Structural and functional heterogeneity of single-stranded DNA-binding proteins from calf thymus

A new purification technique for ‘single-stranded DNA-binding proteins’ from calf thymus permits the demonstration of a considerable heterogeneity within these proteins. Several molecular species are obtained with M_r between 24·10³ and 30·10³ and pI values between 6 and 8, showing significant differences with regard to the following functional properties: (1) strength of binding to single-stranded DNA; (2) lowering of melting temperature of poly[d(A-T)]; (3) stimulation of DNA polymerase α on a poly[d(A-T)] template. Analysis of trypsin digestion products demonstrates that the different molecular species share extensive primary sequence homology. Experiments with antibodies show that the different molecular species are antigenically related and that a 31 kDa protein present in low amounts in our preparations is very cross-reactive.     

High Temperature Stabilization of DNA in Complexes with Cationic Lipids

The influence on the melting of calf thymus and plasmid DNA of cationic lipids of the type used in gene therapy was studied by ultraviolet spectrophotometry and differential scanning calorimetry. It was found that various membrane-forming cationic lipids are able to protect calf thymus DNA against denaturation at 100°C. After interaction with cationic lipids, the differential scanning calorimetry melting profile of both calf thymus and plasmid DNA revealed two major components, one corresponding to a thermolabile complex with transition temperature, T_m(labile), close to that of free DNA and a second corresponding to a thermostable complex with a transition temperature, T_m(stable), at 105 to 115°C. The parameter T_m(stable) did not depend on the charge ratio, R(±). Instead, the amount of thermostable DNA and the enthalpy ratio ΔH_(stable)/ΔH_(labile) depended upon R(±) and conditions of complex formation. In the case of O-ethyldioleoylphosphatidylcholine, the cationic lipid that was the main subject of the investigation, the maximal stabilization of DNA exceeded 90% between R(±) = 1.5 and 3.0. Several other lipids gave at least 75% protection in the range R(±) = 1.5 to 2.0. Centrifugal separation of the thermostable and thermolabile fractions revealed that almost all the transfection activity was present at the thermostable fraction. Electron microscopy of the thermostable complex demonstrated the presence of multilamellar membranes with a periodicity 6.0 to 6.5 nm. This periodic multilamellar structure was retained at temperatures as high as 130°C. It is concluded that constraint of the DNA molecules between oppositely charged membrane surfaces in the multilamellar complex is responsible for DNA stabilization.     

Spectroscopic study of the interaction of Ni-5-triethyl ammonium methyl salicylidene ortho-phenylendiiminate with native DNA

The interaction of native calf thymus DNA with the cationic Ni(II) complex of 5-triethyl ammonium methyl salicylidene ortho-phenylendiimine (NiL²⁺), in 1 mM Tris-HCl aqueous solutions at neutral pH, has been monitored as a function of the metal complex-DNA molar ratio by UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The dramatic modification of the DNA CD spectrum, the appearance of a broad induced CD band in the range 350-400 nm, the strong increase of the DNA melting temperature (T_m) and the fluorescence quenching of ethidium bromide-DNA solutions, in the presence of increasing amounts of the NiL²⁺ metal complex, support the existence of a tight intercalative interaction of NiL²⁺ with DNA, analogous to that recently reported for both ZnL²⁺ and CuL²⁺. The intrinsic binding constant (K_b) and the interaction stoichiometry (s), determined by UV spectrophotometric titration, are equal to 4.3 × 10⁶ M⁻¹ and 1.0 base pair per metal complex, respectively. Interestingly, the value of K_b is slightly higher and 10 times higher than that relative to the CuL²⁺-DNA and the ZnL²⁺-DNA systems, respectively. Speculations can be performed to rationalize the observed trend, on the basis of the electronic and geometrical structures of the three complexes of the same ligand. Analogously to what previously observed for CuL²⁺, the shape of the CD of the NiL²⁺-DNA system at NiL²⁺-DNA molar ratios higher than 0.5 is indicative of the formation of supramolecular aggregates in solutions, as a possible consequence of the electrostatic interaction between the cationic complex and the negatively charged phosphate groups of DNA.     

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase α from both calf thymus and human lymphoma cells and DNA polymerase β from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG⁴ lesions when Mg²⁺ is the divalent cation. Substitution of Mn²⁺ for Mg²⁺ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase α inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase β is specific, inserting mainly C even in the presence of Mn²⁺. The K_m for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn²⁺ is about 0.5 mm. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg²⁺ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn²⁺. dNTPαS and recA protein increase only the insertion of C.We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.     

Temperature dependence of CD spectra of DNA from various sources

The CD spectra of DNA from various sources (T2; T4; C_d; Escherichia coli; calf thymus; Streptomyces chrysomalis) were investigated. A new band Δ_ε210 in the CD spectra of glucosylated DNA of the T even phages was found. The temperature dependence of the CD spectra of DNA was obtained over a wide range of temperatures, including those of the helix–coil transition. The band Δ_ε275 for all DNAs does not appreciably change in the range of the helix–coil transition. The monotonic increase of this band before melting, and its decrease after melting is observed with an increase in temperature. The amplitude of the CD band Δ_ε245 for all the DNAs studied and Δ_ε210 (glucosylated DNA) parallels the change of E₂₆₀ absorbance.     

Formation of adducts in the reaction of glyoxal with 2'-deoxyguanosine and with calf thymus DNA

The reactions of glyoxal with 2′-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, ¹H and ¹³C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2′-deoxy-β-d-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx₂-dG) and N²-(carboxymethyl)-9-(2′-deoxy-β-d-erythro-pentofuranosyl)-purin-6(9H)-one (Gx₁-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx₂-dG were found to be unstable and partly transformed to Gx₁-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2′-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2′-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx₁-dG was the only stable adduct. The transformation of Gx-dG to Gx₁-dG seemed to take place in single-stranded DNA and therefore, Gx₁-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.     

Influence of the tritium labeling of calf thymus DNA by the Wilzbach method on its physical properties

The adsorption at the air-water interface of calf thymus H³-DNA labeled in the dry state by the Wilzbach method was studied by measuring surface concentration, surface tension, and surface potential. It was found that, correspondingly to the behavior of E. coli H³-DNA labeled by incorporation of thymine H³ and described in another paper, both the rate of adsorption and the amount of material adsorbed increased with increase in DNA concentration, salt concentration, or in the valency of the counterion. Surface pressure and potential did not change in the course of adsorption, and this is also in accordance with the properties found for E. coli DNA. However, while the surface concentration of the E. coli DNA corresponds approximately to monomolecular layer adsorption, the radiation from the adsorbed layer of the calf thymus H³-DNA indicated apparently multilayer adsorption. On comparing the physical properties of the H³-DNA labeled by the Wilzbach method to those of nonlabeled DNA, it is found that while the chemical composition and the bihelical structure is essentially maintained in the labeled material, exposure to tritium gas results in a reduction in molecular weight and produces random breaks in the strands of the bihelix. The H³-DNA produced by Wilzbach's method is not labeled homogeneously. The more the molecule is exposed to the gaseous tritium, the more efficient is the isotopic, exchange and the greater the alteration in physical properties. The defects in the labeled H³-DNA molecule make it more surface active, thus H³-DNA of higher specific radioactivity concentrates at the interface, conveying the impression of multilayer formation although actually the adsorbed layer is approximately monomolecular.     

The interaction of native calf thymus DNA with Fe(III)-dipyrido[3,2-a:2',3'-c]phenazine

The mono and bis dipyrido[3,2-a:2′,3′-c]phenazine (dppz) adducts of iron(III) chloride, i.e. [Fe(dppz)]Cl₃ and [Fe(dppz)₂]Cl₃, have been synthesized and characterized. The interaction of the Fe^IIIdppz hydrolyzed aquo complex with native calf thymus DNA has been monitored as a function of the metal complex-DNA molar ratio, by variable temperature UV absorption spectrophotometry, circular dichroism (CD) and fluorescence spectroscopy. The results obtained in solution at various ionic strength values give support for a tight intercalative binding of the Fe^IIIdppz cation with DNA. In particular, the appearance of induced CD bands, caused by the addition of Fe^IIIdppz, indicate the existence of a rigid metal complex-DNA-binding leading to dominating chiral organization of Fe^IIIdppz species within the DNA double helix. The trend of selected CD bands with the molar concentration of Fe^IIIdppz emphasizes that the presence of high amounts of metal complex induces also the formation of DNA-Fe^IIIdppz supramolecular aggregates in solution. The analysis of fluorescence measurements allowed us to calculate a value of the intercalative binding constant comparable to that obtained by UV spectrophotometric titration. Finally, the temperature dependence of the absorbance at 258 nm shows that the metal complex strongly increases the DNA melting temperature already at metal complex-DNA molar ratio equal to 0.25 suggesting that metal complex intercalation effectively hinders DNA denaturation. Overall, the results of the present study point out that the Fe^IIIdppz aquo complex has DNA-binding properties analogous to those previously reported for the tris-chelate Fe^II(phen)₂dppz complex (phen = 1,10-phenantroline).     

Exploring the intercalation binding of tiamulin with DNA using multispectroscopy and a molecular modelling approach

The binding of tiamulin with calf thymus DNA was systematically investigated using multispectroscopy and molecular modelling techniques. For DNA, once tiamulin was added, viscosity (η) and melting temperature (T_m) both exhibited an uptrend. The fluorescence performance of the tiamulin–DNA complex did not change with the ionic strength changes. The binding constant (K_a) of tiamulin for single-stranded DNA (ssDNA, 1.48 × 10⁴ M⁻¹) was obviously higher than that for double-stranded DNA (dsDNA, 9.51 × 10³ M⁻¹) at 291 K. The helix structure became looser and the base stack force became stronger for DNA due to the presence of tiamulin as seen from circular dichroic (CD) spectra. The intercalation binding mode of tiamulin with DNA was disclosed. Molecular modelling also revealed tiamulin inserting into the base pairs with the lowest binding free energy of −18.73 kJ mol⁻¹ using van der Waals forces as well as hydrogen bonds.     

pH- and DNA-induced dual molecular light switches based on a novel ruthenium(II) complex

A novel Ru(II) complex, [Ru(bpy)₂(btppz)]Cl₂, where bpy = 2,2′-bipyridine and btppz = benzo[h]tripyrido[3,2-a:2′,3′-c:2″,3″-j]phenazine, has been synthesized and characterized. The pH effects on UV-visible (UV-vis) absorption and emission spectra of the complex have been studied and ground- and excited-state ionization constants of the complex have been derived. The calf thymus DNA (ct-DNA) binding properties of the complex were investigated with UV-vis absorption and luminescence spectrophotometric titrations, steady-state emission quenching by [Fe(CN)₆]⁴⁻, DNA competitive binding with ethidium bromide, DNA melting experiments, reverse salt titrations and viscosity measurements. The complex was demonstrated to act as dual molecular switches: pH-induced “on-off” emission switch with an on-off intensity ratio of ∼54 which is favorably compared with those reported for structurally analogous Ru(II) complexes, and a DNA molecular light switch with a luminescence enhancement factor of 22 as it intercalatively bound to the DNA.     

In vitro study of DNA interaction with clodinafop-propargyl herbicide

The interaction of native calf thymus DNA with clodinafop-propargyl (CP), in 10 mM HEPES aqueous solutions at neutral pH 7.2, has been investigated by spectrophotometric, circular dichroism (CD), spectrofluorometric, melting temperature (Tm), and viscosimetric techniques. It was found that CP molecules could intercalate between base pairs of DNA as evidenced by hyperchromism in UV absorption band of DNA, an increase in melting temperature, a sharp increase in specific viscosity of DNA, induced CD spectral changes, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of CP, which indicates that it is able to release the intercalated MB completely. All results suggest that the CP interacts with calf thymus DNA by an intercalative mode of binding.     

Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides

In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [³H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O₂ or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radioactivity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [³H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.     

The interaction of [Ru(NH3)5Cl]2+ and [Ru(NH3)6]3+ ions with DNA

The interaction of [Ru(NH3)5Cl]2+ and [Ru(NH3)6]3+ complex ions with calf thymus DNA has been studied at various r values (r = [Mn+]/[DNA-P]). Electronic spectra of metal-DNA solutions have been recorded and compared to the spectra of metal, as well as of DNA, solutions. Melting curves have been taken for the determination of DNA melting temperature (Tm) in the presence of the above complex ions. The results showed a biphasic melting of the DNA strands for relatively high r values. The Tm for the first phase increased with increasing r values, indicating metal ion interaction with the phosphate moieties of the DNA. The appearance of a second-phase melting, in connection with electronic spectra, pH values, and conductivity measurements of metal ion solutions, is indicative of the initial complexes' transformation to [Ru(NH3)5OH]2+, which binds preferentially to double-stranded rather than single-stranded DNA, thus leading to a second melting curve at a higher temperature than the first one.     

Electron spin resonance melting of chemically spin-labeled nucleic acids.

Conformational transitions of nitroxide labeled and unlabeled nucleic acids were analyzed by esr and uv spectroscopy to evaluate potential perturbation effects caused by chemical modifications of nucleic acids with spin labels. The melting temperature (T_m) determined by uv or esr melting profiles of 2 → 1 or 3 → 1 transitions is similar for labeled and unlabeled polyadenylic acid [(A)_n] and polyuridylic acid [(U)_n] complexes provided spin-labeled (A)_n with a nitroxide to nucleotide ratio of 0.002 is used. Complexes formed with spin-labeled (A)_n of greater spin-labeling extent display a noticeable perturbation of their thermal melting profiles. The studies reconfirm the existence of a low temperature esr transition at about 20 °C with calf thymus and T4 DNA duplexes spin-labeled with a nitroxide to nucleotide ratio of about 0.006. The uv melting profiles of the spin-labeled duplexes reveal no low-temperature discontinuity, but the T_m values reflecting the 2 → 1 transitions were reduced by several degrees versus those of the unlabeled duplexes. Thus, these studies suggest that with homopolymers, chemically modified to a low extent with nitroxides, the monitoring of local conformational transitions of duplexes or triplexes reflect the overall 2 → 1 or 3 → 1 transitions. In the case of the heteropolymers the possibility that the chemical modification is responsible for the low-temperature phenomenon cannot be ruled out.     

Interaction of 2‐Chloro‐N10‐Substituted Phenoxazine with DNA and Effect on DNA Melting

Five N¹⁰‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid _PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

One‐Pot Solvent Free Synthesis and DNA Binding Studies of Thieno[2,3‐b]‐1,8‐Naphthyridines

Abstract

With the aim of evaluating interaction between double‐stranded calf thymus (ds)DNA and sulphur containing fused planar rings, the derivatives of 1,8‐naphthyridine containing thiono groups were synthesized by the condensation of 2‐mercapto‐3‐formyl[1,8]naphthyridines using 1‐chloroacetone, 2‐chloroacetamide, chloroaceticacid, and 2‐chloro‐1‐phenylethanone in the presence of anhydrous potassium carbonate as s catalyst under solvent free microwave irradiation. The structures of the compounds were elucidated on the basis of elemental analysis, IR, ¹H NMR, and mass spectra. The interaction of thieno[2,3‐b]‐1,8‐naphthyridine‐2‐carboxylic acid (TNC) (3a) with ct‐DNA was studied by UV‐Vis spectrophotometry, viscosity, thermal denaturation, as well as cyclic voltammetry experiments. On binding to DNA, the absorption spectrum underwent bathochromic and hypochromic shifts. Binding parameters, determined from spectrophotometric measurements indicated a binding constant of K _b =2.1×10⁶ M^?1. The thieno[2,3‐b]‐1,8‐naphthyridine‐2‐carboxylic acid (3a) increases the viscosity of sonicated rod‐like DNA fragments. The binding of TNC to DNA increased the melting temperature by about 4°C. The decrease in peak current heights and shifts of peak potential values are observed by the addition of calf thymus DNA in cyclic voltammetry studies.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.