Structural transitions of deoxyribonucleic acid in aqueous electrolyte solutions. II. The role of hydration.

The data and approach reported in paper I (Hanlon et al., 1975, preceding paper) have been used to calculate the fractional changes in secondary structure of calf thymus deoxyribonucleic acid which occur in aqueous solutions as a function of the concentration of NaCl, KCl, LiCl, CsCl, and NH4Cl. There is a continuous loss in the "B" character of the nucleic acid with concomitant production of the C and, in some instances, an A form, as well, as the salt concentration increases. Sedimentation velocity studies suggest that there is an accompanying change in the hydrodynamic characteristics of the DNA molecules, as well. Utilizing the existing hydration data in the literature (Hearst and Vinograd, 1961a,b; Hearst, 1965; Tunis and Hearst, 1968a; Cohen and Eisenberg, 1968; Falk et al., 1962, 1963a,b), we have found that a gradual loss of "B" character and a decrease in the frictional coefficient of DNA occur as the net hydration of DNA is reduced from the fully hydrated from (60-80 mol of H2O/mol of nucleotide) to values of ca. 12-14 mol of H2O/mol of nucleotide. Below that value, a more precipitous decrease in these properties occurs. Extrapolation of the linear relationship observed between the fractional B content and the net hydration in the latter regions yield values of ca. 18 mol of H2O/mol of nucleotide at 100% B and ca. 4 mol of H2O/mol of nucleotide at 0% B (i.e., 100% C or C + A) for the alkali metal salts of DNA. The ammonium salt retains somewhat more H2O in the C and A forms (ca. 7). These results together with the hydration site assignments of Falk et al. (1962, 1963a,b) are interpreted in terms of a hydration model for DNA in aqueous solution in which an intact primary hydration shell of ca. 18 mol of H2O/mol of nucleotide is required for the maintenance of the "B" conformation. Removal of all but those water molecules solvating the phosphate groups results in the conversion to the C forms, predominantly, with a small amount of A structure formed as well in some salts. The accompanying changes in the sedimentation coefficients suggest that the DNA molecule assumes a more compact and/or flexible form under these conditions in which it is mainly in the C and A structures. The combination of these two events which ensue upon dehydration create a polymeric structure which can be more easily packaged in biological systems.     

Binding of netropsin to several DNA constructs: evidence for at least two different 1:1 complexes formed from an -AATT-containing ds-DNA construct and a single minor groove binding ligand

Isothermal titration calorimetry, ITC, has been used to determine the thermodynamics (DeltaG, DeltaH, and -TDeltaS) for binding netropsin to a number of DNA constructs. The DNA constructs included: six different 20-22mer hairpin forming sequences and an 8-mer DNA forming a duplex dimer. All DNA constructs had a single -AT-rich netropsin binding with one of the following sequences, (A(2)T(2))(2), (ATAT)(2), or (AAAA/TTTT). Binding energetics are less dependent on site sequence than on changes in the neighboring single stranded DNA (hairpin loop size and tail length). All of the 1:1 complexes exhibit an enthalpy change that is dependent on the fractional saturation of the binding site. Later binding ligands interact with a significantly more favorable enthalpy change (partial differential DeltaH(1-2) from 2 to 6 kcal/mol) and a significantly less favorable entropy change (partial differential (-TDeltaS(1-2))) from -4 to -9 kcal/mol). The ITC data could only be fit within expected experimental error by use of a thermodynamic model that includes two independent binding processes with a combined stoichiometry of 1 mol of ligand per 1 mol of oligonucleotide. Based on the biophysical evidence reported here, including theoretical calculations for the energetics of "trapping" or structuring of a single water molecule and molecular docking computations, it is proposed that there are two modes by which flexible ligands can bind in the minor groove of duplex DNA. The higher affinity binding mode is for netropsin to lay along the floor of the minor groove in a bent conformation and exclude all water from the groove. The slightly weaker binding mode is for the netropsin molecule to have a slightly more linear conformation and for the required curvature to be the result of a water molecule that bridges between the floor of the minor groove and two of the amidino nitrogens located at one end of the bound netropsin molecule.     

Entropy and heat capacity of DNA melting from temperature dependence of single molecule stretching

When a single molecule of double-stranded DNA is stretched beyond its B-form contour length, the measured force shows a highly cooperative overstretching transition. We have measured the force at which this transition occurs as a function of temperature. To do this, single molecules of DNA were captured between two polystyrene beads in an optical tweezers apparatus. As the temperature of the solution surrounding a captured molecule was increased from 11 degrees C to 52 degrees C in 500 mM NaCl, the overstretching transition force decreased from 69 pN to 50 pN. This reduction is attributed to a decrease in the stability of the DNA double helix with increasing temperature. These results quantitatively agree with a model that asserts that DNA melting occurs during the overstretching transition. With this model, the data may be analyzed to obtain the change in the melting entropy DeltaS of DNA with temperature. The observed nonlinear temperature dependence of DeltaS is a result of the positive change in heat capacity of DNA upon melting, which we determine from our stretching measurements to be DeltaC(p) = 60 +/- 10 cal/mol K bp, in agreement with calorimetric measurements.     

Intercalative binding to DNA of antitumour drugs derived from 3-nitro-1,8-naphthalic acid.

Two new antitumour drugs, imide derivatives of 3-nitro-1,8-naphthalic acid having different basic side chains linked to the imide nitrogen, have been shown to bind to double-helical DNA by intercalation. At ionic strength 0.01 mol/litre, pH 7, their intrinsic association constants are about 1.45 x 10(5) M-1 and each bound ligand molecule occludes about 3.4 nucleotides of the DNA lattice. They remove and reverse the supercoiling of closed circular duplex PM2 DNA with apparent unwinding angles of 11-12 degrees per bound drug molecule, referred to an assumed unwinding angle of 26 degrees for ethidium. They increase the viscosity of sonicated rod-like DNA fragments, each bound drug molecule producing a calculated increment in length of 2.2 - 2.5 A. No important differences between the DNA-binding characteristics of the two drugs were detected, though one appears marginally more active than the other in certain biological tests.     

Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.     

Simian virus 40 DNA replication: characterization of gaps in the termination region.

A class of precursor DNA (pDNA) II molecules has been identified as the immediate precursor of simian virus 40 DNA I. A pDNA II molecule contains a strand of newly synthesized DNA with an interruption located in the region where DNA synthesis terminates (4). These pDNA II molecules have been isolated and further characterized. They are converted to covalently closed structures (simian virus 40 DNA I) only when they are treated in vitro with both T4 DNA polymerase and Escherichia coli ligase. After in vitro repair of pDNA II with T4 DNA polymerase and nucleoside triphosphates, approximately 7 mol of alpha-[32P]dATP is incorporated per mol of DNA II. Alkaline sucrose analysis of these gap-filled molecules, after they have been cleaved with Eco RI restriction endonuclease, has demonstrated that gaps are specifically located in the termination region. alpha-[32P]dATP is incorporated equally into the two labeled products that are generated by RI cleavage of these molecules. This indicates the presence of gaps in both the newly synthesized plus the minus strands. Electrophoretic analysis of the gap-filled molecules, after they have been cleaved with endonuclease Hind, has shown that gaps are localized in Hind fragments G and B and to a minor degree in fragment J. pDNA II molecules have the following properties. There is a gap in the newly synthesized linear DNA strand contained in the pDNA II molecule. Nicked pDNA II molecules cannot be detected. The two molecules that arise by segregation contain gaps in both of the complementary strands. Based on the amount of alpha-[32P]dATP incorporated and the rate of exonuclease III digestion of gap-filled molecules, it is estimated that the size of the gaps is between 22 and 73 nucleotides. Models for termination of DNA synthesis are proposed based on these findings.     

Atomic force microscopic study on topological structures of pBR322 DNA

Plasmid pBR322 DNA (0.5mg/mL) isolated from Escherichia coli HB101 was suspended in Tris-HCl-EDTA (1 mol/L - 0.1 mol/L, pH8.5); then a drop of the above solution was deposited on freshly cleaved mica substrate. After adsorption for about 1 min, the sample was stained with phosphotungstic acid. The residua] solution was removed with a piece of filter paper. Afterwards the sample was imaged with a home-made atomic force microscope (AFM) in air. The AFM images of pBR322 DNA with a molecular resolution have been obtained. These images show that pBR322 DNA exists in several different topological structures: (i) relaxed circular DNA with a different diameter; (ii) supercondensed DNA with different particle sizes; (iii) dimeric catenane connected by one relaxed circular molecule and another dose-compacted molecule which might be either supercoiled or intramolecular knotted form; (iv) oligomeric catenane with multiple irregular molecules in which DNA is interlocked into a complex oligomer; (v) possibly-existing     

The binding of echinomycin to deoxyribonucleic acid.

Echinomycin is a peptide antibiotic which binds strongly to double-helical DNA up to a limit of approximately one molecule per five base-pairs. There is no detectable interaction with rRNA and only extremely feeble non-specific interaction with poly(rA)-poly(rU). Heat denaturation of DNA greatly decreases the binding, and similarly limited interaction is observed with naturally occurring single-stranded DNA. Association constants for binding to nine double-helical DNA species from different sources are presented; they vary by a factor of approximately 10, but are not simply related to the gross base composition. The interaction with DNA is ionic-strength-dependent, the binding constant falling by a factor of 4 when the ionic strength is raised from 0.01 to 0.10mol/litre. From the effect of temperature on the association constant for calf thymus DNA, the enthalpy of interaction is calculated to be about -13kJ/mol (-3kcal/mol). Binding of echinomycin persists in CsCl gradients and the buoyant density of nicked bacteriophage PM2 DNA is decreased by 25 mg/ml. Echinomycin interacts strongly with certain synthetic poly-deoxynucleotides, the binding constant decreasing in the order poly(dG)-poly(dC) greater than poly(dG-dC) greater than poly(dA-dT). For the latter two polymers the number of base-pairs occluded per bound antibiotic molecule is calculated to be three, whereas for poly(dG)-poly(dC) it is estimated to be four to five. Poly(dA)-poly(dT) and poly(dI)-poly(dC) interact only very weakly with the antibiotic. Poly(dI-dC) interacts to a slightly greater extent, but the binding curve is quite unlike that seen with the three strongly binding synthetic polynucleotides. Echinomycin affects the supercoiling of closed circular duplex bacteriophage PM2 DNA in the characteristic fashion of intercalating drugs. At low ionic strength the unwinding angle is almost twice that of ethidium. Likewise the extension of the helix, determined from changes in the viscosity of rod-like sonicated DNA fragments, is nearly double that expected for a simple (monofunctional) intercalation process. On this basis the interaction process is characterized as bifunctional intercalation. At higher ionic strength the unwinding angle relative to that of ethidium and the helix extension per bound echinomycin molecule fall, indicating a smooth progression towards more nearly monofunctional intercalation. Two simpler compounds which act as analogues of the quinoxaline chromophores of echinomycin, quinoxaline-2-carboxamide and the trypanocidal drug Bayer 7602, interact with DNA very much more weakly than does echinomycin, showing that the peptide portion of the antibiotic plays an essential role in determining the strength and specificity of the interaction.     

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA-protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.     

Chemical modification of simian virus 40 DNA by reaction with a water-soluble carbodiimide.

Superhelical simian virus 40 (SV40) DNA I can be modified with N-cyclohexyl-N'-beta-(4 methylmorpholinium)ethylcarbodiimide (CMC). The reaction produces an increase in the sedimentation velocity of DNA I from 21 to 22.5S and a decrease in its buoyant density in CsCl from 1.694 to 1.688. A comparable shift in buoyant density is observed in a saturated ethidium bromide-cesium chloride gradient where form II, which has been exposed to CMC, shows no shift. The CsCl-buoyant density data allows us to estimate that 108 mol of CMC are bound per mol of SV40 DNA I. In the subsequent paper an alternative procedure has been used to locate CMC sites, and the extent of the regions available to bind CMC have been measured.     

Different modes of interaction between hydrated magnesium ion and DNA functional groups: database analysis and ab initio studies

Role of Magnesium ion is well substantiated in DNA structure and function though the appropriate nature of DNA magnesium interaction is still not fully established. We have analyzed available DNA crystal structures in presence of magnesium ion, which show the experimental evidences for various interaction modes between DNA molecule and magnesium ion. Two preferred modes are found: direct coordinating interaction between magnesium ion and electronegative DNA atoms, and the secondary mode of interaction via formation of hydrogen bonds. This qualitative data is further supported by ab initio quantum chemical calculations using restricted Hartree-Fock and Density Functional Theory. We have analyzed the energies and partial charges of different DNA fragments and hydrated magnesium ions, following restrained and unrestrained geometry optimizations along the reaction coordinate. The restrained optimizations for the systems generally show two energy minima separated by an energy barrier, the height ranges from about 5 to 15 kcal/mol, which is in agreement with experimental observations. All these analyses suggest that both modes of interactions occur almost with equal probability, although water mediated secondary mode of interaction is preferred in most cases, which was so far neglected.     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

Kinetic mechanism of cytosine DNA methyltransferase MspI.

A kinetic analysis of MspI DNA methyltransferase (M.MspI) is presented. The enzyme catalyzes methylation of lambda-DNA, a 50-kilobase pair linear molecule with multiple M.MspI-specific sites, with a specificity constant (kcat/KM) of 0.9 x 10(8) M-1 s-1. But the values of the specificity constants for the smaller DNA substrates (121 and 1459 base pairs (bp)) with single methylation target or with multiple targets (sonicated lambda-DNA) were less by an order of magnitude. Product inhibition of the M.MspI-catalyzed methylation reaction by methylated DNA is competitive with respect to DNA and noncompetitive with respect to S-adenosylmethionine (AdoMet). The S-adenosylhomocysteine inhibition of the methylation reaction is competitive with respect to AdoMet and uncompetitive with respect to DNA. The presteady state kinetic analysis showed a burst of product formation when AdoMet was added to the enzyme preincubated with the substrate DNA. The burst is followed by a constant rate of product formation (0.06 mol per mol of enzyme s-1) which is similar to catalytic constants (kcat = approximately 0.056 s-1) measured under steady state conditions. The isotope exchange in chasing the labeled methyltransferase-DNA complex with unlabeled DNA and AdoMet leads to a reduced burst as compared with the one involving chase with labeled DNA and AdoMet. The enzyme is capable of exchanging tritium at C-5 of target cytosine in the substrate DNA in the absence of cofactor AdoMet. The kinetic data are consistent with an ordered Bi Bi mechanism for the M.MspI-catalyzed DNA methylation where DNA binds first.     

Thermostability of double-stranded deoxyribonucleic acids: effects of covalent additions of a psoralen

We have carried out a thermodynamic study on the effects of covalent additions of the psoralen derivative HMT, 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, on the stability of double-stranded deoxyoligonucleotides. This was done with two systems. The first was a double-stranded DNA formed by two non-self-complementary oligonucleotides, 5'-GAAGCTACGAGC-3' and 5'-GCTCGTAGCTTC-3', where we site specifically placed an HMT molecule on the thymidine residue in oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The second was a double-stranded DNA formed by a self-complementary oligonucleotide, 5'-GGGTACCC-3', where we placed an HMT molecule on the thymidine residue of each strand as a furan-side monoadduct or cross-linked the two strands with an HMT molecule linked to the two thymidines. We found that HMT cross-linking of the two strands stabilizes the double helix formed by 5'-GGGTACCC-3', as one might expect. Less predictable results were that the monoaddition of a psoralen stabilizes the double helix formed by the two non-self-complementary oligonucleotides by as much as 1.3 kcal/mol as a furan-side monoadduct and 0.7 kcal/mol as a pyrone-side monoadduct at 25 degrees C in 50 mM NaCl. In contrast, the monoaddition of a psoralen on each of the two thymidines in the double helix formed by 5'-GGGTACCC-3' destabilizes the helix by 1.8 kcal/mol at 25 degrees C in 1 M NaCl. This destabilization arises from an unfavorable enthalpy change (8.6 kcal/mol) and a favorable entropy change (23 cal/K X mol) due to the two HMT molecules at the centers of each strand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stoichiometric uptake of molecular oxygen and consumption of sulfhydryl groups by neocarzinostatin chromophore bound to DNA

In the presence of DNA, and under conditions which resulted in efficient DNA degradation, the reaction of the neocarzinostatin chromophore with sulfhydryl groups was accompanied by a rapid drop in the oxygen tension of the solution. The total extent of oxygen uptake indicated that, consistently, 1 mol of O2 was consumed/mol of chromophore. The rate of oxygen uptake, however, was strongly dependent on the sulfhydryl concentration, and uptake occurred within a few seconds of the sulfhydryl-induced increase in 420-nm fluorescence of the chromophore. Parallel experiments, in which the sulfhydryl concentration of the solution was monitored, showed that approximately 2 mol of sulfhydryl groups were consumed/mol of chromophore, with kinetics similar to those of O2 uptake. Under anaerobic conditions, only 1 mol of sulfhydryl was consumed, but the sulfhydryl-induced fluorescence increase was not inhibited. These results suggest that (i) a reaction with a single sulfhydryl group converts the chromophore to an activated form, (ii) in the presence of DNA this activated chromophore participates in a subsequent reaction which consumes 1 mol of O2 followed by an additional mole of sulfhydryl, and (iii) each chromophore molecule undergoes only one such reaction cycle. In the absence of sulfhydryl groups, the chromophore slowly degraded, giving a product with intense 490-nm fluorescence. This spontaneous degradation reaction, which does not result in DNA damage, was also accompanied by uptake of nearly 1 mol of O2/mol of chromophore.     

Plastid origin of an extrachromosomal DNA molecule from Plasmodium, the causative agent of malaria.

Several species of Plasmodium have been shown to contain a circular extrachromosomal DNA molecule which is widely supposed to be mitochondrial DNA. However, it has recently been shown to have a number of features in common with chloroplast DNA. Here, a phylogenetic analysis of RNA polymerase coding sequences from the Plasmodium molecule has been carried out using distance matrix, maximum likelihood, parsimony and operator invariant methods. The analysis indicates that the molecule is in fact derived from an oxygenic photosynthetic organism and should be regarded as plastid DNA. This suggests that Plasmodium originated from a phototroph that has lost the capacity to photosynthesize.     

The killer toxin of Kluyveromyces lactis: characterization of the toxin subunits and identification of the genes which encode them.

The killer character of the yeast Kluyveromyces lactis is associated with the presence of the linear DNA plasmids k1 and k2 and results from the secretion of a protein toxin into the growth medium. We find that toxin activity co-purifies with three polypeptides which we have termed the alpha- (mol. wt 99,000), beta- (mol. wt 30,000) and gamma- (mol. wt 27,500) subunits. The alpha-subunit appears to contain a single asparagine-linked oligosaccharide chain but neither of the smaller subunits is glycosylated. The N-terminal amino acid sequence of each subunit has been determined. Comparison of these data with the DNA sequence of plasmid k1 indicates that it encodes all three subunits. The alpha- and beta-subunits must be processed from the primary translation product of a single gene by an enzyme related to the KEX2 endopeptidase of Saccharomyces cerevisiae.