The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.     

Quantitation of supercoiled DNA cleavage in nonradioactive DNA: application to ionizing radiation and synthetic endonuclease cleavage.

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.     

Hydrolysis of linear DNA duplex catalyzed by Co(III) complex of cyclen attached to polystyrene.

To design artificial restriction enzymes, synthetic catalytic centers that effectively hydrolyze linear double-stranded polydeoxyribonucleotides are needed. The Co(III) complex of cyclen (CoCyc) attached to polystyrene derivatives hydrolyzes linearized pUC18 DNA with half-lives as short as 30 min at 25 degrees C. The catalytic activity of CoCyc is enhanced by >150 times on attachment to the resin.     

The inhibition of restriction endonucleases due to Z-DNA in negatively supercoiled plasmid.

Plasmid pGC20 containing the (dGC)9 insert in SmaI recognition site has been used to study the inhibition of cleavage by different restriction endonuclease due to Z-DNA formation in (dCG)10 sequence of the negatively supercoiled plasmid. Data obtained indicate the different sensitivity of restriction endonucleases to DNA conformational perturbations resulted from the Z-DNA formation. Therefore, the inhibition of DNA cleavage by a particular restriction endonuclease cannot serve as a criterion for the estimation of the length of B-Z junctions in circular supercoiled DNAs.     

Efficient, Mg(2+)-dependent photochemical DNA cleavage by the antitumor quinobenzoxazine (S)-A-62176

The quinobenzoxazines, a group of structural analogues of the antibacterial fluoroquinolones, are topoisomerase II inhibitors that have demonstrated promising anticancer activity in mice. It has been proposed that the quinobenzoxazines form a 2:2 drug-Mg(2+) self-assembly complex on DNA. The quinobenzoxazine (S)-A-62176 is photochemically unstable and undergoes a DNA-accelerated photochemical reaction to afford a highly fluorescent photoproduct. Here we report that the irradiation of both supercoiled DNA and DNA oligonucleotides in the presence of (S)-A-62176 results in photochemical cleavage of the DNA. The (S)-A-62176-mediated DNA photocleavage reaction requires Mg(2+). Photochemical cleavage of supercoiled DNA by (S)-A-62176 is much more efficient that the DNA photocleavage reactions of the fluoroquinolones norfloxacin, ciprofloxacin, and enoxacin. The photocleavage of supercoiled DNA by (S)-A-62176 is unaffected by the presence of SOD, catalase, or other reactive oxygen scavengers, but is inhibited by deoxygenation. The photochemical cleavage of supercoiled DNA is also inhibited by 1 mM KI. Photochemical cleavage of DNA oligonucleotides by (S)-A-62176 occurs most extensively at DNA sites bound by drug, as determined by DNase I footprinting, and especially at certain G and T residues. The nature of the DNA photoproducts, and inhibition studies, indicate that the photocleavage reaction occurs by a free radical mechanism initiated by abstraction of the 4'- and 1'-hydrogens from the DNA minor groove. These results lend further support for the proposed DNA binding model for the quinobenzoxazine 2:2 drug-Mg(2+) complex and serve to define the position of this complex on the minor groove of DNA.     

Formation of Cu(II)-brazilin complex in the presence of DNA and its activities as chemical nuclease

Brazilin, a traditional medicine for the treatment of pain and inflammation, forms a complex with Cu(II) in the presence as well as the absence of DNA. The Cu(II)-brazilin complex exhibited the strand cleavage activity for the pBR322 supercoiled DNA, converting supercoiled form to nicked form. The presence of various scavengers for the oxygen species suppresses or reduces the cleavage activity of the complex, indicating that the DNA cleavage is oxidative. The binding mode of the Cu(II)-brazilin complex was studied by absorption and CD spectroscopy. While a large metal-to-ligand charge transfer (MLCT) band was apparent when Cu(II) and brazilin was mixed in the presence and absence of DNA, the CD did not show any signal in the same region in the presence of DNA, suggesting a weak interaction between the Cu(II)-brazilin complex and DNA bases.     

Cleavage of supercoiled circular double-stranded DNA induced by a eukaryotic cambialistic superoxide dismutase from Cinnamomum camphora

Superoxide dismutase (SOD, EC 1.15.1.1) that protectsorganisms from O2?– toxicity is a family of transitionmetal-containing enzyme existing in all oxygen-consu-ming living beings [1,2]. SOD catalyzes the dismutationof the toxic superoxide anion O2?– int…     

The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs

Collisions with DNA tracking systems are critical for the conversion of transient topoisomerase-DNA cleavage complexes to permanent strand breaks. Since DNA is overwound ahead of tracking systems, cleavage complexes most likely to produce permanent strand breaks should be formed between topoisomerases and positively supercoiled molecules. Therefore, the ability of human topoisomerase IIalpha and IIbeta and topoisomerase I to cleave positively supercoiled DNA was assessed in the absence or presence of anticancer drugs. Topoisomerase IIalpha and IIbeta maintained approximately 4-fold lower levels of cleavage complexes with positively rather than negatively supercoiled DNA. Topoisomerase IIalpha also displayed lower levels of cleavage with overwound substrates in the presence of nonintercalative drugs. Decreased drug efficacy was due primarily to a drop in baseline (i.e., nondrug) cleavage, rather than an altered interaction with the enzyme-DNA complex. Similar results were seen for topoisomerase IIbeta, but the effects of DNA geometry on drug-induced scission were somewhat less pronounced. With both topoisomerase IIalpha and IIbeta, intercalative drugs displayed greater relative cleavage enhancement with positively supercoiled DNA. This appeared to result from negative effects of high concentrations of intercalative agents on underwound DNA. In contrast to the type II enzymes, topoisomerase I maintained approximately 3-fold higher levels of cleavage complexes with positively supercoiled substrates and displayed an even more dramatic increase in the presence of camptothecin. These findings suggest that the geometry of DNA supercoils has a profound influence on topoisomerase-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either topoisomerase IIalpha or IIbeta.     

Site-specific cleavage of supercoiled DNA by ascorbate/Cu(II).

We have investigated ascorbate/Cu(II) cleavage of double-stranded DNA in the presence and absence of DNA negative torsion. We found that ascorbate/Cu(II) cleavage shows a site-specificity that is dependent on negative torsion and is influenced by the nature of the salt, ionic strength, and pH. This provides strong evidence for involvement of local DNA conformation in ascorbate/Cu(II) specific cleavage sites, that differs from the previous reports on cleavage of linear double-stranded DNA and secondary structures assumed by single-stranded DNA. The data indicate specific binding of Cu(II) ions to sites in the negatively supercoiled DNA. Fining mapping of the cleavage sites does not reveal any known DNA conformation, nor does it indicate any sequence identity among the sites cleaved. However, identification of a major site of cleavage of supercoiled DNA at physiological ionic strength, pH and temperature, along with fact that ascorbate and Cu(II) are normal cell constituents, suggests the torsion-dependent, site-specific interactions could have biological significance.     

Cleavage of single-stranded DNA by plasmid pT181-encoded RepC protein.

RepC protein encoded by plasmid pT181 has single-stranded endonuclease and topoisomerase-like activities. These activities may be involved in the initiation (and termination) of pT181 replication by a rolling circle mechanism. RepC protein cleaves the bottom strand of DNA within the origin of replication at a single, specific site when the DNA is in the supercoiled or linear (double or single-stranded) form. We have found that RepC protein will also cleave single-stranded DNA at sites other than the origin of replication. We have mapped the secondary cleavage sites on pT181 DNA. When the DNA is in the supercoiled, or linear, double-stranded form, only the primary site within the origin is cleaved. However, when the DNA is present in the single-stranded form, several strong and weak cleavage sites are observed. The DNA sequence at these cleavage sites shows a strong similarity with the primary cleavage site. The presence of Escherichia coli SSB protein inhibited cleavage at all of the secondary nick sites while the primary nick site remained susceptible to cleavage.     

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.     

Synthesis and DNA-cleaving activity of a series of substituted arenediazonium ions

We investigated the reactions of substituted aryl radicals and aryl cations derived from arenediazonium ions and their ability to cause cleavage of supercoiled DNA and their tendency toward free radical or cation formation in the presence and absent of copper (I) chloride. It was found that the substituted arenediazonium salts can cleave supercoiled DNA to the open circular form II DNA and linear form III DNA. Results methodical studies indicate that both carbon-centered radicals and aryl cations participate in the cleavage pathways.     

Hydrolysis of DNA by a Dipeptides Containing Histidine

Three ligands which contain histidine and conjugated by a flexible linker, have been characterized and evaluated as DNA cleavage agents. The cleavage activity of metal complexes were evaluated by monitoring the conversion of supercoiled plasmid DNA (pUC19) (Form I) to nicked circular DNA (Form II) by agarose gel electrophoresis. The results showed that the cleavage activity of Cu(II) complexes was enhanced compared with histidine. Specially, at a high reaction concentration (0.2 mM), Cu(II) complexes can cleave the plasmid DNA with some selectivity.     

Linear adenovirus DNA is organized into supercoiled domains in virus particles.

Electron microscopic analysis of bis-psoralen crosslinked adenovirus type 5 virion DNA revealed supercoiled domains in an otherwise linear DNA. The existence of supercoiled arrangement in all the virion DNA was demonstrated by the sensitivity of Ad5 DNA in pentonless virus particles to the supercoiling-dependent endonucleolytic activity of Bal31 and S1 nucleases. These nucleases were found to cleave Ad5 virion DNA at specific sites. The observation of stable cleavage sites in the limit digestion of virion DNA by Bal31 suggests that cleavage sites represent boundaries of core proteins which impede the exonuclease activity of Bal31. These data suggest that specific arrangement of core proteins on Ad5 virion DNA. Based on this analysis we determined positions of core proteins in viral genome using indirect end labeling technique. The size of supercoiled domains of virion DNA was estimated by electron microscopy and also by boundaries of mutually exclusive Bal31 cleavage sites at limit digestion condition. Our data suggest each supercoiled domain is equal to about 12% of Ad5 genome length and about 8 loops can be accommodated in Ad5 virion. However sequences at two extreme ends of the viral genome were found to be outside of supercoiled domains. An interesting correlation between supercoiled domains and gene domains of Ad5 genome was noticed.     

Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases

In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.     

Regulation of the function of eukaryotic DNA topoisomerase I: analysis of the binding step and of the catalytic constants of topoisomerization as a function of DNA topology

It was previously observed that two steps of the reaction of eukaryotic DNA topoisomerase I (topoisomerization and cleavage) depend upon the conformation of the DNA substrate: in both instances the supercoiled form is a more efficient substrate than the relaxed one. This paper reports the analysis of two other steps of the reaction: the binding of DNA topoisomerase I to DNA and the catalytic constants (Kcs) of topoisomerization as a function of the topology of the substrate. Binding. Competition assays show that supercoiled DNA binds the enzyme with even slower kinetics than the relaxed form. Therefore, the preferential topoisomerization of supercoiled DNA is not due to the binding step. Additional evidence that the rate-limiting step of the topoisomerization reaction is not the binding of the enzyme to DNA is provided by the fact that the kinetics of relaxation is first order. Catalysis. The Kcs of the topoisomerization reaction have been calculated and it was shown that they do not vary as a function of the topology of the substrate or of its size. Taken together, the data on binding, cleavage, topoisomerization, and Kcs suggest that the preferential topoisomerization of torsionally strained DNA is due to the higher availability, on this topological form, of DNA sites that allow the onset of the reaction.     

Site-specific cleavage of double-strand DNA by hydroperoxide of linoleic acid

The breakage of double-strand (ds) DNA by 13-L-hydroperoxy-cis-9,trans-11-octadecadienoic acid (LAHPO) was investigated by agarose gel electrophoresis of supercoiled pBR322 DNA and the site of cleavage on the DNA molecule was determined by the method of DNA sequence analysis using 3'-end and 5'-end-labeled DNA fragments as substrates. LAHPO caused cleavage at the position of guanine nucleotide in dsDNA. LAHPO caused dsDNA breaks at specific sites, but linoleic acid (LA) and 13-L-hydroxy-cis-9,trans-11-octadecadienoic acid (LAHO) have no such effects on dsDNA. The active oxygen atom of the hydroperoxy group of LAHPO was perhaps responsible for the site-specific cleavage of dsDNA.     

Analysis of chemical and enzymatic cleavage frequencies in supercoiled DNA

Chemical and enzymatic probing methods are powerful techniques for examining details of sequence-dependent structure in DNA and RNA. Reagents that cleave nucleic acid molecules in a structure-specific, but relatively sequence-non-specific manner, such as hydroxyl radical or DNase I, have been used widely to probe helical geometry in nucleic acid structures, nucleic acid-drug complexes, and in nucleoprotein assemblies. Application of cleavage-based techniques to structures present in superhelical DNA has been hindered by the fact that the cleavage pattern attributable to supercoiling-dependent structures is heavily mixed with non-specific cleavage signals that are inevitable products of multiple cleavage events. We present a rigorous mathematical procedure for extracting the cleavage pattern specific to supercoiled DNA and use this method to investigate the hydroxyl radical cleavage pattern in a cruciform DNA structure formed by a 60 bp inverted repeat sequence embedded in a negatively supercoiled plasmid. Our results support the presence of a stem-loop structure in the expected location and suggest that the helical geometry of the cruciform stem differs from that of the normal duplex form.     

Dinuclear macrocyclic polyamine zinc(II) complexes linked with flexible spacers: synthesis, characterization, and DNA cleavage

Dinuclear macrocyclic polyamine zinc(II) complexes, which have two cyclen groups linked by flexible spacers, have been synthesized as DNA cleavage agents. The structures of these new dinuclear complexes are consistent with the data obtained from elemental analysis, MS and ¹H NMR spectroscopy. The catalytic activity of these dinuclear complexes on DNA cleavage was studied. The results showed that the dinuclear zinc(II) complexes can catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II).     

Synthesis and DNA cleavage activities of mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes which linked with uracil

Mononuclear macrocyclic polyamine zinc(II), copper(II), cobalt(II) complexes, which could attach to peptide nucleic acid (PNA), were synthesized as DNA cleavage agents. The structures of these new mononuclear complexes were identified by MS and (1)H NMR spectroscopy. The catalytic activities on DNA cleavage of these mononuclear complexes with different central metals were subsequently studied, which showed that copper complex was better catalyst in the DNA cleavage process than zinc and cobalt complexes. The effects of reaction time, concentration of complexes were also investigated. The results indicated that the copper(II) complexes could catalyze the cleavage of supercoiled DNA (pUC 19 plasmid DNA) (Form I) under physiological conditions to produce selectively nicked DNA (Form II, no Form III produced) with high yields. The mechanism of the cleavage process was also studied.