Effect of divalent metal ions on DNA conformational transitions in water-ethanol solutions

The influence of different MgCl2 and MnCl2 concentrations on DNA conformational transitions in water-ethanol solutions was studied. It was shown that the presence of magnesium ions in solution at a concentration of 5 x 10(-4) M did not influence the decrease in the size of DNA without change in its persistent length at an alcohol concentration of about 17 % v/v. In contrast, manganese ions prevent this change in DNA parameters. At sufficiently high ethanol concentrations, the compaction of DNA followed by its precipitation takes place, which is accompanied by an increase of scattering in solution. As the concentration of Mg2+ and Mn2+ in solution increases, this process is observed at lower ethanol concentrations.     

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO₂ stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.     

Effect of magnesium ions on heat denaturation of DNA

The dependence of animal DNA denaturation on magnesium ion concentration has been studied in the range (10(-6)--10(-1) M with sodium ion content of 10(-3) and 10(-2) M. Special attention has been given to the effect of multivalent metallic impurities bound to DNA. An increase of DNA thermal stability has been shown to occur in the magnesium concentration rage of 10(-6)--10(-4) M. At concentrations exceeding 10(-3) M the T M begins to decrease. The dependence of the DNA melting range on magnesium ion concentration has a maximum at approximately 10(-5) M Mg2+. At low magnesium and sodium ion concentrations a strong asymmetry of the melting curves has been observed. This effect can be described in terms of the melting theory for DNA complexed with small molecules and is explained by magnesium ion redistribution from the denatured portions of DNA to native ones. The method for calculation of melting curves in the DNA-ligand system has been proposed. Studies of thermal denaturation parameters have been shown to be an effective method for the estimation of binding constants of ligands to native and denatured DNA.     

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)).     

DNA interaction with Mn2+ ions at elevated temperatures: VCD evidence of DNA aggregation

Interaction of natural calf thymus DNA with Mn(2+) ions was studied at room temperature and at elevated temperatures in the range from 23 degrees C to 94 degrees C by means of IR absorption and vibrational circular dichroism (VCD) spectroscopy. The Mn(2+) concentration was varied between 0 and 1.3M (0 and 10 [Mn]/[P]). The secondary structure of DNA remained in the frame of the B-form family in the whole ion concentration range at room temperature. No significant DNA denaturation was revealed at room temperature even at the highest concentration of metal ions studied. However at elevated temperatures, DNA denaturation and a significant decrease of the melting temperature of DNA connected with a decrease of the stability of DNA induced by Mn(2+) ions occurred. VCD demonstrated sensitivity to DNA condensation and aggregation as well as an ability to distinguish between these two processes. No condensation or aggregation of DNA was observed at room temperature at any of the metal ion concentrations studied. DNA condensation was revealed in a very narrow range of experimental conditions at around 2.4 [Mn]/[P] and about 55 degrees C. DNA aggregation was observed in the presence of Mn(2+) ions at elevated temperatures during or after denaturation. VCD spectroscopy turned out to be useful for studying DNA condensation and aggregation due to its ability to distinguish between these two processes, and for providing information about DNA secondary structure in a condensed or aggregated state.     

The effects of Na+ and Mg2+ on the thermal denaturation of native and acetylated spinach ferredoxins

The effects of metal ions on the thermal denaturation and Mg2+ binding of native spinach ferredoxin and its acetylated derivative were investigated. The denaturation of ferredoxin in a metal-free solution at 40 degrees C was quickly prevented by the addition of Mg2+ or Na+ at appropriate concentrations. The metal concentrations required for 50% protection from thermal denaturation were 1.54 x 10(-4) M Mg2+ or 8.0 x 10(-3) M Na+ for native ferredoxin and 1.05 x 10(-3) M Mg2+ or 6.0 x 10(-2) M Na+ for acetylated ferredoxin. It was also found that native ferredoxin in the presence of over 20 mM Mg2+ was almost completely protected from thermal denaturation at 40 degrees C. The D-form which has been observed in acetylated ferredoxin by Masaki et al. (1977) (J. Biochem. 81, 1-9) was confirmed to be present in native ferredoxin at high temperature (49 degrees C) and is suggested to be an important form in the denaturation processes of the ferredoxin molecule.     

The effect of HCl on the solution structure of calf thymus DNA: A comparative study of DNA denaturation by proton and metal cations using fourier transform IR difference spectroscopy

The interaction of HCl with calf thymus DNA was investigated in aqueous solution at pH 7-2 with H⁺/DNA(P)(P:phosphate) molar ratios (r) of 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1, using Fourier Transform (FTIR) difference spectroscopy. Correlations between spectral changes, proton binding mode, DNA denaturation, and conformational variations are established. A comparison was also made between their spectra of denaturated DNA, in the presence of proton and Cu ions with similar cation concentrations. The FTIR difference spectroscopic results have shown that at low proton concentrations of r = 1/80 and 1/40 (pH 7–5), no major spectral changes occur for DNA, and the presence of H⁺ results in an increased base-stacking interaction and helical stability. At higher proton concentrations of r > 1/40, the proton binding to the cytosine and adenine bases begins with major destabilization of the helical duplex. As base protonation progresses, a B to C conformational conversion occurs with major DNA spectral changes. Protonation of guanine bases occurs at a high cation concentration r > 1/2 (pH < 3) with a major increase in the intensity of several DNA in-plane vibrations. Copper ion complexation with DNA exhibits marked similarities with proton at high cation concentrations (r > 1/10), whereas at low metal ion concentrations, copper–PO₂ and copper–guanine N-7 bindings are predominant. No major DNA conformational transition was observed on copper ion complexation. © 1995 John Wiley & Sons, Inc.     

Effect of magnesium concentration in the culture medium on the trace element requirement of yeasts]

The effect of different magnesium concentrations in the culture liquid (ranging from 2=6 to 220 mg/l) on the accumulation of Mg and Mn ions in the yeast Candida guilliermondii was studied during their continuous cultivation on purified liquid paraffins. A reverse correlation between the accumulation of magnesium and manganese in the yeast biomass was established. The magnesium content in the biomass increased with an increase of its concentration in the nutrient medium. The biomass yield was optimal at a concentration of magnesium ions in the nutrient medium of 10=25 mg/l. Under these conditions the content of magnesium ions was 0.4 mg % and that of manganese ions (upon their concentration in the nutrient medium of 1=2 mg/l) was 20 mg%.     

Effect of divalent copper ions on heat denaturation of DNA

Using the thermal denaturation method the effect of bivalent copper of (4-10(-6)-10(-3)) M concentrations on the helix-coil transition of DNA was studied in the solution of Na+ concentrations 10(-3)-10(-1) M. Unlike the previous studies, this paper makes allowance for the effect of impurity ions present in DNA and deionized water. It has been shown that in the region of low Cu2+ and Na+ concentrations, thermal stability increases, the melting range extends and the denaturation curves become asymmetric. At concentrations more than approximately 3-10(-5) M Cu2+, melting temperature starts to fall, and the range reduces to 1-1.5 degrees at [Cu2+] greater than or equal to 2-10(-4) M. As [Cu2+] reaches these values, the denaturation curve asymmetry and melting range increase again, which is due to the inversion of the relative stability of AT- and GC-pairs. Employing experimental and phase-transition-theory data for homopolymers, the constants of Cu2+ binding with phosphates and DNA bases were calculated. The concentration dependence of the DNA denaturation parameters was shown to be governed by the superposition of binding Cu2+ with phosphates and nucleic acid bases.     

The effects of Cu2+ and Pb2+ on the solution structure of calf thymus DNA: DNA condensation and denaturation studied by fourier transform IR difference spectroscopy

The interaction of calf thymus DNA with Cu²⁺and Pb²⁺ was studied in aqueous solution at pH 6.5 with metal/DNA (P) (P = phosphate) molar ratios (r) 1/80, 1/40, 1/20, 1/10, 1/4, 1/2, and 1, using Fourier Transform ir (FTIR) spectroscopy. Correlations between the ir spectral changes, metal ion binding mode, DNA condensation, and denaturation, as well as conformational features, were established. Spectroscopic evidence has shown that at low metal/DNA (P) molar rations 1/80 and 1/40, copper and lead ions bind mainly to the PO of the backbone, resulting in increased base-stacking interaction and duplex stability. The major copper ion base binding via G-C base pairs begins at r > 1/40, while the lead ion base binding occurs at r > 1/20 with the A-T base pairs. The denaturation of DNA begins at r = 1/10 and continues up to r = 1/2 in the presence of copper ions, whereas a partial destabilization of the helical structure was observed for the lead ion at high metal ion concentration (r = 1/2). Metal-DNA binding also results in DNA condensation. No major departure from the B-family structure was observed, upon DNA interaction with these metal ions. © 1993 John Wiley & Sons, Inc.     

Effects of ethidium bromide and SYBR Green I on different polymerase chain reaction systems

In an in-gel polymerase chain reaction (PCR), the generation of a 1750-bp yeast DNA fragment was inhibited when yeast DNA gel-stabs or gel-slices stained with ethidium bromide (EtBr) or SYBR Green I were used. Similar inhibition occurred to a varying degree in the reamplification of PCR fragments in prokaryotic systems. Inclusion of the dyes in PCR resulted in an inhibition at about 10 microg/ml EtBr and at 10,000-20,000-fold dilution of SYBR Green I in all systems. The effect remained unchanged despite increasing the PCR cycles to 40. However, increasing the magnesium chloride concentration did reverse the inhibitory actions, although the PCR specificity was lost. In an unusual observation, we find that, at higher dye concentrations (50 microg/ml EtBr, or thousand fold dilution of SYBR Green I), the input yeast DNA electrophoretic profile is maintained following 25 PCR cycles (despite a denaturation temperature of 94 degrees C). It varied significantly in different DNA systems and was readily reversed by high Mg++ concentrations. It is concluded that, at low Mg++ concentrations, different PCR systems are inhibited to varying extents by intercalating dyes and, in some PCR systems, intercalating dyes at unusually high concentrations maintain input DNA electrophoretic profile.     

Salt effects on bacteriophage T7-I

The thermal denaturation as a measure of the structural stability of the nucleoprotein in bacteriophage T7 has been studied in dependence of the ionic environment. Optical density and circular dichroism melting curves measured at wavelengths characterizing either the DNA or protein conformational changes were compared to identify different steps of the denaturation and to follow the effect of the ions. Monovalent salts strengthen the helical structure of intraphage DNA logarithmically in the way as they do in the case of isolated double-stranded DNA. Mg2+ and Ca2+ at very low concentrations stabilize the DNA helicity. Higher divalent ion concentrations decrease the stability of the double helix because of the repulsive ionic interactions. The high structural sensitivity of DNA in the presence of Mg2+ and Ca2+ in this "in situ" environment can be related to the biological role of these ions.     

Changes in conformation, stability and condensation of DNA by univalent and divalent cations in methanol-water mixtures

Circular dichroism spectroscopy, absorption spectroscopy, measurements of Tm values, sedimentation analysis and electron microscopy were used to study properties of calf thymus DNA in methanol-water mixtures as a function of monovalent cation (Na+ or Cs+) concentration and also in the presence of divalent cations Ca2+, Mg2+, and Mn2+. In the absence of divalent cations only slight conformational changes occurred and no condensation and/or aggregation could be detected. The Tm values depend on the amount of methanol and on the nature and concentration of cations. In methanol-water mixtures higher thermal stability was observed in solutions containing Cs+ ions. Up to 40% (v/v) methanol the addition of divalent ions leads to DNA stabilization. At methanol concentration higher than 50% the presence of divalent cations causes DNA condensation and denaturation even at room temperature. The denaturation is reversible with respect to EDTA addition indicating that no separation of complementary strands occurred and the resulting form of DNA is probably similar to the P form. DNA destacking appears to be a direct consequence of stronger cation binding by the condensed DNA in methanol-water mixtures.     

Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg (2+)] (0.5)/[Mon (+)] is less than 0.22 M (-1/2), monovalent ions (K (+), Na (+)) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg (2+) ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.     

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.     

西伯利亚蝗基因组DNA提取及RAPD分析条件的优化

以西伯利亚蝗Gomphocerus sibiricus(L.)为研究材料,利用改良的SDS法提取高质量的DNA,分别测试了dNTP浓度、镁离子浓度、TaqDNA聚合酶用量、模板DNA的量等因素对反应结果的影响。通过各因子的组合比较,建立了西伯利亚蝗RAPD优化体系:25μLPCR反应体系,10×buffer2·5μL;dNTP0·24mmol/L;MgCl22·0mmol/L;Taq DNA聚合酶1U;DNA模板45ng;引物30ng。扩增程序为:94℃预变性1min45s、94℃变性30s、35℃退火1min30s、72℃延伸2min,45个循环、72℃延伸10min。结果表明,利用优化的反应条件进行西伯利亚蝗基因组DNA分析,实验有着良好的重复性和稳定性。     

ATP, histidine or magnesium ions can protect DNA against sisomicin-induced damage, following stray Cu(II) binding

The oxidative DNA damage by the cupric complexes of sisomicin was investigated in the presence of varying amounts of histidine, ATP, Mg(II) ions or phosphates. We found that by very low concentrations, the amino acid is able to inhibit the cleavage totally. This occurs both by its competition with antibiotic for copper(II) binding, what was proved by spectroscopic measurements, as well as by ROS scavenging by the imidazole ring. ATP and magnesium also exert an influence on the yield of the DNA destruction by decreasing the amount of the single strand breaks, however only their significant excess is able to break this process. The influence of ATP on the plasmid damage has in this case a similar chemical mechanism to that one observed for histidine. Mg(II) ions, however, interact with DNA and thus prevent the complex binding. Only phosphate anions, in the range of their physiological concentrations, exert no influence on the cleavage process.     

Unwinding of DNA by nonhistone protein HMG1 and HMG2

The enzyme kinetic studies with endonucleases specific for single-stranded DNA and the thermal denaturation analyses of DNA showed that a high mobility group (HMG) nonhistone protein fraction HMG (1 + 2), composed of HMG1 and HMG2, has an activity to unwind DNA partially at low protein-to-DNA weight ratio. Isolated HMG1 and HMG2 have the same activity. Divalent cations such as Mg++ or Ca++ were necessary for the unwinding reaction. A peptide containing high glutamic and aspartic (HGA) region, isolated from the tryptic digest of HMG (1 + 2), unwound DNA depending on the presence of Mg++ or Ca++, suggesting that the HMA region in HMG protein is the active site for the DNA unwinding reaction. Poly-L-glutamic acid, employed as a model peptide of the HGA region, showed the activity. Finally, mechanisms of the DNA unwinding reaction by the HMG protein and possible role of the divalent cations are discussed.