Double-stranded Polydeoxyribonucleotides Research Articles

Interaction of Topotecan, DNA Topoisomerase I Inhibitor, with Double-Stranded Polydeoxyribonucleotides. 5. Topotecan is Capable of Producing Single- and Double-Strand Breaks in Circular Supercoiled DNA in the Absence of the Enzyme

A study was made of the temperature, concentration, and time dependences for the emergence of breaks in the sugar-phosphate backbone of a circular supercoiled DNA (scDNA) in the presence of a campto-thecin derivative topotecan (TPT) and in the absence of DNA topoisomerase I (topo I). The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). The incubation time necessary for the appearance of double-strand breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT–DNA complex. This is the first demonstration that molecules of the camptothecin family can cause double-strand breaks in scDNA in the absence of the enzyme. A model is suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of the TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of different strands of one DNA duplex. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines 5 bp apart in one and the same strand of the second DNA duplex.

Interaction of topotecan, DNA topoisomerase I inhibitor, with double-stranded polydeoxyribonucleotides. III. Binding at the minor groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dG-dC). poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism (LD) signal of TPT bound to DNA was shown to have positive sign in the range 260-295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT molecule form an angle lower 54 degrees with the long axis of DNA, and hence TPT molecule can not intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose all cytosines in the major groove were glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC). poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.

Interaction of topotecan, DNA topoisomerase I inhibitor, with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.

Hydrolysis of linear DNA duplex catalyzed by Co(III) complex of cyclen attached to polystyrene

To design artificial restriction enzymes, synthetic catalytic centers that effectively hydrolyze linear double-stranded polydeoxyribonucleotides are needed. The Co(III) complex of cyclen (CoCyc) attached to polystyrene derivatives hydrolyzes linearized pUC18 DNA with half-lives as short as 30 min at 25 °C. The catalytic activity of CoCyc is enhanced by >150 times on attachment to the resin.

Interaction of Topotecan, DNA Topoisomerase I Inhibitor, with Double-Stranded Polydeoxyribonucleotides. 2. Formation of Topotecan–DNA Complex Containing Several DNA Molecules

This study is a continuation of a series of papers dealing with topotecan interaction with double-stranded polydeoxyribonucleotides. We showed earlier that topotecan molecules form dimers in solution at concentration above 10–5(per base pair). Topotecan interaction with calf thymus DNA in solutions of low ionic strength was studied by fluorescence, circular dichroism, and linear flow dichroism. The data obtained indicate that topotecan forms two types of complex with DNA, DNA molecules combining with each other during formation of one of these complexes. The association constant of two topotecan-filled DNA molecules with each other was estimated at 104M–1(per base pair) in 1 mM sodium cacodylate buffer, pH 6.8, at 20°C. A possibility of modulation of DNA topoisomerase I activity by topotecan due to complexation with several sites of a supercoiled DNA molecule is discussed.

Interaction of Topotecan, DNA Topoisomerase I Inhibitor, with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa ≈ 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10–6M) to 7.1 ([TPT] = 10–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7) · 103M–1.

CDNA cloning, expression and functional characterization of an Arabidopsis thaliana homologue of the Escherichia coli DNA repair enzyme endonuclease III.

Reactive oxygen species (ROS) are ubiquitous DNA-damaging agents, and the repair of oxidative DNA lesions is essential to prevent mutations and cell death. Escherichia coli endonuclease III is the prototype repair enzyme for removal of oxidized pyrimidines from DNA. A database homology search identified a genomic sequence in Arabidopsis thaliana encoding a predicted protein with sequence similarity to E. coli endonuclease III. We cloned, sequenced and expressed the corresponding cDNA, which encodes a 39.1 kDa protein containing several sequence motifs conserved in endonuclease III homologues, including an iron-sulfur cluster domain and critical residues at the active site. The protein, designated AtNTH1, was over-expressed in E. coli and purified to apparent homogeneity. AtNTH1 exhibits DNA-glycosylase activity on different types of DNA substrates with pyrimidine damage, being able to release both urea and thymine glycol from double-stranded polydeoxyribonucleotides. The enzyme also possesses an apurinic/apyrimidinic lyase activity on UV- and gamma-irradiated DNA substrates. The AtNTH1 gene contains 10 introns and 11 exons and is widely expressed in different plant tissues. Our results suggest that AtNTH1 is a structural and functional homologue of endonuclease III and probably plays a major role in plant defence against oxidative DNA damage.

Defibrotide, a single-stranded polydeoxyribonucleotide acting as an adenosine receptor agonist

The binding of single-stranded polydeoxyribonucleotides to adenosine A 1 and A 2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [ 3H]CHA (N 6 -cyclohexyl-adenosine) and [ 3H]NECA (5′-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. K i values of 371±68 and 688±115 μg/ml (mean±S.E.M. of 4–5 replications) were computed for adenosine A 1 and A 2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K +-contracted guinea-pig trachealis muscle (IC 50 = 4001 μg/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A 1/A 2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A 1/A 2 receptor blocker 8-phenyltheophylline, but not by the selective A 1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)- xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.

Fd gene 5 protein binds to double-stranded polydeoxyribonucleotides poly(dA.dT) and poly[d(A-T).d(A-T)

Circular dichroism (CD) data indicated that fd gene 5 protein (G5P) formed complexes with double-stranded poly(dA.dT) and poly[d(A-T).d(A-T)]. CD spectra of both polymers at wavelengths above 255 nm were altered upon protein binding. These spectral changes differed from those caused by strand separation. In addition, the tyrosyl 228-nm CD band of G5P decreased more than 65% upon binding of the protein to these double-stranded polymers. This reduction was significantly greater than that observed for binding to single-stranded poly(dA), poly(dT), and poly[d(A-T)] but was similar to that observed for binding of the protein to double-stranded RNA [Gray, C.W., Page, G.A., & Gray, D.M. (1984) J. Mol. Biol. 175, 553-559]. The decrease in melting temperature caused by the protein was twice as great for poly[d(A-T).d(A-T)] as for poly(dA.dT) in 5 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), pH 7. Upon heat denaturation of the poly(dA.dT)-G5P complex, CD spectra showed that single-stranded poly(dA) and poly(dT) formed complexes with the protein. The binding of gene 5 protein lowered the melting temperature of poly(dA.dT) by 10 degrees C in 5 mM Tris-HCl, pH 7, but after reducing the binding to the double-stranded form of the polymer by the addition of 0.1 M Na+, the melting temperature was lowered by approximately 30 degrees C. Since increasing the salt concentration decreases the affinity of G5P for the poly(dA) and poly(dT) single strands and increases the stability of the double-stranded polymer, the ability of the gene 5 protein to destabilize poly(dA.dT) appeared to be significantly affected by its binding to the double-stranded form of the polymer.

Reaction of nucleic acids and cis-diamminedichloroplatinum(II) in the presence of intercalating agents.

The reaction of cis-diamminedichloroplatinum(II) and several synthetic or natural double-stranded polydeoxyribonucleotides has been carried out in the presence of such intercalating agents as ethidium bromide, proflavine, and acridine. After incubation of the reaction mixtures at 37 degrees C for 24 hr, some ethidium or proflavine, but no acridine, molecules are tightly bound to nucleic acids. Tight binding is defined by resistance to extraction with butanol, assayed by filtration at acid pH or by thin-layer chromatography at basic pH. In the ternary complexes, there is about one tightly bound ethidium (or proflavine) per platinum residue. At 37 degrees C, but not at 4 degrees C, tightly bound ethidium exchanges with free ethidium, whereas platinum residues do not exchange. The binding and the release of tightly bound ethidium are very slow (several hours). It is suggested that in the ternary complexes, nucleic acid-cis-Pt(NH3)2-intercalating agent, a bidentate adduct (guanine-ethidium or -proflavine)-cis-Pt(NH3)2, is formed. No tightly bound ethidium or proflavine is found when cis-diamminedichloroplatinum(II) is replaced by trans-diamminedichloroplatinum(II). Competition experiments between cis-diamminedichloroplatinum(II), poly(dG-dC), and poly(dG)-poly(dC) or poly(dA-dT) show that the presence of ethidium bromide, proflavine, or acridine interferes with the distribution of platinum between the polynucleotides. These results might help to explain the synergism for drugs used in combination with cis-diamminedichloroplatinum(II) and in the design of new chemotherapeutic agents.

Sensitization of the Escherichia coli cyclic AMP receptor protein to trypsin cleavage by polydeoxyribonucleotides and polyribonucleotides.

In the absence of cAMP the cyclic AMP receptor protein (CRP) is relatively resistant to trypsin whereas the cAMP X CRP complex is attacked yielding N-terminal core fragments of 14,300 and 18,500 Da which still bind cAMP. The DNA X CRP complex formed at low ionic strength in the absence of cAMP is cleaved by trypsin with the formation of 9,700- and 6,000-Da fragments and the concomitant loss of cAMP binding activity. DNA X CRP remains as resistant to attack by subtilisin, clostripain, and the Staphylococcus aureus V8 protease as unliganded CRP but is slowly digested by chymotrypsin. All of the double-stranded polydeoxyribonucleotides and several of the single-stranded polydeoxyribonucleotides and polyribonucleotides tested render CRP sensitive to cleavage by trypsin. CRP is less rapidly cleaved by trypsin in the presence of d(A)n, d(I)n, and r(C)n indicative of a weaker affinity of CRP for these polynucleotides. The 9,700-Da fragment is N-terminal in CRP and probably terminates at Lys-89. The loss of cAMP binding activity following trypsin cleavage of DNA X CRP indicates that regions beyond this residue are important in the function of the cAMP-binding domain of CRP. The 6,000-Da fragment extends from Val-131 to Arg-185 or Lys-188 and contains part of the F helix involved in DNA binding by CRP.

A DNA glycosylase for oxidized thymine residues in [formula omitted

A DNA glycosylase activity which excises fragmented thymine residues has been identified in cell extracts from Drosophila melanogaster embryos. The enzyme has an apparent M r = 40,000, acts preferentially on double-stranded polydeoxyribonucleotide substrate and requires no co-factors. The DNA glycosylase presumably acts in excision-repair of pyrimidines damaged by hydroxyl radicals and other oxidizing species. This is the first identification of a DNA glycosylase in Drosophila cells.

Enzymatic excision from gamma-irradiated polydeoxyribonucleotides of adenine residues whose imidazole rings have been ruptured.

The main forms of base damage in polydeoxyadenylic acid gamma-irradiated under hypoxic conditions are due to saturation and fragmentation of the adenine imidazole ring. An irradiated polymer was annealed with an equimolar amount of poly (dT) to generate a double-stranded polydeoxyribonucleotide containing scattered damaged base residues. On incubation of the latter with partially purified cell extracts of E.coli, imidazole ring-opened adenine, i.e. 4,6-diamino-5-formamidopyrimidine, was released in free form by a DNA glycosylase activity. The enzyme has been purified 4,500-fold, has Mr = 29,000, and appears to be identical with the previously described DNA repair enzyme formamidopyrimidine-DNA glycosylase.

Urea--DNA glycosylase in mammalian cells.

Urea-DNA glycosylase, an enzyme presumed to be active in the repair of DNA damage caused by oxidizing agents, has been identified previously in Escherichia coli. This enzyme has now been shown to be present in cell extracts of calf thymus and human fibroblasts. It catalyzes the release of free urea from a double-stranded polydeoxyribonucleotide containing thymine residues fragmented by KMnO4 and NaOH treatment. The calf thymus enzyme has been 400-fold purified and largely separated from previously identified mammalian DNA glycosylases. It has a molecular weight of about 25 000 and requires no cofactors. The identity of the enzymatically released product as unsubstituted urea has been verified by its susceptibility to urease.

Some properties of a mitochondrial endonuclease from yeast.

A mitochondrial endonuclease from Saccharomyces cerevisiae was previously shown to cut both strands of native DNA at opposite or nearby sites. The present studied demonstrate that the endonuclease activity is dependent on the strength of the hydrogen bonds between the DNA strands; the activity was measured at different ionic strengths, with substrates of different base compositions and also with DNA in which the double helix has been locally destabilized by ultraviolet irradiation, by depurination, and by single-stranded nicks. The activity is 30% greater with mitochondrial DNA (mt-DNA) than with nuclear DNA. At 0.08 ionic strength, the relative activities with double-stranded polydeoxyribonucleotides are 2.4:1:0.6 for poly(dA).poly(dU) : poly(dA).poly(dT) : poly(dG). poly(dC). Increasing ionic strength decreases similarly the activity with poly(dA).poly(dU) and poly(dA).poly(dT), but has little effect with poly(dG).poly(dC). The greater activity with poly(dA).poly(dU) than with poly(dA).poly(dT) was confirmed with nick-translated mt-DNA and with DNA synthesized in isolated mitochondria using [3H]TTP and [3H]dUTP in both cases. The endonuclease cuts modified DNA preferentially in the thymine dimer regions, at the apurinic sites, and at sites opposite to nicks. The possible involvement of this endonuclease in the degradation of mitochondrial DNA during "petite" induction is discussed.

444 — Electrochemical analysis of polynucleotides

Natural and synthetic polynucleotides were studied by means of differential and normal pulse polarography (d.p.p., n.p.p.) and by cyclic voltammetry (c.v.) in connection with a d.m.e. or h.m.d.e.oaIt was shown that the d.p.p. behaviour of synthetic double-stranded polydeoxyribonucleotides is markedly influenced by the nucleotide sequence; homopolymer pairs (poly(dA) · poly(dT) and poly(dG) · poly(dC)) yield almost no signal under the given conditions, whereas polynucleotides with the alternating nucleotide sequence (poly(dA-dT) · poly(dA-dT) and poly(dG-dC) · poly(dG-dC)) produce well-developed peaks. This difference may be connected with differences in the secondary structures of the two types of polynucleotides.bIt was demonstrated that both synthetic and natural polynucleotides containing guanine produce a characteristic anodic c.v. peak at about −0.2 V (vs s.c.e.). It is necessary to polarize the h.m.d.e. to potentials of about −1.8 V in order to obtain this peak. Polynucleotides which do not contain guanine (poly(A) · poly(C) and poly(U)) do not yield this anodic peak. Similar to cathodic responses, the anodic peak is substantially higher in denatured (single-stranded) DNA than in its native double-helical form.cVarious polynucleotides were modified with osmium tetroxide in an attempt to introduce a new electroactive centre into the polynucleotide chain. It is known that OsO4 reacts with pyrimidine bases in single-stranded polynucleotides. It does not react, however, with bases incorporated in an intact Watson-Crick DNA double helix; this agent can thus act as a chemical probe of DNA structure. It was found that the introduction of osmium into singlestranded poly(C), poly(U), poly(dT) and denatured DNA resulted in the appearance of several reduction signals. It was shown that the d.p.p. behaviour of synthetic double-stranded polydeoxyribonucleotides is markedly influenced by the nucleotide sequence; homopolymer pairs (poly(dA) · poly(dT) and poly(dG) · poly(dC)) yield almost no signal under the given conditions, whereas polynucleotides with the alternating nucleotide sequence (poly(dA-dT) · poly(dA-dT) and poly(dG-dC) · poly(dG-dC)) produce well-developed peaks. This difference may be connected with differences in the secondary structures of the two types of polynucleotides. It was demonstrated that both synthetic and natural polynucleotides containing guanine produce a characteristic anodic c.v. peak at about −0.2 V (vs s.c.e.). It is necessary to polarize the h.m.d.e. to potentials of about −1.8 V in order to obtain this peak. Polynucleotides which do not contain guanine (poly(A) · poly(C) and poly(U)) do not yield this anodic peak. Similar to cathodic responses, the anodic peak is substantially higher in denatured (single-stranded) DNA than in its native double-helical form. Various polynucleotides were modified with osmium tetroxide in an attempt to introduce a new electroactive centre into the polynucleotide chain. It is known that OsO4 reacts with pyrimidine bases in single-stranded polynucleotides. It does not react, however, with bases incorporated in an intact Watson-Crick DNA double helix; this agent can thus act as a chemical probe of DNA structure. It was found that the introduction of osmium into singlestranded poly(C), poly(U), poly(dT) and denatured DNA resulted in the appearance of several reduction signals. After the treatment of native (double-helical) DNA with OsO4 this DNA yielded d.p.p. peaks which are substantially smaller than peaks of single-stranded polynucleotides treated in the same manner. The peaks of the modified native DNA increased if DNA was damaged by various agents such as ionizing and U.V. radiations, which introduce minor local changes into the double helix of DNA.

444 — Electrochemical analysis of polynucleotides

Natural and synthetic polynucleotides were studied by means of differential and normal pulse polarography (d.p.p., n.p.p.) and by cyclic voltammetry (c.v.) in connection with a d.m.e. or h.m.d.e. ( a) It was shown that the d.p.p. behaviour of synthetic double-stranded polydeoxyribonucleotides is markedly influenced by the nucleotide sequence; homopolymer pairs (poly(dA) · poly(dT) and poly(dG) · poly(dC)) yield almost no signal under the given conditions, whereas polynucleotides with the alternating nucleotide sequence (poly(dA–dT) · poly(dA–dT) and poly(dG–dC) · poly(dG–dC)) produce well-developed peaks. This difference may be connected with differences in the secondary structures of the two types of polynucleotides. ( b) It was demonstrated that both synthetic and natural polynucleotides containing guanine produce a characteristic anodic c.v. peak at about −0.2 V ( vs s.c.e.). It is necessary to polarize the h.m.d.e. to potentials of about −1.8 V in order to obtain this peak. Polynucleotides which do not contain guanine (poly(A) · poly(c) and poly(U)) do not yield this anodic peak. similar to cathodic resonses, the anodic peak is substantially higher in denatured (single-stranded) DNA than in its native double-helical form. ( c) Various polynucleotides were modified with osmium tetroxide in an attempt to introduce a new electroactive centre into the polynucleotide chain. It is known that OsO 4 reacts with pyrimidine bases in single-stranded polynucleotides. It does not react, however, with bases incorporated in an intact Watson–Crick DNA double helix; this agent can thus act as a chemical probe of DNA structure. It was found that the introduction of osmium into single-stranded poly(C), poly(U), poly(dT) and denatured DNA resulted in the apearance of several reduction signals. After the treatment of native (double-helical) DNA with OsO 4 this DNA yielded d.p.p. peaks which are substantially smaller than peaks of single-stranded polynucleotides treated in the same manner. The peaks of the modified native DNA increased if DNA was damaged by various agents such as ionizing and U.V. raditions, which introduce minor local changes into the double helix of DNA.

Electrochemical analysis of polynucleotides

Summary Natural and synthetic polynucleotides were studied by means of differential and normal pulse polarography (d.p.p., n.p.p.) and by cyclic voltammetry (c.v.) in connection with a d.m.e. or h.m.d.e. o a It was shown that the d.p.p. behaviour of synthetic double-stranded polydeoxyribonucleotides is markedly influenced by the nucleotide sequence; homopolymer pairs (poly(dA) · poly(dT) and poly(dG) · poly(dC)) yield almost no signal under the given conditions, whereas polynucleotides with the alternating nucleotide sequence (poly(dA-dT) · poly(dA-dT) and poly(dG-dC) · poly(dG-dC)) produce well-developed peaks. This difference may be connected with differences in the secondary structures of the two types of polynucleotides. b It was demonstrated that both synthetic and natural polynucleotides containing guanine produce a characteristic anodic c.v. peak at about −0.2 V ( vs s.c.e.). It is necessary to polarize the h.m.d.e. to potentials of about −1.8 V in order to obtain this peak. Polynucleotides which do not contain guanine (poly(A) · poly(C) and poly(U)) do not yield this anodic peak. Similar to cathodic responses, the anodic peak is substantially higher in denatured (single-stranded) DNA than in its native double-helical form. c Various polynucleotides were modified with osmium tetroxide in an attempt to introduce a new electroactive centre into the polynucleotide chain. It is known that OsO 4 reacts with pyrimidine bases in single-stranded polynucleotides. It does not react, however, with bases incorporated in an intact Watson-Crick DNA double helix; this agent can thus act as a chemical probe of DNA structure. It was found that the introduction of osmium into singlestranded poly(C), poly(U), poly(dT) and denatured DNA resulted in the appearance of several reduction signals. After the treatment of native (double-helical) DNA with O s O 4 this DNA yielded d.p.p. peaks which are substantially smaller than peaks of single-stranded polynucleotides treated in the same manner. The peaks of the modified native DNA increased if DNA was damaged by various agents such as ionizing and U.V. radiations, which introduce minor local changes into the double helix of DNA.

Hypoxanthine in deoxyribonucleic acid: generation by heat-induced hydrolysis of adenine residues and release in free form by a deoxyribonucleic acid glycosylase from calf thymus.

A slow conversion of adenine residues to hypoxanthine occurs in single-stranded DNA when heated in neutral aqueous buffers. The rate of this reaction at pH 7.6 and 110 degrees C is k = 4 x 10(-8) s-1, as determined by base analysis of heat-treated DNA that contains radioactively labeled adenine residues. It is proposed that adenine deamination is one of several forms of hydrolytic damage that may occur as spontaneous premutagenic lesions in DNA in vivo. Cell extracts from calf thymus and human fibroblasts contain a DNA glycosylase activity with specifically catalyzes the release of free hypoxanthine from DNA or polydeoxyribonucleotides that contain dIMP residues. Several properties of the purified enzyme from calf thymus are described: It has an approximate molecular weight of 31 000. No cofactors are required for activity. The enzymatic release of hypoxanthine occurs readily from double-stranded polydeoxyribonucleotides that have either thymine or cytosine residues in the complementary strand. Single-stranded polymers are 10-20-fold more slowly attacked, and there is no detectable cleavage of monomeric dIMP. Hypoxanthine is liberated from DNA directly as a free base. Thus, when poly(dI) x poly(dC) containing both [3H]-dIMP and [32P]dIMP residues was employed as the substrate, 3H-labeled hypoxanthine but no 32P-labeled material was released in ethanol-soluble form. The hypoxanthine-DNA glycosylase presumably acts in DNA repair by preventing deaminated adenine residues from being expressed as mu.

Interaction of cis- and trans-dichlorodiammineplatinum(II) with synthetic polydeoxyribonucleotides: Effect on equilibrium buoyant densities

Equilibrium buoyant densities in Cs 2SO 4 of four synthetic double-stranded polydeoxyribonucleotides were compared following reaction with either cis-Pt(NH 3) 2Cl 2, trans-Pt(NH 3) 2Cl 2 or PtCl 4. While both Pt(NH 3) 2Cl 2 isomers caused significant increases in the buoyant densities (ϱ) of the two G + C polymers, the cis- isomer increased the ϱ of poly[d(G−C)] and the trans- isomer increased the ϱ of poly(dG) · poly(dC) more sharply than the corresponding isomer at low molar ratios of metal ion to polymer. Chloroplatinic acid was without effect. Only cis-Pt(NH 3) 2Cl 2 produced substantial increases in the ϱ of poly[d(A−T)] and poly(dA) · poly(dT) with increasing metal ion concentrations. These increases were accompanied by a proportional decrease in the thermal transition temperature (T m) of poly(dA)-poly(dT) and a sharp increase in the T m of poly[d(A−T)]. Our results indicate that the cis- isomer reacts with all four polymers to produce ϱ changes clearly distinct from those values observed after reaction with either the trans- isomer or PtCl 4. These results help explain the observation by others that the cis- isomer produces greater ϱ changes in native DNA than the trans- isomer. On the basis of our data and the published results of others, one may rationalize the quantitative differences between the cis- and trans- compounds on the buoyant densities of naturally occurring DNA; however, additional studies are clearly needed.