Ernst Freese

Chemical analysis of DNA alterations: I. Base liberation and backbone breakage of DNA and oligodeoxyadenylic acid induced by hydrogen peroxide and hydroxylamine☆

The chemical reactions between DNA and H2O2 or NH2OH, which at low concentration and in the presence of oxygen produces H2O2, have been investigated using DNA and oligodeoxyadenylic acid. Both agents inactivate transforming DNA and cause an extensive decrease in ultraviolet absorption and melting temperature. In addition, they liberate all four bases from DNA and release both adenine and an altered adenine from oligodeoxyadenylic acid. The base liberation is caused by the oxidation of the C-1 carbon of deoxyribose, producing deoxyribonic acid, which has been isolated and characterized. Base liberation and deoxyribonic acid formation, in turn, results in the breakage of the sugar-phosphate backbone, as has been shown by the examination of reaction products obtained from oligodeoxyadenylic acid. Wherever it can occur, β-elimination is the preferred degradation reaction, whereas cyclization occurs when β-elimination is not possible.

The decrease of guanine nucleotides initiates sporulation of Bacillus subtilis.

Massive sporulation of Bacillus subtilis normally begins when carbon, nitrogen or phosphorus sources able to support rapid growth are no longer available. Sporulation can also be induced in exponentially growing cultures, in the presence of rapidly utilizable ammonia, glucose and phosphate if growth is partially but not completely inhibited either by inhibitors of nucleotide synthesis (hadacidin, decoyinine or 6-azauracil) or by purine deprivation in purine and especially in guanine auxotrophs. All these conditions allowing sporulation result in a decrease in the intracellular concentration of guanosine di- and tri-phosphates and usually uridine di- and triphosphates while other nucleotides decrease in some but increase in other cases. A decrease of uracil nucleotides alone, in a uracil auxotroph, does not produce massive sporulation. Our results demonstrate that the partial reduction of a guanine nucleotide, probably relative to some other compound, suffices to initiate sporulation. This reduction may always play a decisive role in the initiation of sporulation, as we have observed it under all conditions so far known to produce massive sporulation.

Inactivating DNA alterations induced by peroxides and peroxide-producing agents

Abstract Agents containing a free-NOH group such as hydroxylamine, N -methylhydroxylamine, hydroxyurea, hydroxyurethan, and hydrazines produce H 2 O 2 on exposure to oxygen. All these agents, H 2 O 2 itself, and disuccinyl peroxide, predominantly induce inactivating DNA alterations, whereas their mutagenic effect (per lethal hit) on transformung DNA is small. Chemicals containing NH 2 in place of NOH, such as urea and urethan (= ethylcarbamate) do not inactivate DNA. Formaldehyde and its oxidation products, formic acid, performic acid, and di-hydroxymethyl peroxide do not inactivate or mutate transforming DNA at a significant rate.

Induction of glycolipid biosynthesis by sodium butyrate in HeLa cells

Summary Butyrate inhibits the growth of HeLa cells and markedly alters their morphology as the cells acquire a more fibroblast-like shape by greatly extending cellular processes. Butyrate simultaneously causes an increase in cellular sialylactosylceramide content by elevating CMP-sialic acid: lactosylceramide sialyltransferase activity (7–24 fold); whereas other glycolipid glycosyltransferase activities do not increase. Induction of this specific sialyltransferase is blocked by cycloheximide or actinomycin D. This first report on the induced synthesis of glycolipid components suggests that these complex carbohydrates have a role in cell growth and morphology.

Induction of sporulation in Bacillus subtilis by decoyinine or hadacidin.

Summary Bacillus subtilis , growing exponentially in the presence of rapidly metabolizable carbon, nitrogen and phosphate sources, can be induced to sporulate by the addition of decoyinine, a specific inhibitor of GMP synthesis, or of hadacidin, a specific inhibitor of AMP synthesis. Optimal sporulation is obtained at inhibitor concentrations causing only partial inhibition of growth.

DNA damage caused by antidepressant hydrazines and related drugs.

Abstract Antidepressant hydrazines ( e.g. isocarboxazid, nialamide, and phenelzine) and the anti-tubercle agent isoniazid react with oxygen, producing hydrogen peroxide. The radicals formed in this reaction inactivate DNA. These compounds, as well as carbamates that can be enzymatically converted to reactive N -hydroxycarbamates, may therefore cause chromosomal breaks and mutations.

Purification and chemical characterization of alanine dehydrogenase of Bacillus subtilis

Abstract 1. 1. Bacillus subtilis alanine dehydrogenase ( L -alanine: NAD oxidoreductase (deaminating), EC1.4.1.1. has been purified by column chromatography using calcium phosphate gel, DEAE-Sephadex and Ecteola-cellulose; it has been obtained in crystalline form. 2. 2. The sedimentation pattern indicated a homogenous preparation. The sedimentation constant at infinite dilution was 8.8 S. The molecular weight estimated by the sedimentation equilibrium method was 228 000. 3. 3. Amino acid analysis gave the following ratios of amino acid residues: Asp, 43; Thr, 34; Ser, 20; Glu, 51; Pro, 25; Gly, 51; Ala, 65; CySH, 2; Val, 49; Met, 11; iLeu, 28; Leu, 42; Tyr, 15; Phe, 7; Try, 1; Lys, 28; His, 9; Arg, 14.

Chemical analysis of DNA alterations: II. Alteration and liberation of bases of deoxynucleotides and deoxynucleosides induced by hydrogen peroxide and hydroxylamine

Abstract During the reaction of H2O2 with deoxyribonucleotides or their components, the ultraviolet absorption decreased at rates which were for cytosine > thymine > adenine > guanine . The chromatographic separation of the reaction mixtures revealed that all four bases were partially removed from both deoxynucleotides and deoxynucleosides, the reaction rates being for thymine > adenine > cytosine > guanine . Since no deoxynucleosides were formed from deoxynucleotides, H2O2 apparently cleaved only the N- glycosidic linkage, the phosphate group not being involved. dpA, dA, and adenine gave rise to an additional ultraviolet-absorbing base, A1. Kinetic arguments showed that dpA was first altered to dpA1 and then A1 was liberated. A quantitative analysis of the ultraviolet-absorbing products showed that the base liberation was only partially responsible for the loss of ultraviolet absorption: some of the purine and the majority of the pyrimidine bases must have been converted to non-ultraviolet-absorbing products, the relative effectiveness of alteration being for cytosine > thymine > adenine > guanine . Experiments with nucleotides labeled in the base moiety showed that most of the bases which lost their ultraviolet absorption remained attached to the deoxyribose phosphate. The relative frequency with which the doexynucleotide molecules were decomposed was for adenine > thymine > cytosine > guanine . 0.1 M NH2OH also liberated and destroyed bases, similar to H2O2, but it produced a different altered adenine: A2.

Commitment to sporulation and induction of glucose-phosphoenolpyruvate-transferase

Abstract Glucose suppresses sporulation of Bacillus subtilis when it is present during exponential growth in nutrient sporulation medium but not when it is added at any time after T 0 , the time at which growth has started to decline from the exponential rate. After this time, B. subtilis cells are committed with respect to glucose to sporulate. While toward the end of growth no significant changes in the specific activities of the glycolytic enzymes occur which could explain this resistance to glucose suppression, the glucose-phosphoenolpyruvate-transferase system does change drastically. The latter enzyme system is inducible by glucose during the exponential growth phase and so is the uptake of glucose, whereas the specific activity of the ATP-dependent glucokinase remains constant throughout growth and is not influenced by glucose addition. The phosphoenolpyruvate-transferase (but not the glucokinase) also phosphorylates α-methylglucoside which can be more advantageously used for assaying the enzyme. Toward the end of growth, the uptake of glucose and the activity of glucose-phosphoenolpyruvate-transferase decline in correlation with the inability of glucose to suppress sporulation of committed cells. This decline occurs also in pre-induced cells, again in correlation with the fact that preinduced cells can still enter the commitment of sporulation with respect to glucose after glucose is withdrawn from the medium. In confirmation of these findings, a mutant, which grows not on fructose or glucosamine, scarcely on glycerol, and slowly on glucose, and which lacks phosphoenolpyruvate-transferase activity, can sporulate when glucose is present throughout growth and sporulation.

Chemical analysis of DNA alterations IV. Reactions of oligodeoxynucleotides with monofunctional alkylating agents leading to backbone breakage

Abstract The mechanisms leading to inactivating DNA alterations by the monofunctional alkylating agents methyl and ethyl methane sulfonate have been studied using oligodeoxythymidylic and oligodeoxyadenylic acids of different chain length as well as mononucleotides and their components. Thymine did not react and deoxythymidylic acid was alkylated exclusively at the phosphate group, about twice as fast as guanine at the 7-position. The alkylation of oligodeoxythymidylic acids produced products, separated by chromatography, which proved the existence of triester breakage. During alkylation of dGMP and dAMP, the amount of free alkylated guanine and adenine both increased with the square of time, which shows a two-step reaction of first alkylation and then depurination. The identification of certain reaction products (dAMP) of oligodeoxyadenylic acid furthermore proved the existence of a small but significant amount of backbone breakage which accompanied alkylation at neutral pH. A quantitative evaluation of the results concluded that triester breakage and depurination of DNA should occur with about equal frequency, both increasing with the square of the time during treatment and linearly afterwards. In contrast, backbone breakage caused by depurination should increase with the third power of time during treatment and with the square of the time afterwards. The results are significant for the explanation of the effects of deoxyribonucleases or repair mechanisms on alkylated DNA.