The early melting of closed duplex DNA: analysis by banding in buoyant neutral rubidium trichloroacetate.

Aqueous RbTCA permits the buoyant banding of both native and denatured DNA at room temperature and neutral pH. A unique property of this solvent is the bouyant resolution of closed circular, underwound DNA (I) from the corresponding nicked (II) species. Conditions are reported here in which PM-2 DNA I is physically resolved from native PM-2 DNA II, the buoyant separation being 1.27 mq/ml in 3.3 M RbTCA at 25 degrees C. The separation between nicked and closed DNAs increases with temperature up to 35.5 degrees C, at which PM-2 DNA II cooperatively melts and subsequently pellets. The isothermal buoyant density of a cloed DNA increases linearly as the linking number (Lk) of the closed DNA decreases. The early melting of closed DNA may be monitored with high precision by buoyant banding in RbTCA, it being possible to detect the disruption of as few as 40 base pairs in PM-2 DNA (10,000 base pairs). The constraint that the linking number be conserved in closed DNA requires that a change in duplex winding be accompanied by a compensating change in supercoiling. We estimate the linking number deficiency of PM-2 DNA I to be 0.094 turns per decibase pair. This result permits the estimation of the EtdBr unwinding angle, phi, by comparison with alternative determinations of the linking number deficiency which depend upom the value of phi. The result obtained here is that phi = 27.7 degrees +/- 0.5 degrees and is approximately independent of temperature over the range 15 degrees-35 degrees.     

Effect of salt-dependent stiffness on the conformation of a stressed DNA loop containing initially coplanar bends

Closed DNA loops containing one or more bent regions are important structures that occur in the regulation of gene expression. We analyze the response of structures of this type to a change in applied rotation (change in linking deficiency, delta Lk). Our results apply to a closed loop formed from an elastic rod that is intrinsically bent in Nb discrete, 20 degrees steps up to a maximum of 240 degrees, the bent regions being initially coplanar with the plane of the relaxed DNA loop. We determine the effect of changing the intrinsic elastic resistance of the DNA loop to bending and torsional deformations. This relative resistance is expressed by Poisson's ratio v, which depends upon the ratio of bending stiffness to torsional rigidity. Poisson's ratio is primarily a function of salt type and concentration. We find that the tertiary structure of DNA loops changes with delta Lk, but that the geometric response can be either of two quite different types, depending upon the precise (Nb, v) pair. For combinations of Nb and v that are above a critical curve (the Fickel curve), the response to increasing delta Lk is nonmonotonic (NMT region): the distance between the loop closure point and its diametric opposite first decreases, then increases, as delta Lk increases. For combinations of Nb and v that are below the Fickel curve (NMT region), the corresponding diameter never increases, but always decreases with increasing delta Lk. In addition to these results, we define and implement a new measure of tertiary structure in closed DNA: the absolute writhe, AWr.     

Strand opening-deficient Escherichia coli RNA polymerase facilitates investigation of closed complexes with promoter DNA: effects of DNA sequence and temperature

Formation of the strand-separated, open complex between RNA polymerase and a promoter involves several intermediates, the first being the closed complex in which the DNA is fully base-paired. This normally short lived complex has been difficult to study. We have used a mutant Escherichia coli RNA polymerase, deficient in promoter DNA melting, and variants of the P(R) promoter of bacteriophage lambda to model the closed complex intermediate at physiologically relevant temperatures. Our results indicate that in the closed complex, RNA polymerase recognizes base pairs as double-stranded DNA even in the region that becomes single-stranded in the open complex. Additionally, a particular base pair in the -35 region engages in an important interaction with the RNA polymerase, and a DNase I-hypersensitive site, pronounced in the promoter DNA of the open complex, was not present. The effect of temperature on closed complex formation was found to be small over the temperature range from 15 to 37 degrees C. This suggests that low temperature complexes of wild type RNA polymerase and promoter DNA may adequately model the closed complex.     

Physical studies of chloroacetaldehyde labelled fluorescent DNA

The reaction of chloroacetaldehyde with denatured DNA produces a fluorescent DNA where both the adenine and cytosine bases are modified. The rate of modification of DNA by chloroacetaldehyde was measured using the absorption spectrum shift. The depolarization and quantum yield of native DNA and denatured DNA were investigated as a function of temperature.The melting points and the renaturation rates of a series of derivative DNA's were investigated. The melting point was decreased by 1.3°C for each base modified per 100 base pairs corresponding to a 2.8 Kcal destabilizing free energy per mismatched base pair. The renaturation rate of the derivative DNA is reduced by a factor 2 when the melting temperature is lowered by 13°C.     

Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis.

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing urea-formamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.     

Sequence dependence of the stability of RNA hairpin molecules with six nucleotide loops

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the types GCGXUAAUYCGC and GGUXUAAUYACC with Watson-Crick loop closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.1 to 5.1 kcal/mol. These results agree with the model previously developed [Vecenie, C. J., and Serra, M. J. (2004) Biochemistry 43, 11813] to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on the 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Thermodynamic parameters are also reported for hairpin formation in 1 M NaCl by RNA sequence of the types GACGXUAAUYUGUC and GGUXUAAUYGCC with GU base pair closure, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates the free energy of loop formation at 37 degrees C varies from 3.6 to 5.3 kcal/mol. These results allow the development of a model for predicting the stability of hairpin loops closed by GU base pairs. DeltaG degrees (37L(n) (kcal/mol) = DeltaG degrees (37i(n) - 0.8 (if the first mismatch is GA) - 0.8 (if the first mismatch is GG and the loop is closed on the 5' side by a purine). Note that for these hairpins, the stability of the loops does not depend on DeltaG degrees (37MM). For hairpin loops closed by GU base pairs, the DeltaG degrees (37i(n) values, when n = 4, 5, 6, 7, and 8, are 4.9, 5.0, 4.6, 5.0, and 4.8 kcal/mol, respectively. The model gives good agreement when tested against six naturally occurring hairpin sequences. Thermodynamic values for terminal mismatches adjacent to GC, GU, and UG base pairs are also reported.     

Melting experiment concerning the topological structure of closed circular double-stranded DNA

A melting experiment was performed on the whole set of populations of the replicative form of ?X174 DNA, which can be obtained treating this DNA with rat liver nicking-closing enzyme in the presence of ethidium bromide. Gel electrophoresis performed by loading the DNA samples at neutral and alkaline pH allows separation of these populations in discrete sets of bands, which can then be compared. The outcome of the experiments indicates that in the range of electrophoretic mobilities which can be explored, no band is formed exclusively by circular complementary strands which can be separated by alkaline denaturation. These results are compared with what would be expected if double-stranded closed circular DNA had structures other than the canonical double helix. Under nonrestrictive hypotheses, the experiments reported allow one to obtain a minimum estimate of the absolute value of the linking number of a closed circular double-stranded DNA: for native ?X174 RF DNA, the linking number appears to be greater than 12 (in absolute value). Some data on the electrophoretic mobility of denatured closed circular duplexes are reported, which still wait for a physicochemical interpretation.     

Stability of RNA hairpin loops closed by AU base pairs

Thermodynamic parameters are reported for hairpin formation in 1 M NaCl by RNA sequence of the type GCAXUAAUYUGC, where XY is the set of 10 possible mismatch base pairs. A nearest-neighbor analysis of the data indicates that the free energy of loop formation at 37 degrees C varies from 3.2 to 5.0 kcal/mol. These results combined with the model previously developed [Dale et al. (2000) RNA 6, 608] allow improvements in the model to predict the stability of RNA hairpin loops: DeltaG degrees (37L(n) = DeltaG degrees (37i(n)) + DeltaG degrees (37MM) - 0.8 (if first mismatch is GA or UU) - 0.8 (if first mismatch is GG and loop is closed on 5' side by a purine). Here, DeltaG degrees (37i(n) is the free energy for initiating a loop of n nucleotides, and DeltaG degrees (37MM) is the free energy for the interaction of the first mismatch with the closing base pair. Hairpins with GG first mismatches were found to vary in stability depending upon the orientation of the closing base pair (5' or 3' purine relative to the loop). The model gives good agreement when tested against four naturally occurring hairpin sequences.     

Binding of diclofenac sodium with bovine serum albumin at different temperatures, pH and ionic strengths

The study was designed to examine the binding of diclofenac sodium with bovine serum albumin (BSA) at different temperatures (20 degrees, 30 degrees and 40 degrees C), pH (6.4, 7.4 and 8.4) and ionic strengths (micro = 0.1, 0.2 and 0.3) by means of equilibrium dialysis method. The concentration of diclofenac sodium was maintained at wider range from 15 to 900 micromole/l and BSA concentration was maintained at 61.5 micromole/l. The data obtained were interpreted by nonlinear regression method using Graphpad prism software. The analysis showed that the interaction between diclofenac sodium with BSA results in two-site saturable binding. A decrease in association constant was observed with increasing temperature. The average standard free energy change (deltaGdegrees) value was -7.07 (site I) and -4.2 (site II) Kcal/mol. The standard enthalpy change (deltaHdegrees) and the standard entropy change (deltaSdegrees) were -7.8 Kcal/mole, -2.35 cal/mole (site I) and -7.4 Kcal/mole, -10.5 cal/mole (site II), respectively. The negative enthalpy change suggested the binding between diclofenac sodium and the binding sites of BSA were spontaneous and exothermic. The negative value of deltaHdegrees and deltaSdegrees indicated hydrogen bonding and van der Waal's force was the major mechanism for diclofenac sodium and BSA interaction. Increase in pH and ionic strength also caused decrease in association constant of diclofenac sodium and BSA binding.     

DNA Topological Context Affects Access to Eukaryotic DNA Topoisomerase I

Abstract

We have analyzed the reactivity of a 217 base pair segment of the intrinsically curved Crithidia fasciculata kinetoplast DNA towards eukaryotic DNA topoisomerase I. The substrates were open [linear fragment and nicked circle] and closed minidomains [closed relaxed circle and circles with linking differences of ?1 and ?2], We interpreted the results with the aid of a model that was used to predict the structures of the topoisomers. The modelling shows that the ΔLk(?l) form is unusually compact because of the curvature in the DNA. To determine the role of sequence-directed curvature in both the experimental and modeling studies, controls were examined in which the curved Crithidia sequence was replaced by an uncurved sequence obtained from the plasmid pBR322.

Reactivity of the Crithidia DNA [as analyzed both by the cleavage and the topoisomerization reactions] markedly varied among the DNA forms: (i) the hierarchy of overall reactivity observed is: linear fragment > nicked circular, closed circular [ΔLk(O)], interwound [ΔLk(?2)] > bent interwound [ΔLk(?l)]; (ii) the intensity of several cleavage positions differs among DNA forms.

The results show that eukaryotic DNA topoisomerase I is very sensitive to the conformation of the substrates and that its reactivity is modulated by the variation of the compactness of the DNA molecule. The C. fasciculata sequence contains a highly curved segment that determines the conformation of the closed circle in a complex way.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

Thermodynamics of RNA internal loops with a guanosine-guanosine pair adjacent to another noncanonical pair

Thermodynamic parameters measured by optical melting are reported for formation of RNA duplexes containing tandem noncanonical pairs with at least one guanosine-guanosine (GG) pair. For selected sequences, imino proton NMR provides evidence that the desired duplex forms and that the structure of a GG pair adjacent to a noncanonical pair depends on context. A GG pair next to a different noncanonical pair is more stable than expected from measurements of adjacent GG pairs. This is likely due to an unfavorable stacking interaction between adjacent GG pairs, where areas of high negative charge probably overlap. The results suggest a model where tandem noncanonical pairs closed by two GC pairs are assigned the following free energy increments at 37 degrees C: 0.8 kcal/mol for adjacent GG pairs, 1.0 kcal/mol for GG next to UU, and -0.3 kcal/mol for all others. These values are adjusted by 0.65 kcal/mol for each closing AU pair.     

The calculated free energy effects of 5-methyl cytosine on the B to Z transition in DNA

We have examined the free energy effects of 5-methylation of cytosine on the B in equilibrium Z conformational equilibrium in DNA. Free energy differences were calculated using the free energy perturbation approach, which uses an easily derived equation from classical statistical mechanics to relate the free energy difference between two states to the ensemble average of the potential energy difference between the states. Calculations were carried both in explicit solvent and (for comparison) in vacuo. The free energy values obtained for the explicit solvent systems are total free energies, with contributions from all parts of the system (solvent + solute), and so are relevant to the B in equilibrium Z transitions observed under real (physiological) conditions. We calculate that in solution, methylation makes the B in equilibrium Z transition more favorable by about -0.4 kcal/mole base pair (bp) in free energy. This value compares well with approximate experimentally derived values of about -0.3 kcal/mole-bp. We also discuss a method for determining the free energy difference between conformational states poorly maintained by a potential energy model. Finally, the effects of methylation on the melting temperature of DNA are examined.     

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.     

Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides

A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non-Watson-Crick base pair in SECIS element plays an important role in the selenocysteine expression by UGA codon.     

Thermodynamic dependence of DNA/DNA and DNA/RNA hybridization reactions on temperature and ionic strength

The thermodynamics of 5'-ATGCTGATGC-3' binding to its complementary DNA and RNA strands was determined in sodium phosphate buffer under varying conditions of temperature and salt concentration from isothermal titration calorimetry (ITC). The Gibbs free energy change, DeltaG degrees of the DNA hybridization reactions increased by about 6 kJ mol(-1) from 20 degrees C to 37 degrees C and exhibited heat capacity changes of -1.42 +/- 0.09 kJ mol(-1) K(-1) for DNA/DNA and -0.87 +/- 0.05 kJ mol(-1) K(-1) for DNA/RNA. Values of DeltaG degrees decreased non-linearly by 3.5 kJ mol(-1) at 25 degrees C and 6.0 kJ mol(-1) at 37 degrees C with increase in the log of the sodium chloride concentration from 0.10 M to 1.0 M. A near-linear relationship was observed, however, between DeltaG degrees and the activity coefficient of the water component of the salt solutions. The thermodynamic parameters of the hybridization reaction along with the heat capacity changes were combined with thermodynamic contributions from the stacking to unstacking transitions of the single-stranded oligonucleotides from differential scanning calorimetry (DSC) measurements, resulting in good agreement with extrapolation of the free energy changes to 37 degrees C from the melting transition at 56 degrees C.     

Statistical mechanical theory of melting transition in supercoiled DNA

The melting curve for a covalently closed circular DNA has been analyzed on the basis of an expression for the supercoiling energy derived in terms of the elastic parameters of the macromolecule, treated as a homopolymer. The result obtained by applying the usual methods of statistical mechanics indicate close agreement with the available experimental data. It is found that the elevation of the melting temperature, as compared to that of the nicked circular or linear DNA, is a natural consequence of the fact that the supercoiled molecule is constrained by an invariant linking number. The flattening of the melting curve, on the other hand, arises as the closed circular duplex melts into a loose helix rather than random coils.     

Effect of nucleosome distortion on the linking deficiency in relaxed minichromosomes

The wrapping of closed circular DNA on a protein surface, followed by relaxation with a topoisomerase and removal of proteins, produces a characteristic DNA linking deficiency, delta Lk. We show that the magnitude of delta Lk depends upon the surface shape, and we calculate changes in delta Lk caused by particular distortions of the protein wrapping surface. If the DNA remains attached to the surface during distortion, the DNA winding number, phi, is not altered. The change in delta Lk is then equal to the change in the surface linking number, SLk, which is a straightforward measure of the wrapping of the DNA around the surface. For left-handed wrapping, as in a nucleosome, SLk = -n, the number of times that the DNA axis winds around the axis of the protein complex. We calculate values of SLk for the helical wrapping of a constant length of DNA on protein surfaces having the shapes of cylinders and of ellipsoids and hyperboloids of revolution. If the equatorial radius of the protein is fixed, change in shape from a cylinder to a hyperboloid increases SLk, while the corresponding change to an ellipsoid reduces SLk. We apply the general results to the interpretation of experiments in which minichromosomes are relaxed with topoisomerase at various temperatures and delta Lk is determined. The result is that a distortion of the nucleosome core by at most 5% (the change in the radius at the axial extremity relative to the equator) is sufficient to explain the observed delta Lk changes.