Single-strand Cleavage Research Articles

Recognition of single-stranded DNA by the bacteriophage T4-induced type II topoisomerase.

The bacteriophage T4-induced type II DNA topoisomerase has been shown previously to make a reversible double strand break in DNA double helices. In addition, this enzyme is shown here to bind tightly and to cleave single-stranded DNA molecules. The evidence that the single-stranded DNA cleavage activity is intrinsic to the topoisomerase includes: 1) protein linkage to the 5' ends of the newly cleaved DNA; 2) coelution of essentially homogeneous topoisomerase and the DNA cleavage activity; 3) inhibition of both single-stranded DNA cleavage and double-stranded DNA relaxation by oxolinic acid; and 4) inhibition of duplex DNA relaxation by single-stranded DNA. The major cleavage sites on phi X174 viral DNA substrates have been mapped, and several cleavage sites analyzed to determine the exact nucleotide position of cleavage. Major cleavage sites are found very near the base of predicted hairpin helices in the single-stranded DNA substrates, suggesting that DNA secondary structure recognition is important in the cleavage reaction. On the other hand, there are also many weaker cleavage sites with no obvious sequence requirements. Many of the properties of the single-stranded DNA cleavage reaction examined here differ from those of the oxolinic acid-dependent, double-stranded DNA cleavage reaction catalyzed by the same enzyme.

Correlation of enzyme-induced cleavage sites on negatively superhelical DNA between prokaryotic topoisomerase I and S 1 nuclease

Negatively superhelical pNS1 DNA with a molecular weight of 2.55 MDa (4 kbp) was found to contain 13 specific, unbasepaired sites that are sensitive to a single-strand-specific S 1 nuclease cleavage. The S 1-cleavage occurred once at these sites. In the absence of added Mg 2+, the topoisomerase I purified from Haemophilus gallinarum formed a complex with the superhelical pNS1 DNA which has a hidden strand cleavage. Extensive proteinase K digestion of the complex led to cleavage of the DNA chain. Then the proteinase K-cleaved product was digested with S 1, which can cut the opposite strand at the preexisting strand cleavage to generate unit-length linear DNA. Restriction endonuclease analysis of the linear DNA shows that the topoisomerase-induced cleavage occurred once at ten specific sites on the DNA. The topoisomerase caused mainly single-strand cleavage at these sites, but infrequently also caused double-strand cleavage at the same sites. Of interest is the fact that these sites considerably coincide with the S 1-cleavable, unbasepaired sites.

A site and strand spedfic nuciease activity with analogies to topoisomerase I frames the rRNA gene ofTetrahymena

Exposure of macronuclear chromatin from Tetrahymena thermophila to sodium dodecyl sulfate causes an endogenous nuclease to cleave the extra-chromosomal rDNA at specific sites. All cuts are single-strand cleavages specific to the non-coding strand. Three cleavages map in the central non-transcribed spacer of the palindromic molecule at positions -1000, -600 and -150 bp with respect to the transcription initiation point. A fourth site is located close to the transcription termination point, while no cleavage is observed in the coding region. The position of each cleavage is in the immediate neighbourhood of DNAse I hypersensitive sites. Additionally, certain DNA sequence motifs are repeated in the region around the cleavages. Upon cleavage induction a protein becomes attached to the rDNA. Our results indicate covalent binding to the generated 3' end, in analogy to the aborted reaction of topoisomerase I.

Specific recognition of apurinic sites in DNA by a tryptophan-containing peptide.

We have used fluorescence spectroscopy to study the binding of lysyltryptophyl-alpha-lysine (Lys-Trp-Lys) to DNA modified by dimethyl sulfate before and after depurination and strand breakage. Quenching of tryptophan fluorescence increased upon association of the peptide with modified DNA as compared with native DNA. We have demonstrated that this quenching is related to a preferential stacking of the indole ring with nucleic acid bases in damaged regions. Stacking increased in the following order: methylated DNA less than DNA with strand breaks at apurinic sites much less than apurinic DNA. For apurinic DNA, the overall association constant of Lys-Trp-Lys was increased by more than two orders of magnitude as compared to native DNA. Enhancement of the affinity of the tripeptide for an apurinic site requires the integrity of the phosphodiester bond. Single-strand cleavage at an apurinic site leads to a marked decrease of the association constant. The peptide Lys-Trp-Lys is therefore able to recognize destabilized regions in the vicinity of a lesion and to discriminate between different configurations of the damaged region. These results are discussed with respect to the role that stacking interactions could play in the specificity of recognition of DNA alterations by enzymes involved in DNA repair mechanisms.

Single-strand and double-strand cleavage at half-modified and fully modified recognition sites for the restriction nucleases Sau3A and TaqI

The influence of cytosine methylation on the cleavage of DNA by the restriction nucleases Sau3A and TaqI has been investigated. Bovine satellite DNA fragments containing a GATCGA sequence i.e. a Sau3A site overlapping with a TaqI site have been used in this study. The methylation of these fragments has been determined by sequence analysis. It has been found that a TaqI site (TCGA) methylated at cytosine in both DNA strands is still sensitive to double-strand cleavage. A Sau3A site (GATC), however, is rendered resistant to double-strand cleavage by methylation of a single cytosine. Fragments containing the “half-modified” Sau3A site are nicked in the unmethylated DNA strand. It has been shown by sequence analysis of nicked DNA that the single-strand break occurs at the same position which is cleaved in unmodified DNA.

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.

Association of cellular membrane of E. coli minicells with the origins/terminus region of replication of plasmid ColEl DNA

COLICINOGENIC plasmid El (ColEl) can be isolated from Escherichia coli as a supercoiled DNA molecule of 2.14 µm long or as a supercoiled DNA–protein complex1. Certain protein denaturing agents will convert this complex to an open circular (relaxed) DNA form with one single-strand break in the heavy (polyUG binding) strand of the duplex ColEl DNA (refs 2, 3). The induced single-strand break in ColEl DNA is located approximately 19% of the length of ColEl DNA from the single EcoRI restriction endonuclease site in the plasmid4. Analysis of ColEl replicating intermediates has shown that replication proceeds unidirectionally from an origin located near the position of the relaxation complex nick, approximately 19% from the single EcoRI restriction site 5–7. The site of the relaxation-complex nick has been located approximately 300 nucleotides away from the origin of ColEl DNA replication9. The nucleotide sequence of the origin region of ColEl DNA8,9 and of a small ColEl-type plasmid10 have been determined. A model for the possible role of the relaxation complex in ColEl DNA replication and/or conjugal transfer has been presented11. A major feature of this model is that the relaxation complex proteins may help promote association of plasmid DNA with a replicator and/or conjugal transfer site on the bacterial membrane. We describe here an electron microscopic analysis of EcoRI-cleaved ColEl and mini-ColEl DNA that were purified as stable DNA–membrane complexes from E. coli minicells. Evidence is presented that membrane-like structures attach to ColEl DNA and mini-ColEl DNA in a region that includes both the site of the origin/terminus of DNA replication and the site of single-strand cleavage by the relaxation complex.

Endonucleolytic Cleavage of Parental DNA and T4 Late‐Gene Expression: Distribution Analysis of Single‐Strand and Double‐Strand Breaks

In order to investigate the dependency of late transcription on concurrent DNA replication during bacteriophage T4 development, we analyzed the endonucleolytic cleavage kinetics of the DNA of a T4 mutant lacking DNA polymerase, DNA ligase and exonuclease by using the sucrose gradient sedimentation technique. Our results can be summarized as follows. 1. The single-strand endonucleolytic cleavage of the T4 mutant DNA is not a random process. 2. The number of single-strand nicks reaches a plateau level of 10--12 nicks/molecule. 3. The occurrence of a double-strand break is delayed and their number is at any time lower than the number of single-strand nicks. 4. The circular permutation T4 genome, as computer-simulated by the Monte Carlo method, produces a smoothing of the discrete distribution which would be expected if nicks were localized in the promoter sites of late transcription units. We conclude that our findings support the model which relates single-strand DNA nicks to the late transcription initiation sites.

Spliced segments at the 5′ terminus of adenovirus 2 late mRNA

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.

Poly(Adenosine Diphosphate Ribose) Polymerase and Deoxyribonucleic Acid Damage

Several classes of cytotoxic agents are known to affect glycolysis in cells by lowering the cellular concentration of NAD. Early investigations showed that both alkylating agents (Roitt, 1956) and ionizing radiation (Maass et al., 1958) were capable of producing this effect, and subsequent research has largely been confined to these agents. Most of the NAD turnover is confined to the cell nucleus; only 5% of the NAD synthesized is required to maintain the cellular pool during growth (Rechsteiner et al., 1976a,b). The rest is needed to replace that degraded, presumably by the DNA-dependent chromosomal enzyme poly(ADP-ribose) polymerase. We have investigated the relationship of this enzyme to the decrease in NAD after treatment with cytotoxic drugs. The activity of the enzyme increases twofold on treatment of Physarum polycephalum with the alkylating agent streptozotocin (Whish et al., 1975). Inhibition of the enzyme has been shown to prevent the decrease in NAD caused by streptozotocin in mouse leukaemia cells (Davies et al., 1976). We have now extended this study to y-radiation and have discovered another cytotoxic agent with an effect on cellular NAD concentrations. Treatment of a suspension culture of mouse L1210 leukaemia cells with y-radiation causes an immediate decrease in cellular NAD, to a minimum after 15min. This fall is dose-dependent, with a half-maximal effect being produced by 3 krd. The NAD concentration returns to control values after about 8 h. Neocarzinostatin is a polypeptide antitumour antibiotic of known sequence (Meienhofer et al., 1972) isolated from a variant of Streptomyces carzinostaticus (Ishida et al., 1965). When L1210 suspension cells are treated with this agent the NAD concentration falls rapidly. A dose of 5pg/ml is sufficient to decrease the NAD to 20% of control; recovery of the NAD concentration is very slow and is not complete in 24h. A 1 h pulse of the drug elicits the full effect. Poly(ADP-ribose) polymerase is strongly inhibited by 5-methylnicotinamide and the methylxanthines (Davies et al., 1976). The effects of both y-radiation and neocarzinostatin on cellular NAD can be inhibited by simultaneous treatment with 5-methylnicotinamide or theophylline. If these inhibitors are added after a pulse treatment with the agent, the NAD concentration returns to the control value more rapidly. Neocarzinostatin is thought to act by single-strand cleavage of DNA (Beerman & Goldberg, 1974). Both alkylating agents and ionizing radiation have major effects on DNA structure which can be demonstrated by alkaline sucrose gradients as singlestrand breaks. Cytotoxic agents which do not affect the structural integrity of DNA (to date we have only tested colchicine and 5-fluorodeoxyuridine) have been shown not to affect cellular NAD concentrations. Deoxyribonuclease has also been reported as stimulating the activity of poly(ADP-ribose) polymerase (Janakidevi & Koh, 1974; Miller, 1975). We propose that damage to DNA results in an increase in the flux through the poly(ADP4bose) polymerase system which, if acute, is sufficient to lower the cellular concentration of NAD. It is possible that this response is related to DNA repair.

Studies related to antitumor antibiotics. Part VII. Synthesis of streptonigrin analogues and their single strand scission of DNA

The syntheses of a group of 2-(o-nitrophenyl)- and 2-(o-aminophenyl)-5,8-quinolinediones which are structurally related to the antitumor antibiotic streptonigrin are described. Ambiguities in the position of required nucleophilic displacements are resolved by independent synthesis. The rates of single strand cleavage of PM2 ccc-DNA (covalently-closed circular-DNA) induced by these compounds are compared, which correlates with antitumor activity. The 2-(o-nitrophenyl) derivatives give consistently more rapid DNA cleavage than the 2-(o-aminophenyl) compounds. The autoxidations of the dihydroxyquinolines are subject to selective catalysis by Cu2+ on. 2-(o-Aminophenyl)-7-amino-6-methoxy-5,6-quinolinedione which has a substitution pattern most closely resembling streptonigrin also closely parallels the rate of scission of DNA of the latter in the presence of NADPH.

Studies related to antitumor antibiotics. Part VIII. Cleavage of DNA by streptonigrin analogues and the relationship to antineoplastic activity.

A group of substituted 5,8-quinolinequinones which exhibit antineoplastic activity and which are structurally related to the antitumor antibiotic streptonigrin induce single strand cleavage of PM2 covalently-closed circular-DNA (ccc-DNA) when reductively activated. The cleavage which is detected by an ethidium fluorescence assay is specifically enhanced by cuprous and ferrous ion and is selectively inhibited by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) and by free radical scavengers. Independent generation of the superoxide ion by xanthine-xanthine oxidase (EC 1.2.3.2) also cleaves PM2 DNA and therefore a chemical mechanism for the scission process induced by the streptonigrin analogues is formulated. A correlation between rate of PM2 ccc-DNA cleavage and inhibition of Walker carcinosarcoma 256 is observed.

The mechanism of the degradation of DNA by streptonigrin.

The production of single strand cleavage in covalently-closed circular-DNA by the antitumour agent streptonigrin (reduced in situ by NADH) is demonstrated using the ethidium bromide fluorescence assay described previously. The degradation dependent on oxygen is completely inhibited by superoxide dismutase (EC 1.15.1.1) suggesting the intermediacy of the superoxide radical anion in the degradation. However similar complete inhibition of DNA strand breakage by catalase (EC 1.11.1.6) indicates that the hydroxyl radical (formed by interaction of superoxide with hydrogen peroxide) is the primary reactive species. Cupric ion stimulates the cleavage reaction and cobaltous ion has no effect in keeping with model studies using quinolinequinones.

Studies related to antitumor antibiotics. Part V. Reactions of mitomycin C with DNA examined by ethidium fluorescence assay.

The cytotoxic action of the antitumor antibiotic mitomycin C occurs primarily at the level of DNA. Using highly sensitive fluorescence assays which depend on the enhancement of ethidium fluorescence only when it intercalates duplex regions of DNA, three aspects of mitomycin C action on DNA have been studied: (a) cross-linking events, (b) alkylation without necessarily cross-linking, and (c) strand breakage. Cross-linking of DNA is determined by the return of fluorescence after a heat denaturation step at alkaline pH's. Under these conditions denatured DNA gives no fluorescence. The cross-linking was independently confirmed by S1-endonuclease (EC 3.1.4.-) digestion. At relatively high concentrations of mitomycin the suppression of ethidium fluorescence enhancement was shown not to be due to depurination but rather to alkylation, as a result of losses in potential intercalation sites. A linear relationship exists between binding ratio for mitomycin and loss of fluorescence. The proportional decrease in fluorescence with pH strongly suggests that the alkylation is due to the aziridine moiety of the antibiotic under these conditions. A parallel increase in the rate and overall efficiency of covalent cross-linking of DNA with lower pH suggests that the cross-linking event, to which the primary cytotoxic action has been linked, occurs sequentially with alkylation by aziridine and then by carbamate. Mitomycin C, reduced chemically, was shown to induce single strand cleavage as well as monoaklylation and covalent cross-linking in PM2 covalently closed circular DNA. The inhibition of this cleavage by superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), and by free radical scavengers suggests that the degradation of DNA observed to accompany the cytotoxic action of mitomycin C is largely due to the free radical O2. In contrast to the behavior of the antibiotic streptonigrin, mitomycin C does not inactivate the protective enzymes superoxide dismutase or catalase. Lastly, mitomycin C is able to cross-link DNA in the absence of reduction at pH 4. This is consistent with the postulated cross-linking mechansims.

Abnormal synthesis of mitochondrial DNA in the presence of [formula omitted] in vitro

1. The in vitro effect of N- methyl-N′- nitro-N- nitrosoguanidine (MNNG) on mtDNA synthesis was studied using isolated newborn rat liver mitochondria. 2. From the kinetics of the incorporation of [ 3H]thymidine into the acid-insoluble material, MNNG neither stimulated nor inhibited the DNA synthesizing activity of mitochondria. The activity observed in the presence of MNNG was inhibited by N- ethylmaleimide and actinomycin D. 3. By the band velocity sedimentation in CsCl/ethidium bromide, the properties of the nascent mtDNA formed in the presence of MNNG were analyzed. The nascent DNA-containing molecule was not found in the closed-circle fraction, and essentially detected in the open-circle fraction. This change of the template was blocked by N- ethylmaleimide but not by actinomycin D, suggesting a conversion of the closed-circular template to the open-circular one by single-strand cleavage(s). From the band sedimentation in alkaline CsCl, the number of nascent higher molecular DNAs was increased but the molecules were all of relatively lower molecular weight. On the other hand, the formation of nascent fragments was inhibited. 4. The alkaline CsCl equilibrium centrifugation analysis revealed that the nascent DNA synthesized in the presence of MNNG consisted of both light and heavy components. 5. Present results suggest that MNNG exerts its effect on the mtDNA synthesis by modifying the intrinsic mechanism of discontinuous synthesis, since the conversion of the template DNA molecule from the closed- to open-circular form and the continuous polymerization of the nascent higher-molecular DNA on such a relaxed template were characteristic events in vitro.

Site-specific cleavage of single-stranded DNA by a Hemophilus restriction endonuclease.

Single-stranded viral DNA of bacteriophage f1 is cleaved into specific fragments by endo R-HaeIII, a restriction endonuclease isolated from Hemophilus aegyptius. The sites of the single strand cleavage correspond to those of the double strand cleavage. A single-stranded DNA fragment containing only one HaeIII site is also cleaved by this enzyme. This observation suggests that the reaction of single-stranded DNA cleavage does not require the formation of a symmetrical double-stranded structure that would result from the intramolecular base-pairing between two different HaeIII sites. Other restriction endonucleases may also cleave single-stranded DNA.

Degradation of duplex DNA by S 1 nuclease from Aspergillus

Summary A nuclease from Aspergillus which selectively degrades single-stranded polynucleotides, S 1 , has been purified by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme is still capable of degrading both OX174 RF and native calf thymus DNA. The hydrolysis of double-stranded DNA appears to be endonucleolytic as is single-strand cleavage.

Kinetics of colicin E2-induced solubilization and fragmentation of Escherichia coli DNA in vivo

In exponentially growing cultures of sensitive bacteria treated with colicin E2, solubilization of DNA was detected after 3–5 min, single-strand cleavage after an additional 2–5 min and finally double-strand breaks in DNA were detected after a further 5–10 min. Early addition of 2,4-dinitrophenol completely prevented E2-induced fragmentation of DNA and the uncoupler immediately inhibited further DNA breakdown when added at various times after the commencement of E2 action. Both E2-induced fragmentation and solubilization of DNA proceeded faster in a RecA mutant compared to the wild type but DNA degradation was normal in a UvrA mutant. In contrast to growing cultures, the induction of E2-induced solubilization of DNA commenced without detectable lag in phosphate buffer suspensions and therefore the formation of the E2-cellular complex which actually triggers DNA breakdown appears to be transiently inhibited in growing bacteria. Addition of high salt concentrations to cell suspensions in buffer effectively blocked both fragmentation and solubilization of DNA induced by E2.

Endonuclease I of Proteus mirabilis: II. PROPERTIES OF THE NONCOMPLEXED AND TRANSFER RIBONUCLEIC ACID-COMPLEXED FORMS OF THE ENZYME

Abstract The purified form of endonuclease I of Proteus mirabilis sediments as a 3 S molecule, requires Mg2+ for activity, and degrades double and single stranded DNA to acid-soluble products. This activity has an alkaline pH optimum and is inhibited by Escherichia coli tRNA and high concentrations of sodium chloride. The two faster sedimenting forms of the enzyme, 7.2 and 6 S, that can be generated by the addition of tRNA to the purified enzyme and also are found in a dihydrostreptomycin sulfate precipitate of a P. mirabilis crude extract, degrade the covalently closed circular duplex form of DNA of colicinogenic factor E1, φ X-174, and SV40 DNA to 8 S DNA fragments that are indistinguishable by sucrose gradient centrifugation analysis. Degradation of the supercoiled circular DNA to the 8 S product is independent of substrate concentration over a wide range and is not caused by inactivation of the enzyme under the reaction conditions used. The 8 S DNA product is double stranded, free of a substantial amount of nicks, and, once formed, is resistant to further endonuclease attack over a relatively long period of time under the conditions used. Evidence is presented for a single strand cleavage as the initial endonucleolytic event in the degradation of the native DNA substrate to the 8 S product.