Effect of CPT on the DNA cleavage/religation reaction mediated by calf thymus Topoisomerase I: evidence of an inhibition of DNA religation

The uncoupling of the calf thymus Topoisomerase I-mediated forward DNA cleavage reaction from the religation event by a rapid shift of cleavage temperature either from 37 °C to 0 °C or from 37 °C to 56 °C has been studied and utilized to elucidate the molecular mechanism by which camptothecin, a clinically relevant antineoplastic agent, influences the half reactions of the enzyme. Results of heating and cooling religation-inducing treatments have been compared: both temperature extremes reduce the amount of protein-linked DNA breaks to background levels, thereby affecting cleavage reversal. Camptothecin is found to stabilize the enzyme-DNA intermediate, by inhibition of the Topoisomerase I-mediated rejoining of cleaved DNA, even when the drug is added after formation of the complex. We conclude that:

1. Heating and cooling treatments show a pronounced effect on the DNA cleavage-religation equilibrium. The efficacy of cold is more pronounced than that of heat.
2. Reversal of the enzyme-DNA intermediate favors the DNA resealing versus the closed relaxed form.
3. Camptothecin affects the heat or cold induced religation: in fact in both cases the drug delays the religation step.

     

Optimum DNA relaxation reaction conditions for calf thymus DNA-Topoisomerase I are determined by specific enzyme features

Reactivity and chemical properties of calf thymus Topoisomerase I have been investigated with respect to enzyme ability to relax supercoiled DNA. The relaxation rate has been analyzed at optimum and relatively high salt concentration. Catalysis is processive at optimum salt concentration and distributive at a higher one; camptothecin decreases the initial rate of reaction in both salt conditions, but more so at the higher one. We conclude that:

Influence of polyethylene glycol on the ligation reaction with calf thymus DNA ligases I and II

High concentrations of the nonspecific macromolecule polyethylene glycol 6000 (PEG 6000) enabled DNA ligases I and II from calf thymus to catalyze intermolecular blunt-end ligation of duplex DNA. Intermolecular cohesive-end ligation with these enzymes was markedly stimulated in the presence of 10-16% (w/v) PEG 6000. The effect of PEG 6000 (4-16%) on the sealing of single-stranded breaks in duplex DNA with DNA ligases I and II was not appreciably stimulatory but rather inhibitory. PEG 6000 (15%) enhanced more twofold the rate of DNA ligase II-AMP complex formation, but moderately suppressed the rate of formation of DNA ligase 1-AMP complex. Polyamines and KCl inhibited blunt-end and cohesive-end ligations with DNA ligases I and II in the presence of PEG 6000.     

Initiation of DNA synthesis by the calf thymus DNA polymerase-primase complex

The calf thymus DNA polymerase-alpha-primase complex purified by immunoaffinity chromatography catalyzes the synthesis of RNA initiators on phi X174 single-stranded viral DNA that are efficiently elongated by the DNA polymerase. Trace amounts of ATP and GTP are incorporated into products that are full length double-stranded circular DNAs. When synthetic polydeoxynucleotides are used as templates, initiation and DNA synthesis occurs with both poly(dT) and poly(dC), but neither initiation nor DNA synthesis was observed with poly(dA) and poly(dI) templates. Nitrocellulose filter binding and sucrose gradient centrifugation studies show that the DNA polymerase-primase complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Using d(pA)-50 with 3'-oligo(dC) tails and d(pI)-50 with 3'-oligo(dT) tails, initiator synthesis and incorporation of deoxynucleotide can be demonstrated when the average pyrimidine sequence lengths are 8 and 4, respectively. These results suggest that purine polydeoxynucleotides are used as templates by the DNA polymerase only after initiation has occurred on the oligodeoxypyrimidine sequence and that the pyrimidine stretch required by the primase activity is relatively short. Analysis of initiator chain length with poly(dC) as template showed a series of oligo(G) initiators of 19-27 nucleotides in the absence of dGTP, and 5-13 nucleotides in the presence of dGTP. The chain length of initiators synthesized by the complex when poly(dT) or oligodeoxythymidylate-tailed poly(dI) was used can be as short as a dinucleotide. Analysis of the products of replication of oligo(dC)-tailed poly(dA) shows that initiator with chain length as low as 4 can be used for initiation by the polymerase-primase complex.     

Processivity of the DNA polymerase alpha-primase complex from calf thymus

The processivity of the DNA polymerase alpha-primase complex from calf thymus was analyzed under various conditions. When multi-RNA-primed M13 DNA was used as the substrate, the DNA polymerase alpha-primase complex was found to incorporate 19 +/- 3 nucleotides per primer binding event. This result was confirmed by product analysis on sequencing gels following DNA synthesis on poly(dT) X (rA)10. The processivity depends strongly on the assay conditions but does not correlate with enzymic activity. Lowering the concentration of Mg2+ ions to less than 2 mM increases the processivity to 60. Replacing Mg2+ by 0.2 mM Mn2+ results in 90 nucleotides being incorporated per primer binding event. Neither the presence of ATP nor the addition of noncognate deoxynucleotide triphosphates affects the processivity of the DNA polymerase alpha-primase complex. Lower processivity was induced by lowering the reaction temperature, by adding spermine, spermidine, or putrescine, in the presence of the antibiotics novobiocin and ciprofloxacin, by adding Escherichia coli single-stranded DNA binding protein, or by adding calf thymus topoisomerase II and RNase H. Three single-stranded DNA binding proteins from calf thymus, including unwinding protein 1, do not affect processivity to any significant extent. Freshly prepared DNA polymerase alpha-primase complex exhibits in addition to its processivity of 20 further discrete processivities of about 55, 90, and 105. This result suggest that further subunits of the polymerase alpha-primase complex are necessary to reconstitute the holoenzyme form of the eukaryotic replicase.     

Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex

Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.     

Reactions of malonaldehyde and acetaldehyde with calf thymus DNA: formation of conjugate adducts

Our previous work has shown that treatment of nucleosides with malonaldehyde simultaneously with acetaldehyde affords stable conjugate adducts. In the present study we demonstrate that conjugate adducts are also formed in calf thymus DNA when incubated with the aldehydes. The adducts were identified in the DNA hydrolysates by their positive ion electrospray MS/MS spectra, by coelution with the 2'-deoxynucleoside standards, and, in the case of adducts exhibiting fluorescent properties, also by LC using a fluorescence detector. In the hydrolysates of double-stranded DNA (ds DNA), two deoxyguanosine and two deoxyadenosine conjugate adducts were detected and in single-stranded DNA (ss DNA) also, the deoxycytidine conjugate adduct was observed. The guanine base was the major target for the malonaldehyde-acetaldehyde conjugates and 2'-deoxyguanosine adducts were produced in ds DNA at levels of 100-500 adducts/10(5) nucleotides (0.7-3 nmol/mg DNA).     

Zinc (II) coordination in Escherichia coli DNA topoisomerase I is required for cleavable complex formation with DNA.

Escherichia coli DNA topoisomerase I catalyzes relaxation of negatively supercoiled DNA. The reaction proceeds through a covalent intermediate, the cleavable complex, in which the DNA is cleaved and the enzyme is linked to the DNA via a phosphotyrosine linkage. Each molecule of E. coli DNA topoisomerase I has been shown to have three tightly bound zinc(II) ions required for relaxation activity (Tse-Dinh, Y.-C., and Beran-Steed, R.K. (1988) J. Biol. Chem. 263, 15857-15859). It is shown here that Cd(II) could replace Zn(II) in reconstitution of active enzyme from apoprotein. The role of metal was analyzed by studying the partial reactions. The apoenzyme was deficient in sodium dodecyl sulfate-induced cleavage of supercoiled PM2 phage DNA. Formation of covalent complex with linear single-stranded DNA was also reduced in the absence of metal. However, the cleavage of small oligonucleotide was not affected, and the apoenzyme could religate the covalently bound oligonucleotide to another DNA molecule. Assay of noncovalent complex formation by retention of 5'-labeled DNA on filters showed that the apoenzyme was not inhibited in noncovalent binding to DNA. It is proposed that zinc(II) coordination in E. coli DNA topoisomerase I is required for the transition of the noncovalent complex with DNA to the cleavable state.     

Effect of camptothecin on the DNA-relaxing and DNA-cleavage activity of calf thymus topoisomerase I

Nucleotide sequences that are cleaved by calf thymus type I topoisomerase have been determined using cloned human Ha-ras and p53 genes. Localization and relative frequency of single-strand cleavages within these sequences were observed to change in the presence of the cytotoxic alkaloid camptothecin.     

Spectroscopic studies on the interaction of quercetin-terbium(III) complex with calf thymus DNA

The interaction of native calf thymus DNA (CT-DNA) with quercetin-terbium(III) [Q-Tb(III)] complex at physiological pH was monitored by UV absorption spectrophotometry, circular dichroism, fluorescence spectroscopy, and viscosimetric techniques. The complex displays binding properties to the CT-DNA and was found to interact with CT-DNA through outside binding, demonstrated by a hypochromic effect of Q-Tb(III) on the UV spectra of CT-DNA and the calculated association constants (K). Also, decrease in the specific viscosity of CT-DNA, decrease in the fluorescence intensity of Q-Tb(III) solutions in the presence of increasing amounts of CT-DNA, and detectable changes in the circular dichroism spectrum of CT-DNA are other evidences to indicate that Q-Tb(III) complex interact with CT-DNA through outside binding.     

Study on interactions of aminoglycoside antibiotics with calf thymus DNA and determination of calf thymus DNA via the resonance Rayleigh scattering technique

A simple and sensitive resonance Rayleigh scattering (RRS) spectra method was developed for the determination of calf thymus DNA (ctDNA). The enhanced RRS signals were based on the interactions between ctDNA and aminoglycoside antibiotics (AGs) including kanamycin (KANA), tobramycin (TOB), gentamicin (GEN) and neomycin (NEO) in a weakly acidic medium (pH 3.3–5.7). Parameters influencing the method were investigated. Under optimum conditions, increments in the scattering intensity (?I) were directly proportional to the concentration of ctDNA over certain ranges. The detection limit ranged from 12.2 to 16.9 ng/mL. Spectroscopic methods, including RRS spectra, absorption spectra and circular dichroism (CD) spectroscopy, coupled with thermo‐denaturation experiments were used to study the interactions, indicating that the interaction between AGs with ctDNA was electrostatic binding mode. Copyright © 2015 John Wiley & Sons, Ltd.     

Purification and characterization of a type I DNA topoisomerase from calf thymus mitochondria

A type I topoisomerase has been purified more than 4000-fold from calf thymus mitochondria. The enzyme is membrane associated and is effectively solubilized by 1% Triton X-100 treatment of purified mitochondrial inner membranes. This ATP-independent enzyme relaxes positively and negatively supercoiled DNA with delta LK = 1. At low ionic strength, the native enzyme appears to be a monomer (sedimentation coefficient of 4.3 S and Stokes radius of 34 A), but it can form a weakly associated dimer at higher salt concentrations (sedimentation coefficient of 7.0 S and Stokes radius of 47.5 A). The mitochondrial type I topoisomerase is distinguishable from the nuclear enzyme by its (1) pH profile, (2) thermal stability, (3) response to dimethyl sulfoxide and Berenil, and (4) molecular weight. The mitochondrial enzyme is inhibited by elevated concentrations of the bacterial DNA gyrase inhibitor novobiocin, but not nalidixic or oxolinic acids. Sensitivity to N-ethylmaleimide indicates the importance of cysteine for catalytic activity. It is estimated that there are at least five copies of topoisomerase I per mammalian mitochondrion or a minimum of one to two per mitochondrial genome. In a manner similar to that observed with leukemia (nuclear and mitochondrial), calf thymus (nuclear), and HeLa (nuclear) cell type I topoisomerase, the calf thymus mitochondrial enzyme is inhibited by physiological concentrations of ATP.     

Effect of Mg2+ and Mn2+ on hydrolysis of calf thymus DNA by pancreatic deoxyribonuclease I

Divalent cations are activators for DNA hydrolysis by pancreatic deoxyribonuclease I. Apparent Vm and Km changes have been studied in presence of Mn2+ or Mg2+. The activation process modifies both Vm and Km, their relationship with Mg2+ or Mn2+ being a linear one. Deoxyribonucleotides inhibit the DNA hydrolysis, whether Mg2+ or Mn2+ is the activator; the purine deoxyribonucleotides are more effective as inhibitors than the pyrimidine ones. The effect of some derivatives of adenine has been studied: the inhibition is maximum with 5'-dAMP and minimum with adenine or adenosine. A kinetic mechanism of enzymatic activation by Mn2+ or Mg2+ is discussed.     

Studies on the interaction of isoxazolcurcumin with calf thymus DNA

The interaction of isoxazolcurcumin (IOC), a synthetic derivative of curcumin, with calf thymus-DNA (ct-DNA) has been investigated by UV-Vis, fluorescence, circular dichroism spectroscopies, viscosity measurements and docking studies. From these analyses, the binding constant, number of binding sites and mode of binding of IOC to ct-DNA has been determined. The binding constant of IOC to DNA calculated from both UV-Vis and CD spectra was found to be in the 10(4)M(-1) range. Analyses of fluorescence spectra, viscosity measurements and molecular modeling of IOC-DNA interactions indicate that IOC is a minor groove binder of ct-DNA and preferentially binds to AT rich regions. Ethidium bromide displacement studies revealed that IOC did not have any effect on ethidium bromide bound DNA which is indicative of groove binding. To elucidate the preferred region of binding of IOC to DNA, docking studies have been performed and changes in accessible surface area (DeltaASA) of nucleobases determined due to IOC-DNA complexation.     

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.     

Formation of 6-dimethylcarbamyloxy-dGuo, 6-dimethylamino-dGuo and 4-dimethylamino-dThd following in vitro reaction of dimethylcarbamyl chloride with calf thymus DNA and 6-diethylcarbamyloxy-dGuo following in vitro reaction of diethylcarbamyl chloride with calf thymus DNA

The rodent carcinogens dimethylcarbamyl chloride (DMCC) and diethylcarbamyl chloride (DECC) react with dGuo (pH 7.0–7.5, 37°C, 4 h) to form the O⁶-acyl derivatives 6-dimethylcarbamyloxy-2′-deoxyguanosine (6-DMC-dGuo) and 6-diethylcarbamyloxy-2′-deoxyguanosine (6-DEC-dGuo), respectively. Reaction of DMCC with dThd under identical conditions yielded 4-dimethylamino-thymidine (4-DMA-dThd). Compounds 6-DMC-dGuo and 6-DEC-dGuo undergo a nucleophilic aromatic substitution reaction with dimethylamine (DMA) to form 6-dimethylamino-2′-deoxyguanosine (6-DMA-dGuo) via displacement of the C-6 dialkylcarbamyloxy moiety. The substitution reaction did not take place when diethylamine or NH₃ were substituted for DMA. The structures of the new compounds 6-DMC-dGuo, 6-DEC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were deduced from chemical analyses and syntheses, UV and nuclear magnetic resonance (NMR) spectra and electron impact, isobutane chemical ionization and source insertion isobutane chemical ionization mass spectra. It was postulated that 4-DMA-dThd was formed following reaction of the transient intermediate 4-DMC-dThd with DMA formed by hydrolysis of DMCC. Calf thymus DNA was reacted in vitro with DMCC (pH 7.0–7.5, 37°C, 4 h) and the modified DNA hydrolyzed enzymatically to 2′-deoxynucleosides. Compounds 6-DMC-dGuo, 4-DMA-dThd and 6-DMA-dGuo were identified in the hydrolysate by high-pressure liquid chromatography (HPLC). In an indentical manner 6-DEC-dGuo was identified following in vitro reaction of DECC with calf thymus DNA. Compounds 6-DEC-dGuo and 6-DMC-dGuo possess novel structures with respect to the types of adducts known to be formed between carcinogens and bases in DNA. The implications of these findings with respect to chemical mutagenesis and carcinogenesis is discussed. The structural relationship between N⁴-dimethyl-5-methylcytosine (4-dimethylamino-Thy) formed in DNA following in vitro reaction with DMCC and 5-methylcytosine, the only modified base found in vertebrate DNA is noted.     

Photosensitized damage to calf thymus DNA by a hypocrellin derivative: mechanisms under aerobic and anaerobic conditions

The di-cysteine substituted hypocrellin B (DCHB) derivative has been found to be a potential phototherapeutic agent and exhibit photosensitized damage to DNA. Electronic paramagnetic resonance (EPR) and spectrophotometry demonstrate that one-electron transfer from calf thymus DNA to triplet DCHB induces the generation of the reduced form of DCHB (DCHB*- radical), followed by the second electron transfer from DNA to DCHB*- or the disproportionation of DCHB*- to form the hydroquinone of DCHB (DCHBH2) in anaerobic conditions. This electron transfer process induces the direct damage to DNA in oxygen-free media and contributes partly to the damage of DNA in aerobic media. Superoxide radical and hydroxyl radical are formed with enhanced efficiencies while singlet oxygen is generated with a reduced efficiency from irradiation of DCHB and DNA solution under aerobic conditions as compared with the case in the absence of DNA. All of three reactive oxygen species play an evident role in the photosensitized damage to DNA in aerobic system in addition to the direct electron-transfer damage.     

A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.     

The effects of polyols on the thermal stability of calf thymus DNA

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, (−) and (+) arabitol, -mannitol, -sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (Δ_dH) and temperature of the maximum of the denaturation peak (T_max) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of (−) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for (−) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of (−) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.