Effect of alkylation on the secondary structure of DNA

S1 nuclease hydrolysis and bezoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography have been used to demonstrate that alkylation of DNA by dimethyl sulfate at neutral pH leads to the production of partially denatured molecules under conditions where no significant depurination occurs. DNA was alkylated with increasing concentrations of the alkylating agent, and subjected to enzymatic degradation and binding to BND cellulose. An increasing degree of DNA hydrolysis and adherence to BND cellulose was seen. On hydroxyapatite chromatography the alkylated DNA still eluted at the position of double-stranded molecules suggesting the presence of partially denatured regions. The presence of salt had a preventive effect on such denaturation.     

In vitro studies on mild alkali induced lesions in DNA.

S1 nuclease hydrolysis, benzoylated naphthoylated DEAE cellulose (BND-cellulose) chromatography as well as certain immunological and genetic techniques have been used to evaluate the effect of mild alkali (pH10.0) on the DNA molecule. Native calf thymus DNA after exposure to alkaline pH10.0 when subjected to S1 nuclease hydrolysis released significant amount of acid soluble nucleotides as compared to the untreated control. With pBR322 DNA, the population of linear DNA species increased on S1 digestion with concomitant reduction in the supercoiled form. BND cellulose chromatographic studies also suggested the formation of single strandedness and/or distortions in the alkali treated DNA molecule. Antisera raised against the alkali treated DNA exhibited high cross-reactivity with both single stranded and Z DNA. Moreover, a significant reduction in transformation frequency of the treated DNA molecule compared with the untreated control further ascertained the structural alterations in DNA as a result of exposure to mild alkali.     

Degradation of DNA by silicic acid

S1 nuclease hydrolysis and hydroxyapatite chromatography were used to study the effect of silicic acid on DNA. Native calf thymus DNA was incubated with increasing concentrations of silicic acid (DNA nucleotide/silicic acid molar ratios of 1:0.25, 1:0.5 and 1:1) and subjected to S1 nuclease hydrolysis. An increasing degree of DNA degradation was seen suggesting a destabilization of the secondary structure. A decrease in melting temperature was also observed. Hydroxyapatite chromatography indicated that incubation at the molar ratio of 1:1 resulted in denaturation and degradation of DNA.     

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.     

Kinetic studies of the hydrolysis of platinum-DNA complexes by nuclease S1

The antitumor agent cis-diamminedichloroplatinum(II) (cis-DDP) reacts covalently with DNA and disrupts its secondary structure. Damaged DNA, but not native DNA, is readily digested by S1 nuclease, an endonuclease specific for single stranded polynucleotides. We have measured S1 nuclease digestion of platinated DNA by the release of platinum-DNA adducts and compared it with digestion of unplatinated DNA. The rate of hydrolysis of damaged substrate from platinum-DNA complexes was less than the overall rate of digestion of nucleotides. Similar results were observed for platinum-DNA complexes in native, denatured or renatured conformations. The hydrolysis of denatured platinum-DNA complexes, rb = 0.075 platinum per nucleotide, obeyed Michaelis-Menten kinetics. Taking into account the level of DNA damage, Vm, for the release of platinated adducts was 0.6 times smaller than for digestion of unplatinated DNA. Km values and competition experiments indicated that the enzyme bound equally well to platinated and unplatinated substrates. Similar results were obtained for denatured DNA complexes with trans-DDP while [PtCl(diethylenetriamine)]Cl had no influence on nuclease digestion. These results suggest that bifunctional platinum-DNA lesions have contradictory effects on the hydrolysis of double stranded DNA by S1 nuclease. On one hand they create nuclease sensitive substrate by disrupting DNA secondary structure. On the other, they inhibit digestion of the damaged strand by increasing the activation energy for hydrolysis.     

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.     

Extracellular nuclease from Rhizopus stolonifer: purification and characteristics of - single strand preferential - deoxyribonuclease activity

An extracellular nuclease from Rhizopus stolonifer (designated as nuclease Rsn) was purified to homogeneity by chromatography on DEAE-cellulose followed by Blue Sepharose. The M(r) of the purified enzyme determined by native PAGE was 67? omitted?000 and it is a tetramer and each protomer consists of two unidentical subunits of M(r) 21? omitted?000 and 13? omitted?000. It is an acidic protein with a pI of 4.2 and is not a glycoprotein. The purified enzyme showed an obligate requirement of divalent cations like Mg(2+), Mn(2+) and Co(2+) for its activity but is not a metalloprotein. The optimum pH of the enzyme was 7.0 and was not influenced by the type of metal ion used. Although, the optimum temperature of the enzyme for single stranded (ss) DNA hydrolysis in presence of all three metal ions and for double stranded (ds) DNA hydrolysis in presence of Mg(2+) was 40 degrees C, it showed higher optimum temperature (45 degrees C) for dsDNA hydrolysis in presence of Mn(2+) and Co(2+). Nuclease Rsn was inhibited by divalent cations like Zn(2+), Cu(2+) and Hg(2+), inorganic phosphate and pyrophosphate, low concentrations of SDS, guanidine hydrochloride and urea, organic solvents like dimethyl sulphoxide, dimethyl formamide and formamide but not by 3'- or 5'-mononucleotides. The studies on mode and mechanism of action showed that nuclease Rsn is an endonuclease and cleaves dsDNA through a single hit mechanism. The end products of both ssDNA and dsDNA hydrolysis were predominantly oligonucleotides ending in 3'-hydroxyl and 5'-phosphoryl termini. Moreover, the type of metal ion used did not influence the mode and mechanism of action of the enzyme.     

P1 nuclease defines a subpopulation of active SV40 chromatin--a new nuclease hypersensitivity assay.

Under exhaustive digestion conditions P1 nuclease was found to cleave a subpopulation of intracellular SV40 chromatin only once. The major P1 cleavage site in SV40 DNA was mapped at the origin of DNA replication, and the two minor sites at the SV40 enhancers. The P1-sensitive SV40 chromatin subpopulation was found to have higher superhelical density than the bulk of the intracellular SV40 chromatin. Furthermore, pulse labeled SV40 DNA which had higher superhelical density than that of the steady state viral DNA (S.S.Chen and M.T.Hsu, J.Virol 51:14-19, 1984) was also found to be preferentially cleaved by P1 nuclease. These results are consistent with a supercoil-dependent alteration of chromatin conformation near the regulatory region of the viral genome that can be recognized by P1 nuclease. Since P1 nuclease cleaves the subpopulation of SV40 chromatin only once without further degradation, this nuclease can be used as a general tool to define viral or cellular chromatin fraction with altered chromatin conformation and to map nuclease hypersensitive sites. Preliminary studies indicate that P1 makes limited double stranded cleavages in cellular chromatin to generate large DNA fragments.     

Interaction of quercetin with DNA

I n vitro experiments to study interaction of the mutagenic flavonoid quercetin with DNA are described. Calf thymus DNA treated with quercetin for various time periods was subjected to S₁ nuclease hydrolysis. Thermal melting profles of treated DNA were also determined using St nuclease. The rate of DNA hydrolyzed after 1 hr of pre-treatment with quercetin was found to be only about 50% of that in its absence. However, after 10 and 24hrs of treatment with the drug, the rate of S₁ nuclease hydrolysis was observed to be greater than that of native DNA. Thermal melting profiles of DNA, treated with quercetin for 10 and 24 hrs, indicated a slight decrease in melting temperatures. Gel filtration of native DNA, which had been digested with S₁ nuclease after preincubation with quercetin for 24 hrs, indicated the production of various sized degraded molecules. The results suggest that the initial interaction of quercetin with DNA may have a stabilizing effect on its secondary structure, but prolonged treatment leads to an extensive disruption of the double helix.     

Effect of alkylated and intercalated DNA on the generation of superoxide anion by riboflavin

Superoxide anion (O ₂ ^.– ) was photogenerated upon illumination of riboflavin in fluorescent light. The rate of O ₂ ^.– formation was stimulated by double stranded DNA but not by denatured DNA or RNA. Depurinated DNA, which was predominantly depleted in guanine residues, did not exhibit the stimulatory effect, indicating an interaction of riboflavin, or active oxygen species derived from it, with guanine bases. Also, the stimulation of O ₂ ^.– photogeneration was not observed with ethidium bromide but was seen with proflavin-intercalated DNA. Since ethidium bromide intercalates preferentially between purines and pyrimidines, and proflavin prefers dA-dT rich sites, these results were interpreted to suggest that the interaction of riboflavin with DNA is mainly with GC or CG base pairs.     

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.     

Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.     

Application of oligonucleoside methylphosphonates in the studies on phosphodiester hydrolysis by Serratia endonuclease.

The endonuclease from Serratia marcescens is a non-specific enzyme that cleaves single and double stranded RNA and DNA. It accepts a phosphorylated pentanucleotide as a minimal substrate which is cleaved in the presence of Mg2+ at the second phosphodiester linkage. The present study is aimed at understanding the role of electrostatic and hydrogen bond interactions in phosphodiester hydrolysis. Towards this objective, six pentadeoxyadenylates with single stereoregular methylphosphonate substitution within this minimal substrate (2a-4b) were synthesized following a protocol described here. These modified oligonucleotides were used as substrates for the Serratia nuclease. The enzyme interaction studies revealed that the enzyme failed to hydrolyze any of the methylphosphonate analogues suggesting the importance of negative charge and/or hydrogen bond acceptors in binding and cleavage of its substrate. Based on these results and available site-directed mutagenesis as well as structural data, a model for nucleic acid binding by Serratia nuclease is proposed.     

S1 nuclease does not cleave DNA at single-base mis-matches

Three assays have been designed to detect the cleavage of duplex phi X174 DNA at single-base mis-matches. Studies with S1 nuclease failed to detect cleavage at mis-matches. S1 nuclease digestion at 37 and 55 degrees C failed to produce a preferential degradation of a multiply mis-matched heteroduplex when compared to a mis-match-free homo-duplex as analyzed by sedimentation on sucrose gradients. Other heteroduplex templates were not cleaved by S1 nuclease at a defined single-base mis-match when assayed by gel electrophoresis or by marker rescue. In all cases, the amount of S1 nuclease employed was at least 10-times more than that required to render a single-stranded phi X174 DNA molecule completely acid soluble. The rate of hydrolysis of single-base mis-matches by S1 nuclease was estimated to be less than 0.016% of the rate at a base in single-strand phi X174 DNA. In no instance did we detect activity by S1 nuclease directed at mis-matched sites in our template molecules. Similarly, the single-strand specific endonuclease from Neurospora crassa does not cleave heteroduplex templates at a defined single-base mis-match when assayed by marker rescue.     

Aggregation of fragmented chromatin associated with the synthesis of products of its treatment with nuclease

Isolated cell nuclei were incubated with nucleases followed by extraction of chromatin with a low salt buffer. With an increase of nuclear chromatin degradation with DNAse I or micrococcal nuclease, solubilization of deoxyribonucleoprotein (DNP) by a low salt buffer increases, reaching a maximum upon hydrolysis with 2-4% nuclear DNA and then decreases appreciably after extensive treatment with nucleases. Soluble fragmented chromatin aggregates in the course of treatment with DNAase. I. Addition to gel chromatin preparations of exogenous products of nuclease treatment of isolated nuclei leads to its aggregation. Pretreatment of nuclear chromatin with RNAase prevents solubilization of DNP by low ionic strength solutions. Some experimental data obtained with the use of severe nuclease treatment are discussed; for a correct interpretation of these data the aggregation of fragmented chromatin by products of its nuclease degradation should be taken into consideration.     

Interarm interaction of DNA cruciform forming at a short inverted repeat sequence

A novel interarm interaction of DNA cruciform forming at inverted repeat sequence was characterized using an S1 nuclease digestion, permanganate oxidation, and microscopic imaging. An inverted repeat consisting of 17 bp complementary sequences was isolated from the bluegill sunfish Lepomis macrochirus (Perciformes) and subcloned into the pUC19 plasmid, after which the supercoiled recombinant plasmid was subjected to enzymatic and chemical modification. In high salt conditions (200 mM NaCl, or 100-200 mM KCl), S1 nuclease cut supercoiled DNA at the center of palindromic symmetry, suggesting the formation of DNA cruciform. On the other hand, S1 nuclease in the presence of 150 mM NaCl or less cleaved mainly the 3'-half of the repeat, thereby forming an unusual structure in which the 3'-half of the inverted repeat, but not the 5'-half, was retained as an unpaired strand. Permanganate oxidation profiles also supported the presence of single-stranded part in the 3'-half of the inverted repeat in addition to the center of the symmetry. Both electron microscopy and atomic force microscopy have detected a thick protrusion on the supercoiled DNA harboring the inverted repeat. We hypothesize that the cruciform hairpins at conditions favoring triplex formation adopt a parallel side-by-side orientation of the arms allowing the interaction between them supposedly stabilized by hydrogen bonding of base triads.     

A DNA helicase from human cells.

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.     

Reaction of T7 DNA with a polycyclic aromatic hydrocarbon. Lack of structural perturbation

Bacteriophage T7 DNA reacts uniformly with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene(anti-BPDE). The reaction product retains the native configuration so that only one site sensitive to S1 nuclease is produced for every 70 anti-BPDE adducts. DNA treated with anti-BPDE is retained on benzoylated naphthoylated DEAE-cellulose even after washing with 1.0 M salt solutions. About 100 adducts per T7 molecule are required for adherence which is not due to breaks or single-stranded regions since adherence is not affected by S1 nuclease treatment. The binding of anti-BPDE reacted DNA to benzoylated naphthoylated DEAE-cellulose is cooperative and requires many residues per bound fragment. Treatment of T7 DNA treated with anti-BPDE with restriction endonuclease yields smaller molecules, still containing adducts, which do not adhere. We interpret these results to mean that reaction with BPDE does not involve deformation of the DNA structure and that the adducts lie in a position which they are readily accessible for interaction with aromatic groups on the column resin.     

Isolation and comparison of two molecular species of the BAL 31 nuclease from Alteromonas espejiana with distinct kinetic properties

The extracellular nuclease from Alteromonas espejiana sp. BAL 31 can be isolated as two distinct proteins, the "fast" (F) and "slow" (S) species, both of which have been purified to homogeneity. The F and S species of the nuclease have molecular weights, respectively, of 109 X 10(3) and 85 X 10(3), and both are single polypeptide chains with an isoelectric pH near 4.2. Both species catalyze the degradation of single-stranded and linear duplex DNAs to 5'-mononucleotides. The degradation of linear duplex DNA occurs through a terminally directed hydrolysis mechanism that results in the removal of nucleotides from both the 3' and 5' ends. Apparent Michaelis constants (Km) have been obtained for the exonuclease activities of both species and for the activity against single-stranded DNA of the S species. The Km for the hydrolysis of single-stranded DNA catalyzed by the F species has not been obtained because the reaction velocity was maximal even at the lowest substrate concentrations accessible in the photometric assay. The ratio of the turnover numbers for the exonuclease activities of the two species indicates that the F species will shorten linear duplex DNA at a rate 27 +/- 5 (S.D.) times faster than an equimolar concentration of the S species in the limit of high substrate concentration, while the corresponding ratio for the activities against single-stranded DNA (1.2 +/- 0.1) shows that the two species are similar with respect to hydrolysis of this substrate. In the limit of high substrate concentrations, the F and S species break phosphodiester bonds in single-stranded DNA at rates 1.3 +/- 0.3 and 33 +/- 2 times those for the exonucleolytic degradation of linear duplex DNA, respectively. It has not been established whether the two species are physically related.     

Surface-located trypsin-activated Streptococcus sanguis strain Wicky endonuclease

A new Streptococcus sanguis strain Wicky endonuclease was isolated, purified and partially characterized. This nuclease acts preferentially on thermally denatured DNA, is not inhibited by RNA and is activated 3-5 times by trypsin. This activation is accompanied by the reduction of molecular weight of the enzyme. These features distinguish the new S, sanguis nuclease from the 3 previously described S. sanguis endonucleases. With covalently closed circular plasmid DNA, the enzyme causes first the appearance of a single stranded nick, then the second nick on the opposite DNA strand, resulting in plasmid DNA linearization. This nuclease most likely is located at the cell surface. The possible relationship of the described nuclease with ability of S. sanguis cells to take up DNA in genetic transformation is discussed.