Breathing and bending fluctuations in DNA modeled by an open-base-pair kink coupled to axial compression

We develop a model designed to show that flexibility in the DNA molecule can arise from relatively improbable transient opening of base pairs. The axial direction changes at the site of an open base pair. The region between open base pairs is a double helix of hydrogen-bonded base pairs with a slightly decreased rise per residue and a slightly increased helical winding angle. An analysis of the model yields several testable predictions. For example, we predict probability 0.026 for a base pair to be open at 25°C, a value close to that measured by hydrogen-exchange experiments. Other predictions involve matters like the variation of persistence length with ionic strength and temperature, the variation of helical winding angle with temperature, and the kinetics of heat denaturation. An additional result of the analysis is an explanation of the high degree of local stiffness of the DNA molecule. Strong resistance to bending fluctuations is provided from two sources: increased polyelectrolyte repulsion among phosphate groups in the axially compressed stacks between open base pairs and the tendency of stacking forces to oppose opening of a base pair. Stacking forces, however, also support compression of the stacks between open base pairs, so that the net effect of stacking forces on elastic bending of DNA is small relative to the polyelectrolyte effect. If the ionic charges on the phosphate groups were absent, DNA would spontaneously fold, driven by the entropy gained when about 1% of its base pairs open.     

Sequence effects on the propeller twist of base pairs in DNA helices.

Molecular mechanics calculations are performed on all the ten base pair steps (duplex dimers) and also a number of trimer and tetramer duplexes comprising them, in an attempt to systematically examine the possible base sequence effects on the magnitudes of propeller twists of base pairs at a given step. The analysis reveals that though propeller twist is a base pair property, it behaves very much like other base step parameters such as slide, roll, twist etc., Hence, it may be necessary to monitor the nature and variation of magnitudes of pt at a step. Calculations performed on 45 out of the 136 unique tetramer combinations involving all the ten unique base steps show that the difference in magnitudes of propeller twists of the base pairs of a given step has been found to be either steep or moderate depending on base pairs that flank the base step. These observations compare very well with the available experimental data. Tetramer sequences, wherein a base pair of a base step repeats in the same direction, exhibit a relatively steep difference in propeller twist at the step. Tetramers other than these exhibit moderate difference in propeller twist. Such sequences are broadly classified as type-I and type-II respectively. Practically all the tetrads considered in the study, excepting those with GT step and a few involving CG and GC steps, conform to the above classification.     

Studies of DNA dumbbells VII: evaluation of the next-nearest-neighbor sequence-dependent interactions in duplex DNA

Melting experiments were conducted on 22 DNA dumbbells as a function of solvent ionic strength from 25-115 mM Na(+). The dumbbell molecules have short duplex regions comprised of 16-20 base pairs linked on both ends by T(4) single-strand loops. Only the 4-8 central base pairs of the dumbbell stems differ for different molecules, and the six base pairs on both sides of the central sequence and adjoining loops on both ends are the same in every molecule. Results of melting analysis on the 22 new DNA dumbbells are combined with our previous results on 17 other DNA dumbbells, with stem lengths containing from 14-18 base pairs, reported in the first article of this series (Doktycz, Goldstein, Paner, Gallo, and Benight, Biopoly 32, 1992, 849-864). The combination of results comprises a database of optical melting parameters for 39 DNA dumbbells in ionic strengths from 25-115 mM Na(+). This database is employed to evaluate the thermodynamics of singlet, doublet, and triplet sequence-dependent interactions in duplex DNA. Analysis of the 25 mM Na(+) data reveals the existence of significant sequence-dependent triplet or next-nearest-neighbor interactions. The enthalpy of these interactions is evaluated for all possible triplets. Some of the triplet enthalpy values are less than the uncertainty in their evaluation, indicating no measurable interaction for that particular sequence. This finding suggests that the thermodynamic stability of duplex DNA depends on solvent ionic strength in a sequence-dependent manner. As a part of the analysis, the nearest-neighbor (base pair doublet) interactions in 55, 85, and 115 mM Na(+) are also reevaluated from the larger database.     

Melting of short DNA fragments. Relation between the nucleation constant of a spiral region and the ionic strength

Melting of two DNA duplexes of known nucleotide sequences containing 14 and 36 base pairs has been investigated within the range of ionic strength from 0.2 to 0.02 M [Na+]. The values of melting enthalpy of base pair delta H were measured for the duplex of 14 base pairs in the solutions of varying ionic strength. The values of delta H were obtained from slopes of linear plots of reciprocal melting temperature versus logarithm of oligonucleotide chains concentration. In the aforementioned range the decrease of the ionic strength causes a 5% decrease of delta H. By fitting the theoretical profiles to the experimental ones the ionic strength dependence of the nucleation constant beta was measured for DNA fragments of various lengths. With the decrease of the ionic strength the value of beta drops 2 times for the short duplex and 8 times for the long one.     

Sequence Effects On The Propeller Twist Of Base Pairs In DNA Helices

Abstract

Molecular mechanics calculations are performed on all the ten base pair steps (duplex dimers) and also a number of trimer and tetramer duplexes comprising them, in an attempt to systematically examine the possible base sequence effects on the magnitudes of propeller twists of base pairs at a given step. The analysis reveals that though propeller twist is a base pair property, it behaves very much like other base step parameters such as slide, roll, twist etc., Hence, it may be necessary to monitor the nature and variation of magnitudes of pt at a step. Calculations performed on 45 out of the 136 unique tetramer combinations involving all the ten unique base steps show that the difference in magnitudes of propeller twists of the base pairs of a given step has been found to be either steep or moderate depending on base pairs that flank the base step. These observations compare very well with the available experimental data. Tetramer sequences, wherein abase pair of a base step repeats in the same direction, exhibit a relatively steep difference in propeller twist at the step. Tetramers other than these exhibit moderate difference in propeller twist Such sequences are broadly classified as type-I and type-II respectively. Practically all the tetrads considered in the study, excepting those with GT step and a few involving CG and GC steps, conform to the above classification.     

Proton nuclear magnetic resonance investigations of fraying in double-stranded d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpApT have been monitored at low ionic strength and in the presence of Mg ions at neutral pH in aqueous solution to determine the thermodynamic parameters associated with fraying (D. J. Patel (1974), Biochemistry 13, 2396) at the terminal and internal base pairs as a function of temperature and pH. From studies in H2O-MeOH (3:2), the fraying process persists down to approximately -20 degrees for the internal TA base pair and down to and probably beyond -30 degrees for the terminal AT base pair. The observed average chemical shift at each of these base pairs as a function of temperature suggests rapid exchange on the nuclear magnetic resonance (NMR) time scale between helix and coil (chemical shift separation of 3.2 ppm) and have been utilized to determine the dissociation constant at the terminal and internal base pairs. Comparison of the reaction enthalpies elucidated from the chemical shift parameters with those reported from optical studies suggests that the symmetry related internal TA base pairs break in a coupled manner at low ionic strength, with the coupling removed in the presence of Mg ions and high salt. By contrast, the symmetry related terminal AT base pairs break independently of each other in the absence and presence of Mg ions and high salt. The terminal base pair exhibits a Tm of 10-15 degrees lower than that of the internal base pair in the hexanucleotide, with divalent Mg ions and high salt stabilizing the double helix as reflected in the Tm values of these base pairs. The observed line width changes as a function of temperature provide an estimate of the exchange rate of the proton from the coil form with water. The exchange reaction from the coil state is base catalyzed with rate constants in the diffusion limit.     

DNA homoduplexes containing no pyrimidine nucleotide

We show using polyacrylamide gel electrophoresis that guanine+adenine repeat strands of DNA associate into homoduplexes at neutral pH and moderate ionic strength. The homoduplexes melt in a cooperative way like the Watson-Crick duplex, although they contain no Watson-Crick base pair. Guanine is absolutely needed for the homoduplex formation and the homoduplex stability increases with the guanine content of the repeat. The present results have implications for the nature of the first replicators, as well as regarding forces stabilizing the duplexes of DNA.     

Dynamics of spiking neurons connected by both inhibitory and electrical coupling

We study the dynamics of a pair of intrinsically oscillating leaky integrate-and-fire neurons (identical and noise-free) connected by combinations of electrical and inhibitory coupling. We use the theory of weakly coupled oscillators to examine how synchronization patterns are influenced by cellular properties (intrinsic frequency and the strength of spikes) and coupling parameters (speed of synapses and coupling strengths). We find that, when inhibitory synapses are fast and the electrotonic effect of the suprathreshold portion of the spike is large, increasing the strength of weak electrical coupling promotes synchrony. Conversely, when inhibitory synapses are slow and the electrotonic effect of the suprathreshold portion of the spike is small, increasing the strength of weak electrical coupling promotes antisynchrony (see Fig. 10). Furthermore, our results indicate that, given a fixed total coupling strength, either electrical coupling alone or inhibition alone is better at enhancing neural synchrony than a combination of electrical and inhibitory coupling. We also show that these results extend to moderate coupling strengths.     

Analysis of cooperativity and ion effects in the interaction of quinacrine with DNA

The interaction of quinacrine with calf thymus DNA was monitored at several different ionic strengths using spectrophotometric and equilibrium dialysis techniques. The binding results can be explained, assuming each base pair is a potential binding site, using a model containing two negative cooperative effects: (1) ligand exclusion at binding sites adjacent to a filled binding site and (2) ligand–ligand negative cooperativity at adjacent filled binding sites. The logarithm of the observed equilibrium constant (K_obs) determined by this model varies linearily with log[Na⁺], as predicted by the ion condensation theory for polyelectrolytes. When the log K_obs plot is correlated for sodium release by DNA in the intercalation conformational change, the predicted number of ion pairs between the ligand and DNA is approximately two, as expected for the quinacrine dication. Even though K_obs depends strongly on ionic strength, the ligand negative cooperativity parameter ω was found to be indpendent of ionic strength within experimental error. This finding is also in agreement with the ion condensation theory, which predicts a relatively constant amount of condensed counterion on the DNA double helix over this ionic strength range. Drugs would, therefore, experience a relatively constant ionic environment when complexed to DNA even though the ionic conditions of the solvent could change considerably.     

Dielectric spectroscopy of single human erythrocytes at physiological ionic strength: dispersion of the cytoplasm.

Usually dielectrophoretic and electrorotation measurements are carried out at low ionic strength to reduce electrolysis and heat production. Such problems are minimized in microelectrode chambers. In a planar ultramicroelectrode chamber fabricated by semiconductor technology, we were able to measure the dielectric properties of human red blood cells in the frequency range from 2 kHz to 200 MHz up to physiological ion concentrations. At low ionic strength, red cells exhibit a typical electrorotation spectrum with an antifield rotation peak at low frequencies and a cofield rotation peak at higher ones. With increasing medium conductivity, both electrorotational peaks shift toward higher frequencies. The cofield peak becomes antifield for conductivities higher than 0.5 S/m. Because the polarizability of the external medium at these ionic strengths becomes similar to that of the cytoplasm, properties can be measured more sensitively. The critical dielectrophoretic frequencies were also determined. From our measurements, in the wide conductivity range from 2 mS/m to 1.5 S/m we propose a single-shell erythrocyte model. This pictures the cell as an oblate spheroid with a long semiaxis of 3.3 microns and an axial ratio of 1:2. Its membrane exhibits a capacitance of 0.997 x 10(-2) F/m2 and a specific conductance of 480 S/m2. The cytoplasmic parameters, a conductivity of 0.4 S/m at a dielectric constant of 212, disperse around 15 MHz to become 0.535 S/m and 50, respectively. We attribute this cytoplasmic dispersion to hemoglobin and cytoplasmic ion properties. In electrorotation measurements at about 60 MHz, an unexpectedly low rotation speed was observed. Around 180 MHz, the speed increased dramatically. By analysis of the electric chamber circuit properties, we were able to show that these effects are not due to cell polarization but are instead caused by a dramatic increase in the chamber field strength around 180 MHz. Although the chamber exhibits a resonance around 180 MHz, the harmonic content of the square-topped driving signals generates distortions of electrorotational spectra at far lower frequencies. Possible technological applications of chamber resonances are mentioned.     

Conformation and interaction of short nucleic acid double-stranded helices. I. Proton magnetic resonance studies on the nonexchangeable protons of ribosyl ApApGpCpUpU.

1H nuclear magnetic resonance (NMR) spectra of a self-complementary ribosyl hexanucleotide, A2GCU2, are investigated as a function of temperature and ionic strength in D2O. Seventeen nonexchangeable base and ribose-H1' resonances are resolved, and unequivocally assigned by a systematic comparison with the spectra of a series of oligonucleotide fragments of the A2GCU2 sequence varying in chain length from 2 to 5. Changes in the chemical shifts of the 17 protons from the hexamer as well as the six H1'-H2' coupling constants are followed throughout a thermally induced helix-coil transition. These sigma vs. T and J vs. T (degrees C) profiles indicate that the transition is not totally cooperative and that substantial populations of partially bonded structures must exist at intermediate temperatures, with the central G-C region being most stable. Transitions in chemical shift for protons in the same base pair exhibit considerable differences in their Tm values as the data reflect both thermodynamic and local magnetic field effects in the structural transition, which are not readily separable. However, an average of the Tm values agrees well with the value predicted from studies of the thermally induced transition made by optical methods. The values of J1'-2' for all six residues become very small (less than 1.5 Hz) at low temperatures indicating that C3'-endo is the most heavily populated furanose conformation in the helix. The sigma values of protons in the duplex were compared with those calculated from the ring current magnetic anisotropies of nearest and next-nearest neighboring bases using the geometrical parameters of the A'-RNA and B-DNA models. The sigma values of the base protons in the duplex calculated assuming the A'-RNA geometry agree (+/- approximately 0.1 ppm) with the observed values much more accurately than those calculated on the basis of B-DNA geometry. The measured sigma values of the H1' are not accurately predicted from either model. The synthesis of 35 mg of A2GCU2 using primer-dependent polynucleotide phosphorylase is described in detail with extensive discussion in the microfilm edition.     

The Myofilament Lattice: Studies on Isolated Fibers : III. The effect of myofilament spacing upon tension

The effect of ionic strength on the generation of tension and upon the interfilament spacing in living intact and skinned single striated muscle fibers from the walking leg of crayfish (Orconectes) were determined by isometric contraction studies correlated with low-angle X-ray diffraction. Sarcomere lengths were determined by light diffraction. Tensions were induced in intact fibers by caffeine in the bathing medium and by ionophoretic microinjection of calcium. Tensions were induced in skinned fibers by a buffered calcium-EGTA solution. The interfilament spacing of intact and skinned fibers over the range of ionic strengths investigated were determined by X-ray diffraction and correlated with the physiological data. It is demonstrated that the ionic strength affects the tension-generating capacity of the muscle as it affects the chemo-mechanical transform of excitation-contraction coupling. It is further demonstrated that interfilament spacing changes encountered during shortening and with variation in the osmotic strength have no effect upon the tension-generating capacity of muscle.     

UvrD helicase unwinds DNA one base pair at a time by a two-part power stroke

Helicases use the energy derived from nucleoside triphosphate hydrolysis to unwind double helices in essentially every metabolic pathway involving nucleic acids. Earlier crystal structures have suggested that DNA helicases translocate along a single-stranded DNA in an inchworm fashion. We report here a series of crystal structures of the UvrD helicase complexed with DNA and ATP hydrolysis intermediates. These structures reveal that ATP binding alone leads to unwinding of 1 base pair by directional rotation and translation of the DNA duplex, and ADP and Pi release leads to translocation of the developing single strand. Thus DNA unwinding is achieved by a two-part power stroke in a combined wrench-and-inchworm mechanism. The rotational angle and translational distance of DNA define the unwinding step to be 1 base pair per ATP hydrolyzed. Finally, a gateway for ssDNA translocation and an alternative strand-displacement mode may explain the varying step sizes reported previously.     

Anisotropic flexibility of DNA and the nucleosomal structure.

Potential energy calculations of the DNA duplex dimeric subunit show that the double helix may be bent in the direction of minor and major grooves much more easily than in other directions. It is found that the total winding angle of DNA decreases upon such bending. A new model for DNA folding in the nucleosome is proposed on the basis of these findings according to which the DNA molecule is kinked each fifth base pair to the side of the minor and major grooves alternatively. The model explains the known contradiction between a C-like circular dichroism for the nucleosomal DNA and the nuclease digestion data, which testify to the B-form of DNA.     

Structure and dynamics of a DNA duplex containing single alpha-anomeric deoxyadenosine residue.

Structure and dynamics of an undecamer DNA duplex containing a single alpha-anomeric deoxyadenosine residue placed in opposition to a thymidine unit have been studied using simulation of molecular dynamics in aqueous solution. Despite several noticeable deviations from the B-DNA duplex structure caused by the anomerisation, such as: West type puckering of the alpha-anomeric sugar, disrupted base stacking pattern and unstable duplex bending, the formation of a non-classical alpha-dA-T pair was observed. A novel way of visual presentation of trajectory data allowing high throughput screening of the conformational parameters is presented.     

The nature of stabilization of the tandem DNA duplex pTGGAGCTG (pCAGC + (Phn-NH-(CH2)3-NH)pTCCA) based on the UV-, CD-, and two-dimensional NMR spectroscopy data

Properties and three-dimensional structure of the tandem DNA duplex pTGGAGCTG.(pCAGC+(PhnL)pTCCA) in aqueous solution, where L is an amino linker and Phn is an N-(2-hydroxyethyl)phenazinium residue, were studied spectrophotometrically and by two-dimensional 1H NMR spectroscopy (COSY and NOESY). When a tandem complex involving a Phn residue-bearing oligonucleotide is formed, the dye aromatic system intercalates into the double helix at the nick site and takes part in two stacking interactions: a strong one (3.5-4 A) with the T5-A12 base pair of its own duplex moiety and a weak one (4-5 A) with the C4-G13 pair of the adjoining duplex (mainly with the C4 base). This arrangement of the dye residue, providing a cross-interaction of the phenazinium moiety with the base pairs of the adjacent duplex structures, results in the stabilization of the whole tandem complex.     

Investigation of the flexibility of DNA using transient electric birefringence

In the preceding article, a Monte Carlo analysis was presented which provides a quantitative numerical relationship between the rotational diffusion coefficients, as measured by the decay of optical anisotropy following an electric field pulse, and the flexibility (persistence length) of short, wormlike chains. In the present article, the results of the foregoing analysis are applied to the observed rates of decay of birefringence for a series of sequenced DNA fragments ranging in size from 104 to 910 base pairs. Under the conditions used in this study, the DNA fragments exist as native, duplex molecules. Furthermore, conditions are defined in which the observed relaxation times are not dependent on DNA concentration, field strength, or the duration of the pulse. It is pointed out that the ionic atmosphere associated with a wormlike polyion does not exert any significant (direct) influence on the rotational diffusion of the polyion and, therefore, that the rotational relaxation times are a true measure of the configurations of the DNA molecules in solution. Moreover, excluded-volume effects are shown not to be significant for the moderately short molecules employed in this study. The major conclusion of this study is that there is no strong ionic strength dependence of the persistence length for ionic strengths above 1 mM and that the persistence length, under conditions where electrostatic contributions are negligible, is approximately 500 Å. For ionic strengths significantly lower than 1 mM, electrostatic contributions to the stiffness of DNA become significant.