Effect of alkylation with streptozotocin on the secondary structure of DNA

S₁ nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of the alkylating antibiotic, streptozotocin, on the secondary structure of DNA. Native calf thymus DNA was alkylatedin vitro with increasing concentrations of streptozotocin and subjected to S 1 nuclease hydrolysis. An increasing degree of DNA degradation was seen, suggesting a destabilization of the secondary structure. Indirect evidence, deduced from alkaline hydrolysis, effect of NaCl on S₁ nuclease hydrolysis, and hydroxyapatite chromatographic analysis of alkylated DNA, suggested a significant alkylation of DNA phosphates in addition to DNA bases. Nictotinamide has been reported to alter the cytotoxic and carcinogenic effects of streptozotocin. Our experiments indicate that in the presence of nicotinamide, streptozotocin causes the formation of a greater proportion of alkylated bases in relation to alkyl phosphotriesters. This may have significance in relation to the differential cytotoxicity of streptozotocin in the absence and presence of nicotinamide.     

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.     

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.     

Electron microscopy of simian virus 40 DNA configuration under denaturation conditions.

After isolation, the DNA of simian virus 40 appeared as a negative supertwist (form I) or as an open circle with at least one single-strand scission (form II). Under the denaturation conditions usually applied, such as heating in the presence of formaldehyde or application of alkali, form I molecules could appear as "relaxed" circles without single-strand scissions (form I') containing denatured sites not visible under the electron microscope. Form II molecules, under these denaturation conditions, showed partial or complete strand separations allowing the construction of denaturation maps. By using a modified denaturation procedure, i.e., heating of isolated SV40 DNA in the presence of dimethyl sulfoxide and formaldehyde followed by keeping the DNA in this denaturation solution at room temperature for periods up to 3 weeks, partially denatured relaxed circles without single-strand scissions were produced (form I'D) in addition to completely denatured form II molecules. The absence of single-strand scissions in form I'D molecules was demonstrated by a second heat treatment, which did not change the configuration of this molecular form. Form I'D molecules, in contrast to form I', contained denatured sites clearly discerible under the electron microscope. This combined application of two subsequent denaturation steps (denaturation by heating followed by denaturation at room temperature and neutral pH) showed that the molecular configuration I'D originated in two steps. The heating procedure produced molecules not distinquishable by electron microscopy from form I. In contrast to form I, these molecules were assumed to possess "preformed" denaturation sites (form I). Further treatment of form I molecules with denaturation solution at room temperature finally transformed them into convalently closed, relaxed, partially denatured circles exhibiting strand separations easily measurable on electron micrographs (form I'D). Denaturation maps of form I'D molecules were constructed by computer and compared with denaturation maps derived from partially denatured form II molecules. From these denaturation maps it can be concluded that the melting of base pairs occurring during the transition of simian virus 40 DNA form I into form I'D also preferentially happened at sites rich in the bases adenosine and thymine.     

Gel electrophoresis of partially denatured DNA. Retardation effect: its analysis and application.

The hypothesis about the role of partial denaturation in DNA retardation during its electrophoresis in denaturing gel /1,2/ was tested. We used partially melted DNA molecules in which the size of the melted regions and their location were known. They were obtained through glyoxal treatment of the melted regions by a procedure allowing the denatured state to be fixed at any point within the melting range. The approach and the availability of the melting maps of DNAs made it possible to investigate DNA molecules differing in length and in the size of the melted regions. The presence of a denatured region at the end of the molecule or inside of it was shown to decrease its electrophoretic mobility, the effect depending on the size of the melted region and on the DNA length. On the basis of the experimental results an explanation is proposed for the cause of retardation in the case of partially denatured DNA.     

Physical map of the DNA of bacteriophage tf of Pseudomonas putida

A physical map has been constructed for P. putida bacteriophage tf DNA containing single-strand breaks (nicks). Localization of cleavage sites for EcoRI, HindIII, HpaI ClaI, BamHI, SalI, XbaI and XhoI restriction endonucleases was determined. Position of single-strand breaks was mapped by electrophoretic analysis of denatured tf DNA and electron microscopy of partially denatured DNA samples. The tf genome is characterized by the presence of two classes of nicks differing in the frequency of their presence in population of bacteriophage DNA molecules.     

A study of DNA denaturation in the ultracentrifuge

The melting transition of DNA in alkaline CsCl can be followed in the analytical ultracentrifuge. Equilibrium partially denatured states can be observed. These partially denatured DNA bands have bandwidths of up to several times those of native DNA. Less stable molecules melt early and are found at heavier densities in the melting region. An idealized ultracentrifuge melting transition is described. The melting transition of singly nicked PM-2 DNA resembles the idealized curve. The DNA profile is a Gaussian band at all points in the melt. DNA's from mouse, D. Melanogaster, M. lysodeikticus, T4, and T7 also show equilibrium bands at partially denatured densities, some of which are highly asymmetric. Simple sequence satellite DNA shows an all-or-none transition with no equilibrium bands at partially denatured densities. The temperature at which a DNA denatures is an increasing function of the (G + C) content of the DNA. The T_m does not show a molecular-weight dependence in the range 1.2 × 10⁶–1.5 × 10⁷ daltons (single strand) for mouse, M. lysodeikticus, or T4 DNA. The mouse DNA partially denatured bands do not change shape as a function of molecular weight. The T4 DNA intermediate band develops a late-melting tail at low molecular weight. M. lysodeikticus DNA bands at partially denatured densities become broader as the molecular weight is decreased. Mouse DNA is resolved into six Gaussian components at each point in the melting transition.     

Pisum sativum endonuclease. Studies on substrate specificity and possible use as a biochemical tool

An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypolynucleotides was in the order poly(dT) greater than denatured DNA greater than poly(dA) greater than poly(dA-dT) = native DNA. Neither poly(dC), poly(dG) nor poly(dC).poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear from III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I degree, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.     

Differences in the binding of methylated albumin to non-replicating, replicating and denatured DNA from Ehrlich ascites cells

The binding of methylated albumin to DNA, the basis of the chromatography on columns of kieselguhr coated with methylated albumin (MAK chromatography), was investigated. Scatchard plots revealed only one mode of interaction with fully double-stranded DNA. The complexes should be completely dissociated by raising the NaCl concentration of the solution to 0.8 M, indicating a binding by electrostatic attraction between the oppositely charged protein and DNA molecules. In complexes with denatured and partially single-stranded replicating DNA an additional kind of binding was found which made these complexes more stable against salt dissociation. These secondary interactions were stronger at 23 degrees C than at 0 degrees C and could be weakened by the addition of 6 M urea. It was therefore concluded that apolar forces were involved in these interactions.     

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.     

Studies on the fate of aldolase molecules in the aging rat lens

It was found that aldolase activity declined considerably in the lens of adult animals with increasing age. Immunoassay showed that defective aldolase C molecules were accumulated. In addition, antibody prepared against denatured enzyme preferentially removes inactive molecules from lens homogenates without affecting active molecules. It is concluded that defective aldolase molecules encountered in aging lenses are at least partially denatured and are inactive.     

Purification and properties of a plant endonuclease specific for apurinic sites.

An endonuclease which hydrolyzes depurinated DNA has been isolated from Phaseolus multiflorus enbryos; it has a molecular weight around 40,000. The enzyme is specific for apurinic sites; it has no action on normal DNA strands or on alkylated sites, and is without exonulcease activity. The rate of phosphoester bond hydrolysis near apurinic sites is far greater in native than in denatured DNA. The endonuclease is not inactivated by 10 mM EDTA, but is activity is however stimulated by Mg2+ or Mn2+. Its optimum pH is 7.5 to 8.0, and its optimum temperature 40degrees although, at this temperature, it is rapidly denatured; even low NaCl concentrations inhibit the enzyme activity. The endonuclease for apurinic sites of P. multiflorus is a non-histone protein of chromatin; the properties (like thermosensitivity of susceptibility to ionic strength) of the enzyme in situ, working on chromatin DNA, might be different from those described for the isolated endonuclease in homogenous aqueous solution.     

Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine, or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet light; low levels of endonuclease activity, obtained when X-irradiated, alkylated, or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.     

The herpes simplex virus type 1 DNA polymerase processivity factor, UL42, does not alter the catalytic activity of the UL9 origin-binding protein but facilitates its loading onto DNA

The herpes simplex virus type 1 UL42 DNA polymerase processivity factor interacts physically with UL9 and enhances its ability to unwind short, partially duplex DNA. In this report, ATP hydrolysis during translocation of UL9 on single-stranded (ss) or partially duplex DNA was examined in the presence and absence of UL42 to determine the effect of UL42 on the catalytic function of UL9. Our studies reveal that a homodimer of UL9 is sufficient for DNA translocation coupled to ATP hydrolysis, and the steady-state ATPase catalytic rate was greater on partially duplex DNA than on ss DNA in the presence or absence of UL42. Although UL42 protein increased the steady-state rate for ATP hydrolysis by UL9 during translocation on either partially duplex or ss DNA, UL42 had no significant effect on the intrinsic ATPase activity of UL9. UL42 also had no effect on the catalytic rate of ATP hydrolysis when UL9 was not limiting but enhanced the steady-state ATPase rate at only subsaturating UL9 concentrations. At subsaturating UL9 to DNA ratios, stoichiometric concentrations of UL42 were shown to increase the amount of UL9 bound to ss DNA at equilibrium. These data support a model whereby UL42 increases the ability of UL9 to load onto DNA, thus increasing its ability to assemble into a functional complex capable of unwinding duplex DNA.     

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.     

Characterization of human enzymes specific for damaged DNA: resolution of endonuclease for irradiated DNA from an apparent N-glycosidase active on alkylated DNA.

An endonuclease partially purified from human lymphoblasts, and active against ultraviolet-irradiated DNA, was found to act additionally on DNA damaged by either x-radiation or methylmethanesulfonate. To determine if these activities were truly endonucleolytic, the reaction products were analyzed under conditions that prevented conversion of apurinic or apyrimidinic sites to single-strand breaks. With either ultraviolet- or x-irradiated DNA, strand breakage remained maximal, hence confirming the endonucleolytic character of the enzyme. By contrast, with DNA alkylated with methylmethanesulfonate, strand breakage was sharply reduced. Additional experiments indicated that the activity for alkylated DNA induces strand breaks only in concert with a purified endonuclease specific for apurinic sites, suggesting that it is an N-glycosidase that depurinates alkylated bases. This enzyme was separated from the endonuclease specific for irradiated DNA, by chromatography on DNA-agarose.     

Co-fractionation of an endonuclease activity during the purification of DNA polymerase-alpha from regenerating rat liver. Properties and separation from DNA polymerase.

The presence of endonuclease activity associated with DNA polymerase was detected during the purification of high-molecular-weight DNA polymerase-alpha from regenerating rat liver by the use of a highly sensitive test. This endonuclease activity co-fractionated with DNA polymerase in a great variety of purification procedures involving ion-exchange chromatographies or molecular weight fractionation, but was further completely separated from DNA polymerase activity by using affinity chromatography on DNA-cellulose. The endonuclease acted on native or denatured DNA by introducing single-strand nicks in the DNA molecules; its enzymatic properties indicate that it could act in polymerisation conditions in vitro.     

Changes in DNA secondary structure afterγ-irradiation

Native calf thymus DNA was gamma-irradiated at 500 mug/ml in 0.01 M NaCl in the presence or absence of oxygen. By irradiation, an increasing amount of DNA becomes reactive with a water-soluble carbodiimide-derivative (CMEC). In the DNA sections reactive with CMEC the nucleotide strands are separated, a phenomenon previously described as radiation-induced denaturation. The dose-effect curve for the formation of denatured DNA shows an upward-bent form; a distinct oxygen effect of about 2 is observed. By a comparative study with DNA samples, degraded partially with DNAse I, it was shown that a minor part of the radiation-induced denaturation results from the formation of the radiation-induced single strand breaks, whereas the major part is a local denaturation independent of the strand breaks. In these locally denatured regions 20 to 50 nucleotide pairs are separated.     

The Presence of a Ribonuclease of High Molecular Weight in Sugar Beet Storage Tissue

Soluble ribonuckasie activity in sliced root tissue or sugar beet (Beta vulgaris) decreased to 30% of initial levels during the first 15 hours of aeration in sterile phosphate buffer. Activity increased with continued aeration reaching a maximum at 30 hours (85% of initial levels). Ribonuclease activity was isolated from unaerated tissue and partially purified by precipitation with acid and ammonium sulfate and by Sephadex chromatography. Enzyme activity was linear with respect to enzyme and substrate concentrations. The enzyme exhibited a substrate preference for RNA with no activity in the presence of native or denatured DNA. Elution of the enzyme from a Sephadex G-150 column indicated a molecular weight of 155,000. This uniquely large ribonuclease had no phosphodiesterase activity, was unaffected by ethylenediamine tetraacetate, and was inhibited by increasing Nig²⁺ concentrations.     

DNA-binding proteins of the malaria vector Anopheles stephensi: purification and characterization of an endonuclease

DNA-binding proteins present in fourth instar larvae of Anopheles stephensi were isolated by affinity chromatography on native and denatured DNA cellulose columns and analyzed by electrophoresis on polyacrylamide gels. A denatured DNA-specific protein with an approximate molecular weight of 30 kDa was the predominant DNA binding protein of larvae. This protein was purified to electrophoretic homogeneity by ammonium sulfate fractionation followed by phosphocellulose chromatography. The purified 30 kDa binding protein showed an endonucleolytic activity capable of converting pBR 322 supercoiled DNA to the circular form. Maximum endonucleolytic activity was observed in the presence of 5 mM Mg(2+) at pH 7.4. Enzyme activity was completely inhibited by EDTA.