The interaction of ribonuclease T 1 with DNA.

The interaction of RNase T1 with calf thymus DNA was studied using uv difference spectroscopy and the effect of the enzyme on DNA melting. There was no indication of RNase T1 binding with native DNA. A prominent difference spectrum for RNase T1 binding with denatured DNA (d-DNA) was observed at pH 5, 25 degrees and low ionic strength (mu = .01 M) which was depressed at higher ionic strength and pH. The normalized difference spectrum at mu = .01 M, pH 5 and 25 degrees can be interpreted as indicating an interaction of an exposed guanine residue directly with the enzyme and a coupling of this process with the "melting" of short folded segments of d-DNA. The apparent association constant calculated per M guanine residues was 2.4 X 10-4 M-1 under these conditions. The results are discussed in reference to comparable studies on the interaction of RNase T1 with RNA and small guanine ligands.     

Features of the damage produced by proflavine on transforming deoxyribonucleic acid.

Proflavine formed a complex with transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae, with optimal formation at a ratio of proflavine to DNA of 0.06. The rate of dissociation of the complex by dialysis increased in the order: native, denatured, renatured DNA. The transforming activity of the DNA was reduced by its interaction with proflavine. This inactivation was dependent on the physical state of the DNA, the proflavine concentration, and the temperature. DNA that had been denatured and renatured was most sensitive; native DNA was much less sensitive. The inactivation remained after dialysis and was stable to prolonged storage. It is concluded that the inactivation of transforming DNA by proflavine takes place by a mechanism different from that of DNA-proflavine complex formation.     

Cyclic voltammetry of DNA at a mercury electrode: an anodic peak specific for guanine

Synthetic homopolyribonucleotides poly(A), poly(U), poly(C), and poly(G), poly(A, G, U), apurinic acid and native and denatured DNA from calf thymus were analyzed by means of cyclic voltammetry (CV) using a hanging mercury drop electrode. It was shown that guanine containing polynucleotides, i.e. poly(G), poly(A, G, U) and DNA yield an anodic peak of guanine in the vicinity of a potential of -0.3 V (against a saturated calomel electrode). The guanine peak appeared only at a sufficiently negative switching potential (about -2 V). The appearance of the guanine peak was conditioned by a reduction of guanine residues in the region of the switching potential and reoxidation of the reduction product in the vicinity of -0.3 V. Native and thermally denatured DNAs were investigated under the conditions of both complete and incomplete coverage of the electrode in various background electrolytes. Both DNA forms yielded anodic CV peaks of guanine with the peak of denatured DNA being always higher than that of native DNA. Irradiation of native DNA with relatively small doses of gamma radiation (5-120 Gy) resulted in an increase of the anodic peak. A comparison of changes induced by gamma radiation in the anodic (guanine) and cathodic (reduction of adenine and cytosine) peaks showed a steeper increase of the cathodic peak as compared to that of the anodic one. It has been concluded that in the given dose range the DNA double-helical structure is mainly damaged in the adenine-thymine rich regions.     

Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of guanine residues, whereas the more positive peak, A, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Alkylation of DNA by melphalan in relation to immunoassay of melphalan-DNA adducts: characterization of mono-alkylated and cross-linked products from reaction of melphalan with dGMP and GMP

A product expected to result from cross-linking of guanine bases in DNA by melphalan (4-(2-(di-guanin-7-yl))ethylamino-L-phenylalanine) was obtained from hydrolysis of melphalan-treated sodium deoxyguanylate at pH7 and characterized by U.V. and mass spectra. When tested in a competitive immunoassay using an antibody specific for melphalan-alkylated DNA it showed an affinity intermediate between that of melphalan-alkylated DNA and melphalan. From this and other assays it seemed possible that the cross-linked moiety in DNA was recognised by the antibody, but that its conformation differed from that of the free base tested, sufficiently to account for the discrepancy. It seemed possible that cross-linked guanine nucleotides would provide a better model, and these were therefore isolated, characterised and tested. Products derived from cross-linking of guanylic acid moieties through N-7 and N-7, and through N-7 and phosphate, had higher affinity than the cross-linked base, approximately the same as for alkylated native DNA, but less than for alkylated denatured DNA or RNA.     

Complexing and denaturation of DNA by methylmercuric hydroxide. II. Ultracentrifugation studies

When increasing concentrations of methylmercuric hydroxide are added to a Cs₂SO₄ solution of native DNA, the buoyant density of DNA is unaltered until a critical concentration is reached above which there is a cooperative transition to denatured DNA which now binds so much CH₃HgOH that it becomes very dense and nonbuoyant. As increasing concentrations of methylmercuric hydroxide are added to a Cs₂So₄ solution of denatured DNA, the buoyant density gradually increases, indicating a gradual increase in the amount of methylmercury cation bound. The denatured DNA methylmercury complex becomes nonbuoyant at the same concentration of methylmercuric hydroxide as does the native DNA. These results support our previous interpretation that CH₃HgOH reacts with the imino NH bonds of thymine and guanine in nucleic acids. The reaction occurs more or less independently at the different binding sites for denatured DNA, but it occurs cooperatively with simultaneous denaturation for native DNA. The nature of the transition of denatured DNA to the nonbuoyant state is not known, but it is probably due to an abrupt decrease in the degree of hydration of the DNA when its density and hydrophobic character are sufficiently increased by the binding of the methylmercury cation. Direct measurements of the amount of methylmercury bound by DNA, as observed by preparative ultracentrifugation, confirm approximately the buoyant density results as to the amount of methylmercury bound. The possibility of using methylmercuric hydroxide as a reagent for the separation of complementary strands, depending on then thymine of their thymine plus guanine content, is discussed.     

Interaction of nucleic acids with electrically charged surfaces. VI. A comparative study on the electrochemical behaviour of native and denatured DNAs at graphite electrodes.

Adsorption and electrochemical oxidation of deoxyribonucleic acid (DNA) at a pyrolytic graphite electrode (PGE) and a paraffin wax-impregnated spectroscopic graphite electrode (WISGE) were studied using differential pulse voltammetry. DNA is adsorbed at the surface of the graphite electrodes in a broad range of potentials including the potentials of electrochemical oxidation of DNA. Both native and denatured DNAs yield two single, well-defined and separated peaks, G and A, on the differential pulse voltammograms at the PGE and WISGE. The more negative peak, G, corresponds to electrochemical oxidation of adenine residues. Peaks G and A of native DNA occur at the same potentials as peaks G and A of denatured DNA. However, electrochemical oxidation of adenine and guanine residues at graphite electrodes is markedly suppressed in native DNA. The heights of the peaks G and A represent a sensitive indicator of the helix-coil transition of DNA. An analysis of the product of interaction of a sample of native DNA with a large pyrolytic graphite electrode in the presence of formaldehyde at approximately neutral pH did not prove changes in the secondary structure of native DNA due to its interaction with the graphite electrode. It is suggested that the decreased differential pulse-voltammetric activity of native DNA is connected with its decreased flexibility.     

Comparison of the inactivation of IgM and IgG complement fixation sites by acid and base.

Rabbit IgM antibodies to denatured mammalian or T6 bacteriophage DNA or poly(A)-poly(U) irreversibly lost complement-(C) fixation reactivity on exposure to low pH and reneutralization, with a halving of the complement-fixation titer occurring after treatment at about pH 3. The titers of IgG antibodies to denatured phage DNA, to poly(A)-poly(U), or to hemocyanin were halved only after exposure to pH 2. Inactivation by acid was enhanced by low protein concentrations, incubation at higher temperatures, and by slow reneutralization; under all these conditions it was more extensive with IgM than with IgG. Inactivation of IgM C-fixation activity at pH 2.5 and room temperature was a first order reaction, with a half-time of about 20 min. Both classes retained antigen-binding activity after exposure to pH 2. In the alkaline range, full C-fixation reactivity was retained by both classes after reneutralization from pH 11.5, some loss occurred at pH 12, and total irreversible inactivation occurred by pH 12.5. In the latter case, antigen-binding activity was also lost. The C-fixation inactivation curves in the alkaline range were similar for IgG and IgM antibodies.     

Single-strand-specific nuclease of pea seeds: glycoprotein nature and associated nucleotidase activity

A single-strand-specific nuclease from germinating pea seeds has been purified to homogeneity. The purification procedure includes affinity chromatography on concanavalin A-Sepharose and gel filtration. The nuclease exhibits its activity at neutral pH and does not have an absolute requirement for a divalent cation. The purified nuclease also possesses a 3'-nucleotidase activity and is a glycoprotein containing about 20% carbohydrate. On native polyacrylamide gels the nuclease activity comigrates with the nucleotidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits in the native enzyme. The nuclease and nucleotidase activities show differential rates of thermal inactivation, the latter following simple first order kinetics and the former exhibiting a more complex reaction. The nucleotidase was also found to be stimulated by DNA, the increase being greater with native DNA than with denatured DNA. These properties are possibly accounted for by the dimeric structure of the enzyme where the nucleotidase catalytic site resides in one subunit while the nuclease site is formed by interaction of both subunits. The enzyme also hydrolyzes double-stranded alkylated DNA and depurinated DNA at a higher rate than native DNA. Experimental evidence suggests that depurinated DNA is hydrolyzed in the region of apurinic sites.     

Partial purification and properties of an endonuclease from germinating pea seeds specific for single-stranded DNA.

An enzyme that rapidly catalyzes the hydrolysis of denatured DNA has been partially purified from germinated pea (Pisum sativum) seeds. The nuclease has been characterised as having endonucleolytic activity degrading single stranded DNA at a 15- to 20-fold higher rate than native DNA. From exclusion chromatography on Sephadex G-200 the molecular weight of the enzyme was calculated to be 42,000. The small extent of hydrolysis of native DNA is suggested to be due to the degradation of partially denatured areas in the native molecule. The enzyme shows activity over a broad range of pH but was most active between pH 6.5 and 8.0. The maximum hydrolysis of denatured DNA was observed at 45 °C while with native DNA the temperature optima was 60 °C. The nuclease does not show an absolute requirement for added divalent cations. However, the addition of Mg²⁺ and Ca²⁺ results in 40 and 60% stimulation, respectively. EDTA has no effect on enzymatic activity, whereas 8-hydroxyquinoline was inhibitory.     

Binding of pyrene to DNA, base sequence specificity and its implication.

Solubilization as well as spectral studies of pyrene in natural DNA and synthetic deoxypolynucleotide solutions at neutral pH reveal at least two binding modes. Sites I are predominant in native DNA and in poly(dA-dT): poly(dA-dT) whereas sites II are found with denatured DNA and other polynucleotides such as poly(dA):poly(dT) and three different types of guanine containing copolymers which solubilize pyrene to a lesser extent. Spectral comparison with the covalent adducts of trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10- tetrahydro-benzo(a)pyrene (anti-BPDE) and the physical complexes of its tetraols lead to the suggestion of a base sequence specific binding model for this carcinogenic metabolite to account for the puzzling fact that although its physical binding is predominantly intercalative, the covalent adducts appear not to be intercalated. It is speculated that in neutral solutions, intercalation may have little, if any, to do with the chemical lesion of this metabolite to the guanine base of the DNA and may, on the contrary, provide an efficient pathway for detoxification.     

Effect of alkylation with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea on the secondary structure of DNA

We have earlier reported that alkylation of DNA by the chemical carcinogen dimethyl sulphate, which mainly alkylates N-7 of guanine and N-3 of adenine, causes the formation of partially denatured regions in double-stranded DNA (Rizvi RY, Alvi NK & Hadi SM, Biosci. Rep. 2, 315-322, 1982). It is known that the major site of alkylation in DNA by N-ethyl-N-nitrosourea (EtNu) are the phosphate groups. N-methyl-N-nitrosourea (MeNu), on the other hand, causes the alkylation of mainly guanine residues. We have therefore studied the effect of these two alkylating carcinogens on the secondary structure of DNA. DNA alkylated with increasing concentrations of EtNu and MeNu was subjected to alkaline and S1 nuclease hydrolysis. Thermal melting profiles of alkylated DNA were also determined using S1 nuclease. The results indicated that alkylation by the two alkylating agents had a differential effect on the secondary structure of DNA. EtNu-alkylated DNA was found to be more thermostable than native DNA at neutral pH. It was however more alkali-labile than MeNu-alkylated DNA. The greater stability of EtNu-alkylated DNA was considered to be due to abolition of negative charges on phosphate alkylation.     

Polarographic reducibility of denatured DNA

The de polarographic behavior of native and denatured DNA at pH 7.0 was studied. Whereas native DNA was polarographically inactive under the given conditions, denatured DNA yielded a reduction polarographic step at the potential of about ?1.4 V. Native DNA produced a single desorption wave on ac polarograms, while denatured DNA yielded, in addition to this wave, another more negative wave approximately corresponding, as to its potential, to the dc polarographic step of denatured DNA. The behavior of apurinic acid was similar to that of denatured DNA. The course of DNA denaturation at elevated temperature was studied by means of the two above techniques and changes at temperatures below the melting temperature observed. This finding is in agreement with earlier results obtained by oscillopolarographic and the pulse-polarographic method.     

The interaction of chromium with nucleic acids

Native and denatured calf thymus DNA, and homopolyribonucleotides were compared with respect to chromium and protein binding after an in vitro incubation with rat liver microsomes, NADPH, and chromium(VI) or chromium(III). A significant amount of chromium bound to DNA when chromium(VI) was incubated with the native or the denatured form of DNA in the presence of microsomes and NADPH. For both native and denatured DNA the amount of protein bound to DNA increased with the amount of chromium bound to DNA. Denatured DNA had much higher amounts of chromium and protein bound than native DNA. There was no interaction between chromium(VI) and either form of DNA in the absence of the complete microsomal reducing system. The binding of chrornium(III) to native or denatured DNA was small and relatively unaffected by the presence of microsomes and NADPH. The binding of chromium and protein to polyriboadenylic acid (poly(A)), polyribocytidylic acid (poly(C), polyri-boguanylic acid (poly(G)) and polyribouridylic acid (poly(U)) was determined after incubation with chromium(VI) in the presence of microsomes and NADPH. The magnitude of chromium and protein binding to the ribo-polymers was found to be poly(G) ? poly(A) ? poly(C) ? poly(U). These results suggest that the metabolism of chromium(VI) is necessary in order for chromium to interact significantly with nucleic acids. The metabolically-produced chromium preferentially binds to the base guanine and results in DNA-protein cross-links. These findings are discussed with respect to the proposed scheme for the carcinogenicity of chromium(VI). Keywords: DNA-protein cross-links — Chromium-guanine interaction-Microsomal reduction of chromate     

INACTIVATION OF CRYSTALLINE TRYPSIN

1. The rate of inactivation of crystalline trypsin solutions and the nature of the products formed during the inactivation at various pH at temperatures below 37°C. have been studied. 2. The inactivation may be reversible or irreversible. Reversible inactivation is accompanied by the formation of reversibly denatured protein. This denatured protein exists in equilibrium with the native active protein and the equilibrium is shifted towards the denatured form by raising the temperature or by increasing the alkalinity. The decrease in the fraction of active enzyme present (due to the formation of this reversibly denatured protein) as the pH is increased from 8.0 to 12.0 accounts for the decrease in the rate of digestion of proteins by trypsin in this range of pH. 3. The loss of activity at high temperatures or in alkaline solutions, just described, is very rapid and is completely reversible for a short time only. If the solutions are allowed to stand the loss in activity becomes gradually irreversible and is accompanied by the appearance of various reaction products the nature of which depends upon the temperature and pH of the solution. 4. On the acid side of pH 2.0 the trypsin protein is changed to an inactive form which is irreversibly denatured by heat. The course of the reaction in this range is monomolecular and its velocity increases as the acidity increases. 5. From pH 2.0 to 9.0 trypsin protein is slowly hydrolyzed. The course of the inactivation in this range of pH is bimolecular and its velocity increases as the alkalinity increases to pH 10.0 and then decreases. As a result of these two reactions there is a point of maximum stability at about pH 2.3. 6. On the alkaline side of pH 13.0 the reaction is similar to that in strong acid solution and consists in the formation of inactive protein. The course of the reaction is monomolecular and the velocity increases with increasing alkalinity. From pH 9.0 to 12.0 some hydrolysis takes place and some inactive protein is formed and the course of the reaction is represented by the sum of a bi- and monomolecular reaction. The rate of hydrolysis decreases as the solution becomes more alkaline than pH 10.0 while the rate of formation of inactive protein increases so that there is a second point at about pH 13.0 at which the rate of inactivation is a minimum. In general the decrease in activity under all these conditions is proportional to the decrease in the concentration of the trypsin protein. Equations have been derived which agree quantitatively with the various inactivation experiments.     

Experimental modelling of thermal inactivation of urease

Thermal inactivation of jack bean urease (EC 3.5.1.5) was investigated in a 0.1 M phosphate buffer with pH 7. An injection flow calorimetry method was adapted for the measurement of the enzyme activity. The inactivation curves were measured in the temperature range of 55 to 87.5 degrees C. The curves exhibited a biphasic pattern in the whole temperature range and they were well fitted with a biexponential model. A simultaneous fit of all inactivation data was based on kinetic models that were derived from different inactivation mechanisms and comprised the material balances of several enzyme forms and the enthalpy balance characterizing the initial heating period of enzyme solution. The multitemperature evaluation revealed that an adequate model had to incorporate at least three reaction steps. It was concluded that the key reaction steps at urease thermal inactivation were the reversible dissociation/denaturation of native form into an inactive denatured form, and irreversible association reactions of both the denatured and native forms.     

Sedimentation and intrinsic viscosity behavior of PM2 bacteriophage DNA in alkaline solution

The sedimentation coefficient and intrinsic viscosity of nicked and closed circular PM2 bacteriophage DNA have been measured as a function of pH in the alkaline region. A gradual increase in the sidimentation coefficient, and a corresponding decrease in the intrinsic viscosity, are observed for the superhelical (closed) circle in the pH region from 10.5 to about 10.9. This has been tentatively interpreted in terms of the known dependence of sedimentation coefficient upon the number of superhelical turns. At slightly higher pH values, the curve passes through the minimum (sedimentation coefficient) and maximum (intrinsic viscosity) expected when the superhelical turns present at neutral pH are unwound by partial alkaline denaturation. Sedimentation studies of the relaxed (nicked) circular species have revealed the existence of DNA forms in the pH region from 11.27 to 11.37 which sediment considerably faster than the closed circle in the same pH region. These have been identified as partially denatured nicked circles, in which varying fractions of the duplex structure have undergone alkaline denaturation, but strand separation has not yet occurred. Varying fractions of a slower species, either undenatured or completely denatured nicked circles, are also observed in some of these experiments. A corresponding result is observed in the intrinsic viscosity vs. pH curve. When nicked circular PM2 DNA is exposed to various alkaline pH's, rapidly neutralized, and sedimented at neutral pH, the expected sharp transition from native to denatured (strand-separated) molecules is seen. However, a very narrow pH range is noted in which native and denatured forms coexist in a single experiment. The above experiments carried out upon the closed form also reveal a narrow pH range in which the bulk of the transition from native closed circles to the collapsed cyclic coil takes place, in acccord with an earlier study on a different DNA. This transition is shown never to be completely effected, however, as there is a fraction (7–8%)of the closed circles which renature to the native form, regardless of the alkaline pH employed. This same phenomenon was not observed in the case of artificially closed λb₂b₅c DNA circles. Possible explanations for some of the above results are discussed.     

Bacteriophage XP-12-induced exonuclease which preferentially hydrolyzes nicked DNA.

An exonuclease has been partially purified from XP-12-infected Xanthomonas oryzae which is not found in uninfected X. oryzae. Although both the phage-induced exonuclease and the major host exonucleolytic DNase released 5'-mononucleotides, these enzymes differed in their chromatographic behavior, pH optimum, salt inhibition, and heat sensitivity. These two exonucleases preferred different substrates. Nicked native DNA was the best substrate for the phage-induced enzyme, whereas denatured DNA was the best substrate for the host enzyme. Also, the host enzyme had a significant preference for denatured or nicked, normal cytosine-containing DNA (e.g., X. oryzae or T7 DNA) over similarly denatured or nicked 5-methylcytosine-rich DNA (namely, XP-12DNA), whereas the phage-induced enzyme hydrolyzed both types of DNA equally well.     

Bindings of RNAse to denatured and native deoxyribonucleic acids

The binding of pancreatic ribonuclease-A by denatured DNA, native DNA, poly-dA, and poly-dT, has been studied by a gel filtration method. With denatured DNA at pH 7.5, ionic strength 0.053M, there is one binding site per 12 nucleotides and the equilibrium binding constant per site is 9.7 × 10⁴ l./mole. The binding constant increases by a factor of 8 as the pH is decreased from 8 to 7. The strength of the binding of denatured DNA increases with decreasing ionic strength. At pH 7.5, native DNA binds about ? as strongly as does denatured DNA. The binding affinity increases in the order poly-dA, denatured DNA, and poly-dT. These results support the view that the binding of denatured DNA involves both electrostatic interactions between the negatively charged polynucleotide and the positively charged protein, and an interaction of the protein with a pyrimidine residue of the denatured DNA, and thus that the binding is basically similar to that between RNAse and its substrate RNA.     

S-adenosylmethionine: DNA-cytosine 5-methyltransferase from a Novikoff rat hepatoma cell line.

Partial purification of DNA methylase from Novikoff rat hepatoma cells is described. Contamination with other proteins persists although the enzyme preparation has a high specific activity and is purified 980-fold over homogenate activity. Evidence suggests, but does not prove, that there may be more than one species of DNA methylase in these cells. The enzyme has two broad pH optima at pH 7.0 and 7.5 and most readily methylates heterologous denatured DNAs although complex reaction kinetics indicate that native DNAs may eventually be methylated to an equal or greater level. The preparation of undermethylated DNA from Novikoff cells is also described. Undermethylated homologous DNA is an 85-fold greater acceptor of methyl groups than fully methylated Novikoff cell DNA. In contrast to other DNA substrates, the enzyme preparation methylates native undermethylated homologous DNA at a 3.5-fold greater than denatured undermethylated homologous DNA.