Short electric-field pulses convert DNA from "condensed" to "free" conformation

Electric-field pulses of e.g. 20 kV/cm and 100 μs induce a strong decrease in the scattered light intensity of DNA condensed by spermine. Analysis of this effect demonstrates that the decrease of the scattered light intensity results from decondensation of DNA. The decondensation reaction requires an electric-field strength exceeding a threshold value. Complete decondensation can be achieved at field strength that are only slightly higher than the threshold value. The decondensation process is strongly accelerated at high electric-field strengths. At 30 kV/cm, the decondensation time constant is ～8 μs, corresponding to an acceleation factor of 10⁵ relative to the field-free decondensation reaction. The dependence of the time constants on the electric-field strength suggests that the field-induced decondensation is due to a dissociation field effect. The condensation process observed after electric-field pulses at low concentrations of DNA and spermine shows a characteristic induction period, which strongly depends on the spermine concentration. This induction period reflects the time required for the binding of spermine to DNA, until the degree of binding is sufficiently high for the condensation reaction. The fast dissociation of condensed DNA by electric-field pulses together with a relatively long lifetime of the free DNA results in a reaction cycle resembling a hysteresis loop.     

Nature of protamine-DNA complexes. A special type of ligand binding co-operativity

The mode of protamine binding to DNA double helices has been analyzed for the example of clupein Z from herring and DNA samples from bacteriophages lambda and PM2 by measurements of light-scattering intensities, ultracentrifugation and kinetics. The light-scattering intensity of DNA increases co-operatively at a threshold clupein concentration suggesting co-operative binding of clupein to double helices. These data are first analyzed in terms of a model with a transition at a threshold degree of binding. The parameters resulting from this analysis appear to be reasonable, but are shown to be in contrast with data on the absolute degree of clupein binding to DNA obtained by centrifugation experiments. An analysis of the kinetics associated with clupein binding to DNA by measurements of the time-dependence of light-scattering intensities in the time range of seconds demonstrates directly that clupein-induced intermolecular interactions of DNA molecules are essential. The rate constants of DNA association increase co-operatively at threshold clupein concentrations, which correspond to those observed in the equilibrium titrations. Above the threshold, the rate constants arrive at a level that is almost constant, but shows some decrease with increasing clupein concentrations. These results are described by a model with a monomer and a dimer state of DNA, which bind ligands with different affinities according to an excluded-site binding scheme. When the ligand binding constant is larger for the dimer than for the monomer state, as should be expected, binding of ligands drives the DNA from the monomer to the dimer state, even if the dimerization equilibrium in the absence of ligands is far in favor of the monomer. The transition from the monomer to the dimer state proves to be strongly co-operative. When the ligand concentration is increased to higher values, the dimers may be converted back to monomers due to an increased extent of ligand binding to the monomer state. The model is consistent with the available experimental data. The analysis of the data by the model indicates the existence of a reaction unit much below the DNA chain length, corresponding to about 80 nucleotide residues. The present model describes ligand driven intermolecular association; an analogous model is applicable to ligand driven intramolecular association. In summary, the co-operativity of clupein binding to DNA double helices is not due to nearest neighbor interactions, but results from thermodynamic coupling of clupein binding with clupein-induced DNA association.     

Spermine Condenses DNA,but Not RNA Duplexes

Interactions between the polyamine spermine and nucleic acids drive important cellular processes. Spermine condenses DNA and some RNAs, such as poly(rA):poly(rU). A large fraction of the spermine present in cells is bound to RNA but apparently does not condense it. Here, we study the effect of spermine binding to short duplex RNA and DNA, and compare our findings with predictions of molecular-dynamics simulations. When small numbers of spermine are introduced, RNA with a designed sequence containing a mixture of 14 GC pairs and 11 AU pairs resists condensation relative to DNA of an equivalent sequence or to 25 bp poly(rA):poly(rU) RNA. A comparison of wide-angle x-ray scattering profiles with simulation results suggests that spermine is sequestered deep within the major groove of mixed-sequence RNA. This prevents condensation by limiting opportunities to bridge to other molecules and stabilizes the RNA by locking it into a particular conformation. In contrast, for DNA, simulations suggest that spermine binds externally to the duplex, offering opportunities for intermolecular interaction. The goal of this study is to explain how RNA can remain soluble and available for interaction with other molecules in the cell despite the presence of spermine at concentrations high enough to precipitate DNA.     

How environmental solution conditions determine the compaction velocity of single DNA molecules

Understanding the mechanisms of DNA compaction is becoming increasingly important for gene therapy and nanotechnology DNA applications. The kinetics of the compaction velocity of single DNA molecules was studied using two non-protein condensation systems, poly(ethylene glycol) (PEG) with Mg(2+) for the polymer-salt-induced condensation system and spermine for the polyamine condensation system. The compaction velocities of single tandem λ-DNA molecules were measured at various PEG and spermine concentrations by video fluorescent microscopy. Single DNA molecules were observed using a molecular stretching technique in the microfluidic flow. The results show that the compaction velocity of a single DNA molecule was proportional to the PEG or spermine concentration to the power of a half. Theoretical considerations indicate that the compaction velocity is related to differences in the free energy of a single DNA molecule between the random coil and compacted states. In the compaction kinetics with PEG, acceleration of the compaction velocity occurred above the overlap concentration while considerable deceleration occurred during the coexistence state of the random coil and the compacted conformation. This study demonstrates the control factors of DNA compaction kinetics and contributes toward the understanding of the compaction mechanisms of non-protein DNA interactions as well as DNA-protein interactions in vivo.     

Studies on the selectivity of DNA precipitation by spermine.

We have examined the selectivity of the precipitation of DNA by spermine. We have found that the intra- and intermolecular condensation of DNA induced by spermine is highly selective even in the presence of added protein or triphosphates. We have also investigated the influence of buffer components on the threshold concentration of spermine required for DNA precipitation. Representative applications exploiting the selectivity of the precipitation reaction are also described.     

Protein-bound water molecule counting by resolution of (1)H spin-lattice relaxation mechanisms

Water proton spin-lattice relaxation is studied in dilute solutions of bovine serum albumin as a function of magnetic field strength, oxygen concentration, and solvent deuteration. In contrast to previous studies conducted at high protein concentrations, the observed relaxation dispersion is accurately Lorentzian with an effective correlation time of 41 +/- 3 ns when measured at low proton and low protein concentrations to minimize protein aggregation. Elimination of oxygen flattens the relaxation dispersion profile above the rotational inflection frequency, nearly eliminating the high field tail previously attributed to a distribution of exchange times for either whole water molecules or individual protons at the protein-water interface. The small high-field dispersion that remains is attributed to motion of the bound water molecules on the protein or to internal protein motions on a time scale of order one ns. Measurements as a function of isotope composition permit separation of intramolecular and intermolecular relaxation contributions. The magnitude of the intramolecular proton-proton relaxation rate constant is interpreted in terms of 25 +/- 4 water molecules that are bound rigidly to the protein for a time long compared with the rotational correlation time of 42 ns. This number of bound water molecules neglects the possibility of local motions of the water in the binding site; inclusion of these effects may increase the number of bound water molecules by 50%.     

An electro-optical study of the mechanisms of DNA condensation induced by spermine

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.     

Condensation prevails over B-A transition in the structure of DNA at low humidity

B-A transition and DNA condensation are processes regulated by base sequence and water activity. The constraints imposed by interhelical interactions in condensation compromise the observation of the mechanism by which B and A base-stacking modes influence the global state of the molecule. We used a single-molecule approach to prevent aggregation and mechanical force to control the intramolecular chain association involved in condensation. Force-extension experiments with optical tweezers revealed that DNA stretches as B-DNA under ethanol and spermine concentrations that favor the A-form. Moreover, we found no contour-length change compatible with a cooperative transition between the A and B forms within the intrinsic-force regime. Experiments performed at constant force in the entropic-force regime with magnetic tweezers similarly did not show a bistable contraction of the molecules that could be attributed to the B-A transition when the physiological buffer was replaced by a water-ethanol mixture. A total, stepwise collapse was found instead, which is characteristic of DNA condensation. Therefore, a low-humidity-induced change from the B- to the A-form base-stacking alone does not lead to a contour-length shortening. These results support a mechanism for the B-A transition in which low-humidity conditions locally change the base-stacking arrangement and globally induce DNA condensation, an effect that may eventually stabilize a molecular contour-length reduction.     

Polyamines stimulate DNA-directed DNA synthesis catalyzed by mammalian type C retroviral DNA polymerases

In the presence of optimal concentrations of Mg2+, rates of activated (gapped) DNA-directed DNA synthesis by purified mammalian type C retroviral DNA polymerases are stimulated greater than 10-fold by the polyamines spermine and spermidine. Such stimulation was not observed using either similar concentrations of the polyamines cadaverine or putrescine or exogenously provided salt or ammonium ions. Avian type C as well as mammalian type B and type D retroviral DNA polymerases, in contrast to the mammalian type C enzyme, were found to be relatively insensitive to spermine and spermidine stimulation. Kinetic analysis of the polyamine stimulation of activated DNA-directed DNA synthesis carried out using spermine and purified Rauscher leukemia virus DNA polymerase revealed at least two distinct mechanisms of activation of DNA synthesis. 1) At DNA concentrations below 2.5 micrograms/ml, spermine appears to interact with the enzyme-DNA complex in order to stimulate synthesis. 2) At DNA concentrations above 2.5 micrograms/ml, increased spermine stimulation is observed which appears to be due to its direct interaction with the activated DNA template resulting in either selective limitation of the formation of "dead-end" enzyme-DNA complexes or its ability to convert such nonproductive enzyme binding sites into productive sites for the initiation of synthetic activity. The addition of spermine to reaction mixtures was found to increase both the apparent Km and Vmax of the activated (gapped) DNA-directed reaction with regard to template concentration.     

Influence of DNA length on spermine-induced condensation. Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Influence of DNA length on spermine-induced condensation: Importance of the bending and stiffening of DNA

Using DNA restriction fragments of 258 to 4362 base-pairs, we have investigated the influence of the DNA length on the condensation process induced by spermine, with the aid of electric dichroism measurements. The 258- and 436 bp fragments condensed into rod-like particles, while the fragments of 748 bp or more condensed into torus-shaped particles. Our results suggest that a DNA molecule longer than the circumference of the toroids observed previously (680 bp) is required to serve as a nucleus for the growth of the condensed particles. The toroids were more stable in the electric field than the rod-shaped particles, suggesting that rapid fluctuations of the bound spermine counterions can provide one of the main attractive forces yielding to the condensation process. Relaxation time data for the 436 bp fragment revealed that the structure of DNA was altered at a spermine concentration as low as one-tenth of that required for condensation: the DNA became bent in the presence of spermine. Moreover, the field strength dependence of the relaxation times, as well as the fitting of the decay curves at 12.5 kV/cm, showed an increase of the stiffness of the DNA double helix upon spermine addition. We estimated that, in the case of DNA condensation by spermine, a decrease in the measured persistence length may occur, irrespective of the DNA flexibility, owing to the bending of the DNA molecule.     

Insight into the molecular recognition of spermine by DNA quadruplexes from an NMR study of the association of spermine with the thrombin‐binding aptamer

The preferred residence sites and the conformation of DNA‐bound polyamines are central to understanding the regulatory roles of polyamines. To this end, we have used a series of selective ¹³C‐edited and selective total correlation spectroscopy‐edited one‐dimensional (1D) nuclear Overhauser effect spectroscopy NMR experiments to determine a number of intramolecular ¹H nuclear Overhauser effect (NOE) connectivities in ¹³C‐labelled spermine bound to the thrombin‐binding aptamer. The results provide evidence that the aptamer‐bound spermine adopts a conformation that optimizes electrostatic and hydrogen bond contacts with the aptamer backbone. The distance between the nitrogen atoms of the central aminobutyl is reduced by an increase in the population of gauche conformers at the C6–C7 bonds, which results in either a curved or S‐shaped spermine conformation. Molecular modelling contributes insight toward the mode of spermine binding of these spermine structures within the narrow grooves of DNA quadruplexes. In each case, the N5 ammonium group makes hydrogen bonds with two nearby phosphates across the narrow groove. Our results have implications for the understanding of chromatin structure and the rational design of quadruplex‐binding drugs. Copyright © 2013 John Wiley & Sons, Ltd.     

Critical amount of oligovalent ion binding required for the B-Z transition of poly (dG-m5dC)

By working at very low Na+ concentrations (1mM and less), the number of bound Mg(2+), cobalt hexamine (3+), and spermine (4+) necessary to induce the B-Z transition of poly (dG-m5dC) has been directly measured. The results show that if as little as 1 cobalt hexamine(3+) or spermine(4+) is bound per 40-50 nucleotides the transition will occur. A greater fraction of bound Mg(2+) is required, 1 bound Mg(2+)/10 nucleotides. The dependence of the transition midpoint concentrations of oligovalent ions on Na+ concentrations suggests that specific ion binding energies, not included in counterion condensation theory, are responsible for the transition.     

Induction of 6-thioguanine resistance in human cells treated with N-acetoxy-2-acetylaminofluorene

Induction of 6-thioguanine resistance was studied in human cells treated with the direct-acting chemical carcinogen N-acetoxy-2-acetylaminofluorene (NA-AAF). At low concentrations (2.5–7.5 μM) induction of resistant clones was linear and followed one-hit kinetics, while at 10 μM the yield of resistant clones was higher and appeared to result from the combination of one-hit and two-hit kinetics. A study of about 50 resistant clones revealed that most had reduced levels of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity (25–85% of controls) and were able to use exogenous hypoxanthine for growth (“Type II mutants,” deMars, 1974); a few had very low HGPRT activity (1–8% of controls) and were unable to use exogenous hypoxanthine (“Type I mutants”). Use of [9-¹⁴C]NA-AAF allowed us to examine the frequency of induction of thioguanine resistance as a function of binding to DNA (μmole AAF/mole DNA-P). Calculations from these data suggest that most “hits” on the HGPRT locus do not result in detectable mutations: At three different levels of binding and induced mutation frequency, the yield was 2.5-3 detectable mutants/10 000 molecules of acetylaminofluorene bound to the HGPRT locus. These data suggest that most bound acetylaminofluorene molecules either produce no change in the primary sequence of DNA (possibly as a result of repair or correct “read through” by the DNA polymerase) or result in changes which are phenotypically undetectable.     

Contribution of hydrophobicity to thermodynamics of ligand-DNA binding and DNA collapse

The importance of understanding the dynamics of DNA condensation is inherent in the biological significance of DNA packaging in cell nuclei, as well as for gene therapy applications. Specifically, the role of ligand hydrophobicity in DNA condensation has received little attention. Considering that only multivalent cations can induce true DNA condensation, previous studies exploring monovalent lipids have been unable to address this question. In this study we have elucidated the contribution of the hydrophobic effect to multivalent cation- and cationic lipid-DNA binding and DNA collapse by studying the thermodynamics of cobalt hexammine-, spermine-, and lipospermine-plasmid DNA binding at different temperatures. Comparable molar heat capacity changes (DeltaC(p)) associated with cobalt hexammine- and spermine-DNA binding (-23.39 cal/mol K and -17.98 cal/mol K, respectively) suggest that upon binding to DNA, there are insignificant changes in the hydration state of the methylene groups in spermine. In contrast, the acyl chain contribution to the DeltaC(p) of lipospermine-DNA binding (DeltaC(p ) = DeltaC(p lipospermine) - DeltaC(p spermine)) is significant (-220.94 cal/mol K). Although lipopermine induces DNA ordering into "tubular" suprastructures, such structures do not assume toroidal dimensions as observed for spermine-DNA complexes. We postulate that a steric barrier posed by the acyl chains in lipospermine precludes packaging of DNA into dimensions comparable to those found in nature.     

Measurement of the absolute temporal coupling between DNA binding and base flipping

The absolute temporal couplings between DNA binding and base flipping were examined for the EcoRI DNA methyltransferase. The binding event (monitored using rhodamine-x fluorescence anisotropy) was monophasic with a second-order on-rate of 1.1 x 10(7) M-1 s-1 相似文献   

Kinetics of the daunomycin--DNA interaction

The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the two slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by two sequential isomerization steps. The six rate constants for this mechanism were extracted from our data by relaxation analysis. The values determined for the six rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using first sodium dodecyl sulfate to dissociate bound drug and second pseudo-first-order conditions to study the fast bimolecular step, provide independent verification of three of the six rate constants. The temperature dependence of four of the six rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. We speculate that the three steps in the proposed mechanism may correspond to a rapid "outside" binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.     

Modeling of DNA condensation and decondensation caused by ligand binding

A theoretical method for computer modeling of DNA condensation caused by ligand binding is developed. In the method, starting (s) and condensed (c) states are characterized by different free energies for ligand free DNA (F(s) and F(c) respectively), ligand binding constants (K(s) and K(c)) and stoichiometry dependent parameters (c(sm) and c(cm) - maximum relative concentration of bound ligands (per base pair) for starting and condensed state respectively). The method allows computation of the dependence of the degree of condensation (the fraction of condensed DNA molecules) on ligand concentration. Calculations demonstrate that condensation transition occurs under an increase in ligand concentration if F(s) < F(c) (i.e. S(sc) = exp [- (F(c) - F(s)) / (RT)], the equilibrium constant of the s-c transition, is low (S(sc) < 1)) and K(s) < K(c). It was also found that condensation is followed by decondensation at high ligand concentration if the condensed DNA state provides the number of sites for ligand binding less than the starting state (c(sm) > c(cm)). A similar condensation-decondensation effect was found in recent experimental studies. We propose its simple explanation.     

Radioprotection of human cell nuclear DNA by polyamines: radiosensitivity of chromatin is influenced by tightly bound spermine

The polyamines putrescine (PUT) and spermine (SPM) were examined for their ability to protect human cell DNA against the formation of radiation-induced double-strand breaks (DSBs). As observed previously, under conditions where polyamines were shown to be almost completely absent, association with nuclear matrix protein into a nucleoid, and organization into chromatin structure, protected DNA from induction of DSBs by factors of 4.5 and 95, respectively. At concentrations below 1 mM, PUT or SPM provided equivalent levels of protection to deproteinized nuclear DNA, consistent with their capacity to scavenge radiation-induced radicals. At constant ionic strength, 5 mM SPM protected deproteinized DNA and nucleoid DNA and DNA in nuclear chromatin by factors of 100 and 26, respectively. At 5 mM, SPM provided 15 times greater protection of deproteinized DNA than did PUT. Under physiologically relevant conditions, 5 mM SPM protected DNA in the intact nucleus from the induction of DSBs by a factor of 2 relative to DNA in the absence of SPM. Studies of SPM binding during cellular fractionation revealed that a significant fraction of the cellular SPM is tightly bound in the nucleus but can be removed by extended washing. Thus the association of SPM with nuclear chromatin appears to be a significant contributor to the resistance of the cell's DNA to the induction of DSBs.     

Distamycin A affects the stability of NF-kappaB p50-DNA complexes in a sequence-dependent manner

The effect of two different DNA minor groove binding molecules, Hoechst 33258 and distamycin A, on the binding kinetics of NF-kappaB p50 to three different specific DNA sequences was studied at various salt concentrations. Distamycin A was shown to significantly increase the dissociation rate constant of p50 from the sequences PRDII (5'-GGGAAATTCC-3') and Ig-kappa B (5'-GGGACTTTCC-3') but had a negligible effect on the dissociation from the palindromic target-kappaB binding site (5'-GGGAATTCCC-3'). By comparison, the effect of Hoechst 33258 on binding of p50 to each sequence was found to be minimal. The dissociation rates for the protein--DNA complexes increased at higher potassium chloride concentrations for the PRDII and Ig-kappaB binding motifs and this effect was magnified by distamycin A. In contrast, p50 bound to the palindromic target-kappaB site with a much higher intrinsic affinity and exhibited a significantly reduced salt dependence of binding over the ionic strength range studied, retaining a K(D) of less than 10 pM at 150 mM KCl. Our results demonstrate that the DNA binding kinetics of p50 and their salt dependence is strongly sequence-dependent and, in addition, that the binding of p50 to DNA can be influenced by the addition of minor groove-binding drugs in a sequence-dependent manner.