Application of tris(2,2'-bipyridyl)ruthenium(III) for the investigation of DNA spatial structure by a chemical modification method

It was shown that the treatment of a DNA fragment and a hepta-decanucleotide having a hairpin structure by Ru(2,2'-bipyridyl)3(3+) complex at 15 degrees C in 1 M NaCl for 10-60 min, and subsequent hydrolysis in 1 M piperidine at 95 degrees C led to the specific cleavage of the polynucleotide chain at guanosine residues in the single-stranded regions. This method can be used for the studying of nucleic acids secondary structure.     

A photoinduced cleavage of DNA useful for determining T residues.

Irradiation of 5'-[32P]-phosphate labeled DNA fragments with ultraviolet light in the presence of primary amines followed by piperidine treatment resulted in base-specific cleavage of the DNA chain at T residues, accompanied by a less intensive G reaction. This simple, T greater than G cleavage offers an alternative method for determining T residues in chemical DNA sequencing.     

Base specific interaction of reductively activated nitroimidazoles with DNA

To exert biological activity, nitroimidazole drugs require reductive activation in vivo. Nucleic acids are susceptible to the activated drug in vitro and are presumably the major target in vivo. We carried out electrolytical reduction of several 5-nitroimidazoles at a controlled potential either in the presence or prior to the addition of DNA. Using a nucleotide sequence specific test to analyse cleavage products, specific interaction of the reduced nitroimidazole intermediate(s) towards the guanine residues is prominent. Since the strand scission depends on subsequent piperidine treatment, it can be concluded that the primary interaction between the activated drug and guanine is a covalent modification weakening the glycosidic bond.     

DNA damage induced by ascorbate in the presence of Cu2+

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.     

Preferential modification of guanine bases in DNA by dimethyldioxirane and its application to DNA sequencing

From gel sequencing experiments with 32P-end-labelled oligodeoxyribonucleotides, it is shown that treatment of DNA with the powerful oxidant dimethyldioxirane, followed by heating in piperidine, causes selective strand scission at the sites of guanine bases. The same specificity for cleavage at guanine was observed with a 45-mer labelled at either the 3'- or 5'-end and with a single and double stranded 34-mer. On account of its speed and operational simplicity, modification with dimethyldioxirane is proposed as a practicable alternative to conventional chemical sequencing procedures for locating guanine bases in DNA.     

Sequence analysis of end-labeled DNA fragments by solvolysis in aqueous solutions of different amines.

Cleavage of 3'-end-labeled DNA in hot aqueous solutions of different amines is comparatively examined for overall rate of DNA scission as well as for potential differences in the preference of the various amines for cleavage at the different bases. Under comparable conditions (0.5 M amine, 0.3 M NaCl, 90 degrees C), piperidine, diethylamine, morpholine, and ethylenediamine produce the same set of labeled fragments, at approximately equal overall cleavage rates. The same set of fragments is also obtained with diisopropylamine, triethylamine, and 1,4-diazabicyclo[2.2.2]octane, but at markedly lower overall cleavage rates. Solvolysis in aqueous piperidine or aqueous diethylamine leads to DNA scission predominantly at A sites, followed by G and C sites, and least frequently at T sites. In contrast, morpholine, ethylenediamine, diisopropylamine, triethylamine, and diazabicyclo[2.2.2]octane cleave the DNA predominantly at G sites. Therefore, use of one of the latter amines allows clear distinction of G bands and C bands, which could not be distinguished by the criterion of band intensity in the original one-lane sequencing method based on cleavage in hot aqueous piperidine (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194-6200). The effect of varying the salt concentration on the cleavage distribution obtained with various amines is also examined, and a rationale is given for the influence of salt concentration and amine basicity on the relative rate of cleavage at G sites.     

Sites of gamma radiation-induced DNA strand breaks after alkali treatment

When DNA is gamma-irradiated in aerated aqueous solution, strand breaks are produced during irradiation or the next few hours. Subsequent piperidine treatment gives rise to further DNA strand ruptures at alkali-labile sites. These different types of DNA chain breaks provoked by gamma-irradiation have been studied with oligonucleotides having defined sequences. The breaks selectively developed inside the DNA chain at alkali-labile sites by piperidine treatment appeared at lower doses preferentially at guanine positions and the order G greater than A greater than T greater than or equal to C was observed. The total contribution of the direct DNA chain ruptures, formed during irradiation and the next few hours, and those obtained by piperidine treatment was studied at doses ranging from 10 to 120 Gy. The chain breaks appeared preferentially at thymine positions and the order T greater than G greater than A greater than or equal to C was shown for the higher doses.     

Solid-phase methods for sequencing nucleic acids. VII. Chemical degradation of synthetic DNA-RNA hybrid fragments using CCS and DE 81 anion-exchange papers

Synthetic oligo(ribo-deoxyribo)nucleotides were analyzed and characterized by different solid-phase chemical degradation procedures, 5'- and 3'-end labelled mixed fragments were degraded by a slightly modified DNA cleavage procedure using 1 and 10% piperidine for the chain scission reaction and CCS anion-exchange paper. Besides the normal degradation products obtained by the usual modification and strand cleavage reactions of both deoxy- and ribonucleotide residues, additional bands were identified in the sequence patterns resulting from the hydrolysis of the RNA moiety induced by piperidine. Since both degradation reactions cleave the backbone of the mixed DNA-RNA fragments differently and produce nucleotide components with different charges, the degradation products do not interfere and can be resolved by gel electrophoresis on polyacrylamide. In addition, 3'-end labelled DNA-RNA oligomers were degraded by a RNA cleavage procedure using DE 81 anion-exchange paper as solid support. The combination of all three degradation methods allows to confirm the nucleotide sequence.     

Reaction of 2-Deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides with Oligonucleotide1

Abstract

Reaction of methyl 2-deoxy-2-C-(3-bromoacetoxypropyl)-α-D-arabinofuranosides, prepared from methyl 2,3-anhydro-α-D-ribofuranoside, with oligodeoxyribonucleotide (21mer) in acetonitrile-H₂O (pH 7) and subsequent treatment with piperidine resulted in the cleavage of the nucleotide chain at the position G, A, and C.     

Photochemical cleavage of DNA by nitrobenzamides linked to 9-aminoacridine

Nitrobenzamido ligands linked to the DNA intercalator 9-aminoacridine via poly(methylene) chains induce single-strand nicks in DNA upon irradiation with long-wavelength ultraviolet light (lambda greater than or equal to 300 nm). Optimal photocleavage activity was found for the reagent 9-[[6-(4-nitrobenzamido)hexyl]amino]-acridine. Removal of the acridinyl ligand or changing the position of the nitro group from the 4- to the 2-position caused a 10-fold decrease in photocleavage efficiency, whereas a change to the 3-position caused a 30-fold reduction. The DNA cleavage was 5-fold enhanced by subsequent piperidine treatment and showed some sequence dependency with predominant cleavage at G and T residues. Furthermore, significant differences in cleavage preference were observed when the poly(methylene) linker length was changed.     

Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator

Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue. On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA. ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides. An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical. These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities.     

A new, convenient method for determining T residues in chemical DNA sequencing by using photoreaction with spermine

Irradiation of 3'-[32p]-labeled DNA fragments with 254-nm light in the presence of spermine resulted in a T specific cleavage of the DNA chain without piperidine treatment. In the presence of methylamine G + T reactions (G greater than T) were observed under the same irradiation conditions. The T reaction with spermine and the G greater than reaction with methylamine may be combined with the standard Maxam-Gilbert's C + T reaction to provide confirmatory determination of DNA sequences.     

The synthesis of a cobalt(II) tetracarboxyphthalocyanine- deoxyribooligonucleotide conjugate as a reagent for the directed DNA modification

The cobalt(II) tetracarboxyphthalocyanine-deoxyribonucleotide pd(TCTTCCCA) conjugate was synthesized. The phthalocyanine N-succinimide ester prepared from phthalocyanine using DCC was mixed in DMF with an aqueous solution of the oligonucleotide bearing a 1,3-diaminopropane linker at the 5'-phosphate. The resulting conjugate was tested in the intraduplex reaction with target 14-mer and 22-mer oligonucleotides containing conjugate-complementary sequences. In the presence of O2 and a thiol (2-mercaptoethanol or DTT) as a coupled reducer or H2O2, sequence-specific DNA modification was observed that caused the cleavage of the target upon treatment with piperidine.     

Sequence analysis of end-labeled DNA fragments by solvolysis in hot aqueous piperidine solutions

One-lane DNA sequencing by solvolysis in hot aqueous piperidine solutions, originally described for 5'-32P-labeled DNA (B. Ambrose and R. Pless (1985) Biochemistry 24, 6194-6200), is extended to 3'-labeled fragments. A salt-free sample for electrophoresis can be obtained by using 1 M LiCl in the solvolysis mixture and removing this salt from the dried hydrolysate by washing with ethanol. Rate and distribution of DNA cleavage in hot aqueous piperidine, containing 0.3 M NaCl, are studied in dependence of temperature, solvent, amine concentration, and reaction time. An increase in temperature strongly accelerates overall DNA degradation, but leaves the distribution of cleavage essentially unchanged. When 50% aqueous ethanol is substituted for water as the reaction solvent, the overall cleavage is slower, and scission at G-sites is enhanced relative to cleavage at the other bases. A rise in the piperidine concentration strongly accelerates the reaction, except at very high amine concentration. Cleavage at A-, G-, and C-sites increases steadily with reaction time, while the T-cleavage observed takes place primarily at the very beginning of the solvolysis.     

Specific binding of 2-amino-1,8-naphthyridine into a single guanine bulge as evidenced by photooxidation of GG doublet

Photoirradiation of 2-amino-1,8-naphthyridines in the presence of duplex DNA containing the GG doublet opposite a single bulge was examined. After hot piperidine treatment, DNA cleavage was observed preferentially at the GG opposite a single bulge. The cleavage efficiency was highly dependent on the nature of bulged base. The G cleavage at the GG opposite a single G bulge was exceptionally weak, suggesting an intercalative binding of 2-amino-1,8-naphthyridine chromophore into the GG step.     

DNA strand scission by activated bleomycin group antibiotics

The bleomycins (BLMs) are a structurally related group of antitumor antibiotics used clinically for the treatment of certain malignancies. The mechanism of action of the BLM is believed to involve DNA strand scission, a process that requires O2 and an appropriate metal ion; the therapeutically relevant metal is probably iron or copper. DNA strand scission by activated Fe X BLM involves oxygenation C-4' of deoxyribose and leads to two sets of products. One set results from scission of the C-3'--C-4' bond of deoxyribose, with concomitant cleavage of the DNA chain. The other set of products consists of free bases and an alkali-labile lesion, the latter of which leads to DNA chain cleavage on subsequent treatment with base. The structures of all of these degradation products have now been established by direct comparison with authentic synthetic samples. Also studied was the activation of BLM with (mono)oxygen surrogates such as iodosobenzene. The chemistry of the activated BLM so formed was remarkably similar to that of activated cytochrome P-450 and structurally related metalloporphyrins, which suggests a mechanistic analogy between the two. Remarkably, both Fe X BLM and Cu X BLM were also shown to be activated by NADPH cytochrome P-450 reductase in a transformation that was dependent on metal ion, O2 and NADPH.     

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a β-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous β-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.     

Sequence selective modification of DNA with muta-carcinogenic 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole

2-Acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole binds covalently to the 8 position of guanine residues in DNA. Treatment of the modified DNA with aqueous piperidine causes the liberation of the modified nucleic acid base, 2-(C8-guanyl)amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole, and cleavage of DNA at the sites of the modified guanylic acid residues. By use of 5'-end 32P-labelled DNA and sequence analysing gel electrophoresis, we discovered the base sequence specificity of DNA modification with 2-acetoxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole. The guanine residues in G-C cluster-like regions are modified more frequently.     

Long-distance radical cation reactions in DNA three-way junctions: inter-arm interaction and migration through the junction

DNA three-way junctions (TWJ) are branched molecules having three ‘arms’. We studied long-distance radical cation migration in these assemblies by incorporating anthraquinone (AQ) groups linked by a covalent tether to one strand of one arm of the TWJ. Excitation of the AQ at 350 nm results in one-electron oxidation of the DNA, which generates a base radical cation. This leads to relatively inefficient (compared with duplex DNA) strand cleavage at guanines following piperidine treatment of the irradiated samples. When the AQ is linked to the 5′-terminus of arm III by a flexible tether, gel electrophoretic analysis shows that strand cleavage occurs at the guanines in all three arms. We also investigated a TWJ in which the anthraquinone is specifically intercalated in arm III. In this case, a different pattern of strand cleavage is detected. We conclude that there are at least two mechanisms for long-distance radical cation migration in TWJs: (i) by inefficient charge hopping through the junction; (ii) by a through-space, cross-arm interaction when the AQ is on a flexible tether.     

Analysis of DNA sequences using a single chemical cleavage procedure

A novel approach to sequence analysis of end-labeled, defined DNA fragments, using a single chemical cleavage procedure and electrophoretic separation in a single lane, has been developed. Prolonged treatment with hot aqueous piperidine results in partial cleavage of the DNA at all positions; the relative propensity for this cleavage is different for the various bases in the DNA. The hydrolysate is resolved on a DNA sequencing gel, and the distribution of radioactivity in the electrophoretic lane is analyzed (a) in terms of differential peak heights of the radioactive bands and (b) in terms of the spacings between successive bands. Simultaneous application of these two base-characteristic criteria allows the deduction of the nucleotide sequence with an accuracy approaching that of the established four-lane methods of DNA sequencing.