gamma torsion angles that alternate between the normal gauche -/ trans / gauche + conformation and an unusual trans / trans / trans conformation. Coupled with this so-called 'alpha/gamma flipping' of the backbone torsion angles, the distance between adjacent phosphorous atoms on the RNA strand systematically varies. Neither of these phenomena are observed on the DNA strand. The structure of the RNA/DNA hybrid presented here differs significantly from that found in solution for this and other sequences. Possible reasons for these differences and their implications for the current model of RNase H activity are discussed.     

DNA adopts normal B-form upon incorporation of highly fluorescent DNA base analogue tC: NMR structure and UV-Vis spectroscopy characterization

The influence of the highly fluorescent tricyclic cytosine base analogue (tC) on duplex DNA conformation is investigated. The duplex properties are characterized by absorbance and circular dichroism (CD) for all combinations of neighbouring bases to tC, and an NMR structure is determined for one tC-containing sequence. For the oligonucleotides with one tC incorporated instead of cytosine, the melting temperature is increased on average by 2.7°C above that for the unmodified ones. CD spectra are practically identical for modified and unmodified sequences, indicating an unperturbed B-DNA conformation. The NMR structure determination of the self-complementary sequence 5′-CTC(tC)ACGTGGAG shows a DNA conformation consistent with B-form for the whole duplex. The root-mean-square distance for the nucleotides of the eight central base pairs between the 10 structures with lowest CYANA target functions and a mean structure is 0.45 ± 0.17 Å. The NMR data confirm correct base pairing for tC by the observation of both intrastrand and interstrand imino proton NOEs. Altogether, this suggests that tC works well as a cytosine analogue, i.e. it is situated in the base stack, forming hydrogen bonds with G in the complementary strand, without distorting the DNA backbone conformation. This first example of an artificial, highly fluorescent DNA base that does not perturb the DNA conformation could have valuable applications for the study of the structure and dynamics of nucleic acid systems.     

Formation of joint DNA molecules by two eukaryotic strand exchange proteins does not require melting of a DNA duplex

We have examined whether DNA strand exchange activities from nuclear extracts of HeLa cells or Drosophila melanogaster embryos have detectable helicase or melting activities. The partially purified recombinases have been shown to recognize homologous single strand and double strand DNA molecules and form joint molecules in a DNA strand exchange reaction. The joint molecule product consists of a linear duplex joined at one end by a region of DNA heteroduplex to a homologous single strand circular DNA. Using two different partially duplex helicase substrates, we are unable to detect any melting of duplex regions under conditions that promote joint molecule formation. One substrate consists of a 32P-labeled oligonucleotide 20 or 30 bases long annealed to M13mp18 circular single strand DNA. The second substrate consists of a linear single strand region flanked at each end by short duplex regions. We observe that even in the presence of excess recombinase protein or after prolonged incubation no helicase activity is apparent. Control experiments rule out the possibility that a helicase is masked by reannealing of displaced single strand fragments. Based on these findings and other data, we conclude that the human and D. melanogaster recombinases recognize and pair homologous sequences without significant melting of duplex DNA prior to strand exchange.