Progressive DNA bending is made possible by gradual changes in the torsion angle of the glycosyl bond.

Structural comparisons have led to the suggestion that the conformational rearrangement that would be required to change A-DNA into the TA-DNA form of DNA observed in the complex with the TATA box binding protein (TBP) could be completed by modifying only the value of the glycosyl bond chi by approximately 45 degrees. The lack of a high number of crystal structures of this type makes it difficult to conclude whether a smooth transition from A-DNA to TA-DNA can occur without disrupting at any point either the Watson-Crick base pairing or the A-DNA conformation of the backbone. To explore the possibility of such a smooth transition, constrained molecular dynamics simulations were carried out for the double-stranded dodecamer d(GGTATATAAAAC), in which a transition from A-DNA to TA-DNA was induced by modifying only the chi angle values. The results demonstrate the feasibility of a continuous path in the A-DNA to TA-DNA transition. Varying extents of DNA curvature are also attainable, by maintaining the A-DNA backbone structure and Watson-Crick hydrogen bonding while changing the chi angle value smoothly from that in A-DNA to one corresponding to B-DNA.     

The influence of tertiary structural restraints on conformational transitions in superhelical DNA.

This paper examines theoretically the effects that restraints on the tertiary structure of a superhelical DNA domain exert on the energetics of linking and the onset of conformational transitions. The most important tertiary constraint arises from the nucleosomal winding of genomic DNA in vivo. Conformational transitions are shown to occur at equilibrium at less extreme superhelicities in DNA whose tertiary structure is restrained than in unrestrained molecules where the residual linking difference alpha res (that part of the superhelical deformation which is not absorbed by transitions) may be freely partitioned between twisting and bending. In the extreme case of a rigidly held tertiary structure, this analysis predicts that the B-Z transition will occur at roughly half the superhelix density needed to drive the same transition in solution, other factors remaining fixed. This suggests that superhelical transitions may occur at more moderate superhelical deformations in vivo than in solution. The influence on transition behavior of the tertiary structural restraints imposed by gel conditions also are discussed.     

A note on sequence-dependence of DNA structure

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.     

A study of the B-A transition in DNA by gel electrophoresis

A procedure is developed for studying the B-A transition in DNA using gel electrophoresis. The starting point has been the idea that the junction between the A and B sections, which appear within the transition interval would increase the mobility of the DNA molecules. Indeed, the mobility of DNA in a gel is shown to increase in the middle of the B-A transition due to the formation of the largest possible number of boundaries between the B and A forms. The middle of the B-A transition in supercoiled DNA appears to be shifted against the middle of the transition in open circular (as well as linear) DNA by about 1.3% towards lower ethanol concentrations under the influence of the superhelical stress.     

Determination of the number of superhelical turns by the hyperchromicity of partially denatured covalently-closed DNA molecules

A novel method of determining the number of superhelical turns of a covalently-closed plasmid DNA is described. It relies on the determination of the hyperchromicity, and hence the proportion of unstacked basepairs, of a partially heat-denatured sample which co-migrates during electrophoresis with nicked circular duplex DNA. The values obtained for plasmid pBR beta G DNA at 4 degrees C (-29.8 and -33.5 in the two buffers used) agree closely with the values obtained in parallel by topoisomer band-counting. Our method is less precise than band-counting but is readily applicable to determining the superhelicity of very large DNA molecules. Our results confirm earlier findings that magnesium-containing buffers cause an increase in the duplex winding angle, and hence an increase in the number of negative superhelical turns.     

A Study of the B-A Transition in DNA by Gel Electrophoresis

Abstract

A procedure is developed for studying the B-A transition in DNA using gel electrophoresis. The starting point has been the idea that the junction between the A and B sections, which appear within the transition interval would increase the mobility of the DNA molecules. Indeed, the mobility of DNA in a gel is shown to increase in the middle of the B-A transition due to the formation of the largest possible number of boundaries between the B and A forms. The middle of the B-A transition in supercoiled DNA appears to be shifted against the middle of the transition in open circular (as well as linear) DNA by about 1.3% towards lower ethanol concentrations under the influence of the superhelical stress.     

Environmental influences on DNA superhelicity. The effect of ionic strength on superhelix conformation in solution

The techniques of small-angle X-ray scattering and analysis that have been developed by the authors are used to investigate the influence of ionic strength on the superhelical conformation of native COP608 plasmid DNA in solution. For salt concentrations below 0.1 M, the superhelicity is partitioned between twisting (Tw) and writhing (Wr) in the ratio delta Tw/Wr = 2. Near the physiological salt concentration, [Na+] = 0.2 M, a co-operative transition is observed in which the pitch angle of the toroidal superhelix is drastically decreased. This results in an almost complete relaxation of writhe. At salt concentrations in excess of the threshold for this transition, the superhelical partitioning occurs in the ratio delta Tw/Wr greater than 25. Energetic considerations support the suggestion that this transition results from co-operative, superhelical B to Z transconformation reactions at susceptible sites. A method is discussed that will enable the direct measurement of this secondary structural transition by means of X-ray scattering.     

Molecular structure of an A-DNA decamer d(ACCGGCCGGT)

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.     

The potentially Z-DNA-forming sequence d(GTGTACAC) crystallizes as A-DNA

(GT)n/(CA)n sequences have stimulated much interest because of their frequent occurrence in eukaryotic DNA and their potential for forming the left-handed Z-DNA structure. We here report the X-ray crystal structure of a self-complementary octadeoxynucleotide, d(GTGTACAC), at 2.5 A resolution. The molecule adopts a right-handed double-helical conformation belonging to the A-DNA family. In this alternating purine-pyrimidine DNA minihelix the roll and twist angles show alternations qualitatively consistent with Calladine's rules. The average tilt angle of 9.3 degrees is between the values found in A-DNA (19 degrees) and B-DNA (-6 degrees) fibers. It is envisaged that such intermediate conformations may render diversity to genomic DNA. The base-pair tilt angles and the base-pair displacements from the helix axis are found to be correlated for the known A-DNA double-helical fragments.     

A reduced set of coordinates for modeling DNA structures: (I). A B-to-A transition pathway driven by pseudorotational angle.

The A-DNA and the B-DNA are two well characterized polymorphous forms of DNA duplex. By using Metropolis Monte Carlo Simulations in a reduced coordinate space, we have shown that the B in equilibrium with A transitions can be induced by forcing pseudorotational angle (W) to change between C3'-endo and C2'-endo puckerings. The energy barrier for the transition pathway is less than 10 Kcal.mol-1. Base-pair parameters x-displacement (Dx) and roll (rho), which have the largest differences between the two forms of structures, cannot drive the transition. Our results support the view that the bistable states of the DNA duplex are due to the bistable structures of the sugar ring.     

Atomic force microscopic measurement of the interdomain angle in symmetric Holliday junctions

The Holliday junction is a key intermediate in genetic recombination. It consists of four DNA strands that associate by base pairing to produce four double helices flanking a junction point. In the presence of multivalent cations, the four helices, in turn, stack in pairs to form two double-helical domains. The angle between these domains has been shown in a number of solution studies to be approximately 60 degrees in junctions flanked by asymmetric sequences. However, the recently determined crystal structure of a symmetric junction [Eichman, B. F., Vargason, J. M., Mooers, B. H. M., and Ho, P. S. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 3971-3976] finds an angle closer to 40 degrees, possibly because of sequence effects. From the crystal structure alone, one cannot exclude the possibility that this unusual angle is a consequence of crystal packing effects. We have formed two-dimensional (2D) periodic arrays of DNA parallelograms with the same junction-flanking sequence used to produce the crystals; these parallelograms are free to adopt their preferred interdomain angle. Atomic force microscopy can be used to establish the interdomain angle in this system. We find that the angle in this junction is 43 degrees, in good agreement with the results of crystallography. We have used hydroxyl radical autofootprinting to establish that the branch point is at the same migratory position seen in the crystals.     

Superhelical turns of closed circular DNA in solution and in a gel: evidence for a conformational difference

We have compared the number of superhelical turns, tau, in circular covalently closed plasmid pBR beta G DNA obtained by four different methods, each based on one particular distinguishing principle. Three of the methods allow an unequivocal determination of tau under gel electrophoresis conditions, whilst the fourth enables us to determine its value in solution. We were able to detect a significant difference between the two environments, corresponding to an unwinding of the DNA duplex angle by 0.3 degrees when a sample is transferred from solution to gel. The possible existence of such an effect has been generally overlooked by previous investigators. Our result suggests that the previously reported value for the number of base-pairs per helical turn should be adjusted downwards by about 0.10, so that it applies to conditions in solution.     

Evidence that a plasmid from a hyperthermophilic archaebacterium is relaxed at physiological temperatures.

A plasmid of 3.45 kb (pGT5) was recently discovered in a strain of hyperthermophilic archaebacterium which was isolated from samples collected in a deep-sea hydrothermal vent. This strain (GE5) grows within a temperature range of 68 to 101.5 degrees C, and we show here that it contains a strong ATP-dependent reverse gyrase activity (positive DNA supercoiling). By comparison with eubacterial plasmids of known superhelical densities, we estimated the superhelical density of the archaebacterial plasmid pGT5 to be -0.026 at 25 degrees C. The equation which relates the change of the rotation angle of the DNA double helix with temperature was validated at 95 degrees C, the optimal growth temperature of the GE5 strain. Considering these new data, the superhelical density of plasmid pGT5 was calculated to be -0.006 at the physiological temperature of 95 degrees C, which is close to the relaxed state. This finding shows that the DNA topology of a plasmid isolated from a hyperthermophilic archaebacterium containing reverse gyrase activity is strikingly different from that of typical eubacterial plasmids.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

Influence of Spermine on DNA Conformation in a Molecular Dynamics Trajectory of d(CGCGAATTCGCG)2: Major Groove Binding by One Spermine Molecule Delays the A→B Transition

Abstract

The effect of spermine on the A-DNA to B-DNA transition in d(CGCGAATTCGCG)₂ has been investigated by five A-start molecular dynamics simulations, using the Cornell et al. potential. In the absence of spermine an A→B transition is initiated immediately and the DNA becomes equidistant from the A- and B-forms at 200ps. In three DNA-spermine simulations, when a spermine is located across the major groove of A-DNA in one of three different initial locations, the time taken to reach equidistance from the A- and B-forms is delayed until 800, 950 or 1000ps. In each case the A-form appears to be temporarily stabilized by spermine's electrostatic interactions with phosphates on both sides of the major groove. The onset of the A→B transition can be correlated with the spermine losing contact with phosphates on one side of the groove and with A-like → B-like sugar pucker transitions in the vicinity of the spermine bridge. However in the fifth trajectory, in which the spermine initially threads from the major groove via the backbone into the minor groove, the B→A transition occurs rapidly once again and the DNA is equidistant between the A- and B-forms within 300ps. This indicates that the mere presence of spermine is insufficient to delay the transition and that major groove binding stabilizes A-DNA.     

Influence of spermine on DNA conformation in a molecular dynamics trajectory of d(CGCGAATTCGCG)(2): major groove binding by one spermine molecule delays the A-->B transition

The effect of spermine on the A-DNA to B-DNA transition in d(CGCGAATTCGCG)(2) has been investigated by five A-start molecular dynamics simulations, using the Cornell et al. potential. In the absence of spermine an A-->B transition is initiated immediately and the DNA becomes equidistant from the A- and B-forms at 200ps. In three DNA-spermine simulations, when a spermine is located across the major groove of A-DNA in one of three different initial locations, the time taken to reach equidistance from the A- and B-forms is delayed until 800, 950 or 1000ps. In each case the A-form appears to be temporarily stabilized by spermine's electrostatic interactions with phosphates on both sides of the major groove. The onset of the A-->B transition can be correlated with the spermine losing contact with phosphates on one side of the groove and with A-like --> B-like sugar pucker transitions in the vicinity of the spermine bridge. However in the fifth trajectory, in which the spermine initially threads from the major groove via the backbone into the minor groove, the B-->A transition occurs rapidly once again and the DNA is equidistant between the A- and B-forms within 300ps. This indicates that the mere presence of spermine is insufficient to delay the transition and that major groove binding stabilizes A-DNA.     

DNA flexibility as a function of allomorphic conformation and of base sequence.

Systematic theoretical modeling of symmetric DNA oligomers, carried out earlier for the B conformation, is now extended to A-DNA. In contrast to the previous results, it is found that A-DNA shows no multiplicity of low-energy substate conformations. The possibilities of the Jumna algorithm are subsequently applied to studying deformations of the oligomers. Controlled winding and stretching deformations are used to study how the two allomorphs and different base sequences absorb such external stress. The results help explain the internal mechanics of the DNA double helix and the extent to which fine structure influences this behavior. The results point to some differences between the A and B double helices, but also to many similarities. Sequence effects on flexibility are relatively limited compared to their impact on optimal energy conformations. It is also shown that the conformational substates detected for B-DNA oligomers are preserved under deformation, but have little influence on its energetics.     

Infrared linear dichroism studies of DNA-drug complexes: quantitative determination of the drug-induced restriction of the B-A transition.

The B-A transition of films or fibers of NaDNA occurs at a relative humidity of 75-85%. The fraction of DNA that changed the conformation from B to A form can be determined quantitatively by infrared linear dichroism. DNA-binding drugs can 'freeze' a fraction of DNA in the B form. This fraction of DNA is in the B form and cannot be converted to A-DNA even at a reduced relative humidity of 54%. The 'freezing' potentiality of various drugs can be described by the 'freezing' index, FI, expressed in base pairs per added drug. Drugs with a high value of FI (more than eight base pairs per drug) were observed among both intercalating and groove-binding drugs. High values of FI imply restriction of the conformational flexibility of DNA significantly going beyond the binding site of the drug. This long-range effect of drugs on the conformational flexibility of DNA may be connected with the molecular mechanism of drug action. The freezing index FI is a new quantitative parameter of drug-DNA interaction that should be considered as a valuable tool for drug design.     

Unpaired bases in the B--Z junction of the supercoiled plasmid

The restriction analysis has been used to establish that O-beta-diethylaminoethylhydroxylamine (OHA) produces modification of unpaired cytidines in the polylinker region adjacent to the Z-insert (dG-dC)10. (dG-dC)10 in the negatively supercoiled plasmid pGC20. The length of the transition region between B- and Z-portions of DNA is not less than 36 bps. The reaction of OHA with the unpaired cytidines in the B-Z junction is a fixing one and produces no secondary despiralling of the neighboring regions. The reaction with DNA proceeds much slower than the one with monomers and single-strand polynucleotides. The structural nonuniformity has been observed, which is manifested in the alternating B and "non-B" form DNA in the B-Z junction. It is suggested that these junctions may contain nucleotide sequences which are stable to violation of the B structure during the change in superhelical density of DNA.