Thermal denaturation of nucleohistones--effects of formaldehyde reaction

Thermal denaturation of native or partially dehistonized nucleohistones shows two melting bands at 66 and 81° in 2.5 × 10^?4 M EDTA, pH 8.0. These correspond to the melting of DNA segments bound by the less basic and the more basic half-molecules of histones, respectively. These two melting bands combine into a broad melting band from around 70 to 85° when these nucleohistones are pre-treated with formaldehyde. A formaldehyde reaction which fixes histones on DNA by covalent bonds account for the effect. Formaldehyde fixation also increases the melting temperature of some free DNA segments from around 42 to around 55°. This is interpreted as a result of closed or rigid boundaries between free DNA and formaldehyde-reacted histone-bound DNA segments. MgCl₂ dissociates histones from DNA more effectively and leaves longer free DNA segments than does NaCl. Thermal denaturation of a formaldehyde-reacted nucleoprotein thus provides an effective tool for comparing the relative size of free DNA regions on nucleoproteins. The effect of reversible binding of ligands on helix-coil transition of DNA is descussed and found not adequate for thermal denaturation of nucleohistones.     

Ultraviolet radiation response to the physical properties of the reconstituted nucleohistone

The effect of UV irradiation on the reconstituted nucleohistone have been studied with reference to its (nucleohistone) changes in physical properties, after irradiation at different UV doses. The rate of fall of specific viscosity ratio of the reconstituted nucleohistone as a function of UV dose decreased gradually with the increasing histone to DNA weight ratio (r). This effect, was not observed when the histones remained dissociated from DNA, in high ionic strength (1.5 M NaCl). Histone-DNA complex (r=0.97) irradiated up to a dose of 3.6×10⁴ J/m² had a stable melting temperature unlike free DNA where UV irradiation lowered the melting temperature and the heterogeneous melting profiles were observed. Rate of formaldehyde reaction, with DNA recovered from the irradiated complex, was slower than that with native DNA treated at the same dose. All this suggested that the effect of UV in the DNA of the nucleohistone was less, compared to that in free DNA.     

Equilibrium melting of plasmid ColE1 DNA: electron-microscopic visualization.

The fine structure of the melting curve for the linear colE1 DNA has been obtained. To find the ColE1 DNA regions corresponding to peaks in the melting curve's fine structure, we fixed the melted DNA regions with glyoxal /12/. Electron-microscopic denaturation maps were obtained for nine temperature points within the melting range. Thereby the whole process of colE1 DNA melting was reconstructed in detail. Spectrophotometric and electron microscopic data were used for mapping the distribution of Gc-pairs over the DNA molecule. The most AT-rich DNA regions (28 and 37% of GC-pairs), 380 and 660 bp long resp., are located on both sides of the site of ColE1 DNA's cleavage by EcoR1 endonuclease. The equilibrium denaturation maps are compared with maps obtained by the method of Inman /20/ for eight points of the kinetic curve of ColE1 DNA unwinding by formaldehyde.     

Compensation of DNA stabilization and destabilization effects caused by cisplatin is partially disturbed in alkaline medium

Binding of the antitumor compound cisplatin to DNA locally distorts the double helix. These distortions correlate with a decrease in DNA melting temperature (Tm). However, the influence of cisplatin on DNA stability is more complex because it decreases the DNA charge density. In this way, cisplatin increases the melting temperature and partially compensates for the destabilizing influence of structural distortions. The stabilization is stronger at low Na+ ion concentration. Due to this compensation, the total decrease in the DNA melting temperature after cisplatin binding is much lower than the decrease caused by the distortions themselves, especially at low [Na+]. It is shown in this study that, besides Na+ concentration, pH also strongly influences the value of a change in the melting temperature caused by cisplatin. In alkaline medium (pH=10.5-10.8), a fall in the melting temperature caused by platination is enhanced several times with respect to neutral medium. Such a stronger drop in Tm is explained by a decrease in pK values of base pairs caused by lowering the charge density under platination that facilitates proton release. At neutral pH, the proton release is low for both control and platinated DNA and does not influence the melting behavior. Therefore, lowering in the charge density under platination, besides stabilization, gives additional destabilization just in alkaline medium. Destabilization caused by structural distortions due to this pH induced compensation of stabilizing effect is more pronounced. In the presence of carbonate ion, destabilization caused by high pH value is strengthened. As a decrease in DNA charge density, interstrand crosslinking caused by cisplatin also increases the DNA stability due to loss in the entropy of the melted state. However, computer modeling of DNA stability demonstrates that interstrand crosslinks formed by cisplatin do not stabilize long DNA. It is shown that the increase in Tm caused by interstrand crosslinking itself is compensated for by a local destabilization of the double helix at the sites of location of interstrand crosslinks formed by cisplatin.     

Activity of endonuclease S1 in denaturing solvents: dimethysulfoxide, dimethylformamide, formamide and formaldehyde.

The endonuclease S₁ from $\text{Aspergillus oryzae}$ is shown to be active in aqueous solutions of greater than 60 percent formamide, 50 percent dimethylsulfoxide, 30 percent dimethylformamide and in 2 percent formaldehyde. The melting profile of T7 DNA is determined by S₁ digestion in 50 percent formamide and is found to agree with the optical melting profile. The single-strand specificity of S₁ is retained in 50 percent formamide at 37°C, 3°C below the DNA melting temperature. S₁ is shown to be active at temperatures greater than 5°C above the DNA melting temperature in this solvent.     

Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.     

Dependence of microwave effect on the secondary structure of DNA on molecular weight of polynucleotide

The effect of ultralow power pulse-modulated electromagnetic radiation (average power density 60 microW/cm2, carrying frequency 1.05; 2.12; or 2.39 GHz; modulating pulses with frequency 4 Hz) on the secondary structure of DNA was investigated. It was established that the exposure of beta-alanine and formaldehyde containing aqueous DNA solution to electromagnetic radiation had activated the process of DNA despiralization under the action of beta-alanine--formaldehyde reaction product. The effect of electromagnetic radiation on the secondary structure of DNA can be removed by lowering of molecular weight of DNA to 0.46 x 10(6) (at carrying frequency 1.05 GHz), or to 0.25 x 10(3) (at carrying frequency 2.39 GHz).     

Melting characteristics of highly supercoiled DNA

The effect of high supercoil densities on the melting characteristics of a supercoiled DNA has been studied. It is found that although the melting temperature increases abruptly on converting a linear DNA merely into the relaxed circular form, it falls back substantially at high supercoil densities. It is further predicted, in such cases, that the number of melted base pairs should be significantly enhanced even at the physiological temperature, which may facilitate the binding of other molecules to the highly supercoiled DNA.     

Evidence for sequence-specific conformational changes in DNA from the melting temperatures of DNA phosphorothioate derivatives.

Analogs of alternating purine-pyrimidine DNA polymers such as poly(dA-dT)-poly(dA-dT) can be made with phosphorothioate groups in the DNA backbone. A phosphorothioate diester at the 5'-purine-pyrimidine-3' step causes a significant lowering of the polymer's melting temperature compared to a phosphorothioate diester at the 5'-pyrimidine-purine-3' step. This may occur because sulfur substitution increases anionic charge density in the DNA minor groove and 5'-purine-pyrimidine-3' steps narrow the minor groove. The ability to modulate charge density in the DNA backbone via sulfur substitution should prove useful in studies of sequence-dependent conformational changes in DNA.     

DNA denaturation in situ. Effect of divalent cations and alcohols

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.     

The interaction of DNA with sperm-specific histones of the H1 family

We have studied the interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea starfish Aphelasterias japonica and bivalve mollusk Chlamis islandicus using circular dichroism and DNA melting analysis. It was shown that echinoderm's sperm H1 protein has additional alpha-helical domains in its C-terminus and it demonstrates stronger DNA compaction. The differential melting curves of DNA-protein complexes have two peaks. The low temperature peak characterized the melting temperature of free DNA within the complex. The higher temperature peak characterizes the melting temperature of DNA bond to protein. DNA is found to be in the most stable state in the complexes with mollusk sperm H1 protein.     

Betaine structure and the presence of hydroxyl groups alters the effects on DNA melting temperatures

Betaine lowers the melting temperature of deoxyribonucleic acid (DNA) and decreases its dependence on base composition. The effects of synthetic betaine analogs on the melting of DNA samples with different GC content were measured. Since many polyhydroxy compounds also lower DNA melting temperatures, hydroxyl-substituted betaine analogs were included. Some synthetic sulfonate analogs of betaine lowered the DNA melting temperatures by twice as much at the same molar concentration. They were up to twice as effective at decreasing the base pair dependence. Some carboxylate homologs of betaine, substituted with hydroxyl groups, increased the melting temperature. This effect was greater with low GC content DNA. Sulfonate analogs of betaine with hydroxyl groups usually destabilize the DNA, while their carboxylate analogs stabilize the DNA. Distances between the charges of these synthetic zwitterionic solutes influence the effect on DNA, with the optimum separation being two or three methylene groups. A betaine with two hydroxyl groups on one N-alkyl group had a greater effect than an isomer with two hydroxyl groups on separate N-alkyl substituents. We suggest that the effect of these solutes depends on structuring the hydration water of DNA, as well as interactions with the DNA structure itself.     

Denaturation maps of DNA: experimental and theoretical maps of phiX174 DNA.

A formaldehyde denaturation map of the replicative form of phiX174 DNA is obtained. The RFI DNA was converted into a linear state by restriction endonuclease pst I which introduces into this DNA a single double-stranded break. The map has four clear-cut peaks. Their positions excellently correlate with the peak positions on the map of equilibrium denaturation theoretically obtained earlier from the known nucleotide sequence of phiX174 DNA. The sequence is also used for a calculation of the maps of smoothed AT-content. The maxima on these maps correlate well with the peaks on the denaturation maps. To reveal the causes of a good correlation between the experimental formaldehyde and theoretical equilibrium denaturation maps, the theoretical formaldehyde denaturation maps are calculated for different conditions (temperature, formaldehyde concentration) using the detailed theory of DNA interaction with formaldehyde developed earlier.     

Mg2+-dependent unwinding of DNA by nonhistone chromosomal protein HMG(1 + 2) from pig thymus as determined by DNA melting temperature analysis

In the previous studies with endonucleases specific for single-stranded DNA, we have indicated that the nonhistone chromosomal protein HMG(1 + 2) prepared from pig thymus has an activity to unwind DNA partially at low protein-to-DNA weight ratios (Yoshida, M. & Shimura, K. (1984) J. Biochem. 95, 117-124). In the present work, we have pursued the unwinding reaction by HMG(1 + 2) by thermal melting temperature analysis of DNA, and by investigating the effect of Mg2+ on the reaction. The melting temperature of DNA in the presence of HMG(1 + 2) at low protein weight ratios decreased in 2 mM Tris-HCl, pH 7.8, whereas it increased at higher ratios. The depressions of melting temperature by HMG(1 + 2) at low ratios were not observed either in the system of 2 mM Tris-HCl, pH 7.8, containing EDTA or in the system containing samples treated in advance with EDTA. An addition of Mg2+ to the system reproduced the depression of melting temperature at low protein-to-DNA ratios as well as the increase at higher ratios. Analysis by Mg2+-equilibrated gel filtration revealed that HMG(1 + 2) is a Mg2+-binding protein. However, the depression of melting temperature at low protein-to-DNA ratios was not due to removal of Mg2+ from DNA by HMG(1 + 2). From these results, it is concluded that HMG(1 + 2) causes a partial DNA unwinding detectable by thermal melting temperature analysis of DNA, and that Mg2+ is necessary for the unwinding reaction.     

87 The melting of DNA in the presence of meso-tetra-pyridyl porphyrins with different peripheral substituents

The influence of water-soluble cationic 3N- and 4N-pyridyl porphyrins with different peripheral substituents (oxyethyl, buthyl, allyl, and metallyl) on melting parameters of DNA has been studied. Results indicate that the presence of porphyrin changes the shape and parameters of DNA melting curve. The increase of porphyrins concentration results in the increase of the melting temperature (Tm) and the melting interval (ΔT) of DNA. At the porphyrin-DNA concentration ratio r?=?0.01, changes in the melting temperature have not been observed. The melting intervals almost do not change upon adding of the 4N-porphyrins, while the decrease of ΔT, in the presence of 3N-porphyrins, is observed. Because the intercalation binding mechanism occurs in GC-rich regions of DNA, we assume that 3N-porphyrins, intercalated in GC-rich regions, reduce the thermal stability of these sites, bringing them closer to the thermal stability of the AT-sites, which is the reason for the decrease in the melting interval. While at the relative concentration r?=?0.01 for 4-N porphyrins, already the external binding mechanism “turns on” and the destabilizing effect of porphyrins on GC-pairs compensates stabilizing effect on AT-pairs, as a result of which change in the melting of DNA upon complexation with these porphyrins is not observed. The decrease of the hypochromic effect also indicates the intercalation of investigated porphyrins in the DNA structure, which weakens the staking interaction of base pairs of DNA. The increase of the hypochromic effect of DNA upon binding with porphyrin depends on the type of peripheral substituents of the porphyrin. The results show that porphyrins with butyl and allyl substituents weaken staking interaction of base pairs less than porphyrins with other substituents. The largest change was observed for metallyl porphyrins. It can be the result of bulky peripheral substituents, which make significant local changes in DNA structure.     

Thermal stability of DNA with interstrand crosslinks

Although many anticancer drugs exert their biological activity by forming DNA interstrand crosslinks (ICLs), the thermodynamics of biologically relevant long crosslinked DNAs has not been intensively studied in contrast to short duplexes. Here, we carry out computer modeling of the shift of melting temperature of long DNAs caused by ICLs taking into account crosslinking effect in itself and concomitant local alterations in the free energy (δG) of the helix-coil transition at sites of ICLs. Depending on δG, DNA interstrand crosslinks at per nucleotide concentration r = 0.05 can change the melting temperature by value from -17 to +47°C, and the influence weakly depends on DNA sequence and GC content. A change in melting temperature caused by introduction of interstrand crosslinking in modified DNA at sites of modifications also depends on δG and varies from 0 to +12°C. Comparison with experiment for the three platinum crosslinking compounds demonstrates utility of the theoretical method for understanding how crosslinking compounds can influence the melting behavior. On the basis of the method, interdependence of local distortions and crosslinking in itself was studied for thermal effect of ICLs. A method for evaluating the nature of the structural alteration that produces a change in thermal stability for short crosslinked DNA is also proposed. The methods can be used for comparative thermodynamic characterization of various DNA crosslinking agents.     

Effect of uridine dethiolation in the anticodon triplet of tRNA(Glu) on its association with tRNA(Phe)

The effect of U(34) dethiolation on the anticodon-anticodon association between E. coli tRNA(Glu) and yeast tRNA(Phe) has been studied by the temperature jump relaxation technique. An important destabilization upon replacement of the thioketo group of s2U(34) by a keto group, was revealed by a lowering of melting temperature of about 20 degrees C. The measured kinetic parameters indicated that this destabilization effect was originated in an increase of dissociation and a decrease of association rate constants by a factor of 4 to 5. Modifications in both stacking interactions and flexibility in the anticodon loop would be responsible for this effect.     

The influence of low-energy millimeter electromagnetic waves on the stability of DNA molecules in solution

The influence of low-energy millimeter electromagnetic waves on aqueous saline solution of DNA from the liver of healthy rats and rats with sarcoma 45 has been investigated. The characteristic parameters of irradiated and unirradiated DNA, melting temperature, and the range of melting were obtained from melting curves. The duration of exposure did not practically affect the range of melting, while the thermostability of DNA increased; as irradiation duration increased to 90 min, the melting temperature of tumor increased by approximately 1.5 degrees C. It was assumped that the increase in the thermostability of DNA is due to a more effective stabilization of the DNA double helix caused by the dehydration of Na(+)- ions present in the solution.     

Denaturation of mouse satellite DNA upon melting of chromatin in solution

The denaturation of mouse satellite DNA upon melting of chromatin in solution of low ionic strength has been studied. A procedure for preparation of partially denaturated chromatin was developed which enabled the isolation of double-stranded (non-denatured) DNA sequences according to their thermal stability in chromatin. The content of mouse satellite DNA in these DNA sequences was determined by hybridization with RNA, complementary to satellite DNA in order to find the temperature interval of denaturation of satellite DNA. It was found that the melting temperature of satellite DNA in chromatin was lower than that of the total DNA. The results are discussed in relation to previously reported anomalous behaviour of satellite DNA upon melting of chromatin on hydroxyapatite.