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PubMed2001

N-propargyl-2-alkynylbenzothiazolium aza-enediynes: role of the 2-alkynylbenzothiazolium functionality in DNA cleavage.

The 2-alkynylbenzothiazolium salts 3a-d incorporating an N-propargyl moiety have been prepared as aza-enediyne analogues. While these aza-enediynes are shown to be modest DNA cleavage agents, DNA cleavage was also observed with the N-methyl-2-alkynylbenzothiazolium salt 4, which lacks the aza-enediyne moiety. The structural requirements for DNA cleavage, and the correlation of DNA cleavage efficiency with the propensity of these compounds to undergo nucleophilic addition by methanol support a proposed DNA cleavage mechanism involving DNA alkylation by appropriate 2-alkynyl-substituted benzothiazolium salts.

Aza Compounds↗

PubMed2003

Photo-induced DNA cleavage reaction characteristics of propargylic sulfones possessing anthraquinone chromophore.

DNA cleavage potency of propargylic sulfones possessing anthraquinone chromophore 1 under UV-irradiation was evaluated in comparison with the dark reaction. 1 showed inefficient DNA cleavage activity, while having considerably strong DNA binding ability. This result is accounted for by spatial conditions that the activated alkylating allenic site of intercalated 1 could not effectively approach to DNA bases, most probably guanine moiety, and thereby led to insufficient DNA strand cleavage. In contrast, the DNA cleavage activity of 1 was notably enhanced upon UV-irradiation (lambda(ex)=365 nm) followed by incubation. Under UV-irradiation, further DNA cleavage were occurred primary at 5'-G of GG steps within DNA. A DNA cleavage mechanism for 1, by which photo-induced one-electron oxidation of 5'-G of GG steps may occur along with ordinary alkylation, has been proposed.

Alkylation↗

PubMed1998

FokI dimerization is required for DNA cleavage.

FokI is a type IIs restriction endonuclease comprised of a DNA recognition domain and a catalytic domain. The structural similarity of the FokI catalytic domain to the type II restriction endonuclease BamHI monomer suggested that the FokI catalytic domains may dimerize. In addition, the FokI structure, presented in an accompanying paper in this issue of Proceedings, reveals a dimerization interface between catalytic domains. We provide evidence here that FokI catalytic domain must dimerize for DNA cleavage to occur. First, we show that the rate of DNA cleavage catalyzed by various concentrations of FokI are not directly proportional to the protein concentration, suggesting a cooperative effect for DNA cleavage. Second, we constructed a FokI variant, FokN13Y, which is unable to bind the FokI recognition sequence but when mixed with wild-type FokI increases the rate of DNA cleavage. Additionally, the FokI catalytic domain that lacks the DNA binding domain was shown to increase the rate of wild-type FokI cleavage of DNA. We also constructed an FokI variant, FokD483A, R487A, which should be defective for dimerization because the altered residues reside at the putative dimerization interface. Consistent with the FokI dimerization model, the variant FokD483A, R487A revealed greatly impaired DNA cleavage. Based on our work and previous reports, we discuss a pathway of DNA binding, dimerization, and cleavage by FokI endonuclease.

Base Sequence↗

PubMed1998

The DNA gyrase-quinolone complex. ATP hydrolysis and the mechanism of DNA cleavage.

Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis which now proceeds through an alternative pathway involving two different conformations of the enzyme (Kampranis, S. C., and Maxwell, A. (1998) J. Biol. Chem. 269, 22606-22614). The kinetics of ATP hydrolysis via this pathway have been studied and found to differ from those of the reaction of the drug-free enzyme. The quinolone-characteristic ATPase rate is DNA-dependent and can be induced in the presence of DNA fragments as small as 20 base pairs. By observing the conversion of the ATPase rate to the quinolone characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone-induced DNA cleavage proceeds via a mechanism consisting of two cleavage events that is modulated in the presence of a nucleotide cofactor. We demonstrate that quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase.

Adenosine Triphosphate↗

PubMed2001

CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I chromatin condensation in apoptosis.

DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic chromatin condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of chromatin condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic chromatin condensation.

Animals↗

PubMed1986

Characterization of acquired epipodophyllotoxin resistance in a Chinese hamster ovary cell line: loss of drug-stimulated DNA cleavage activity.

Recent evidence indicates that type II DNA topoisomerases mediate epipodophyllotoxin-induced DNA damage and may be intrinsic to the drug's antitumor effects. Using an epipodophyllotoxin-resistant cell line, we have now further defined the relationship between DNA damage and cell death and delineated the significance of certain drug-enzyme interactions. When compared to wild-type cells, the mutant Chinese hamster ovary cell line, VpmR-5, exhibits marked resistance to both the cytotoxic and DNA cleavage activities of etoposide (VP-16). Steady-state concentrations of radiolabeled VP-16 are identical in both cell lines. Catalytic activity in crude nuclear extracts from wild-type and VpmR-5 cells is equal and is equally sensitive to inhibition by VP-16. However, using an assay that specifically measures generation of 5' protein-linked breaks in 32P-labeled 3' DNA, we have found that DNA cleavage activity in nuclear extract from the VpmR-5 line is profoundly resistant to stimulation by VP-16. Further, a somatic cell hybrid line of VpmR-5 cells and drug-sensitive EOT-3 cells exhibits recovery of VP-16 sensitivity in concert with reconstitution of DNA cleavage activity. These data indicate that stimulation of enzyme-mediated DNA cleavage, rather than loss of normal topoisomerase function, is responsible for epipodophyllotoxin-induced cytotoxicity.

Aminoacridines↗

PubMed2002

DNA cleavage by aromatic amines.

A series of aryl amines was found to induce cleavage of DNA. Subsequent refinement led to an efficient family of dimeric derivatives capable of cleavage at low concentration. Initial investigations suggest this is an unprecedented mode of DNA cleavage, which may be ultimately applied to the development of sequence-specific agents.

Amines↗

PubMed1988

DNA cleavage based on high voltage electric pulse.

A high voltage electric pulse was applied to DNA cleavage. The DNA cleavage reaction was dependent on the voltage amplitude, pulse number and pulse width. Radical scavengers and ESR data indicated the possibility that active species such as OH radical were strongly related to DNA cleavage.

DNA↗

PubMed1991

Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus.

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.

Amsacrine↗

PubMed1996

DNA cleavage is not required for the binding of quinolone drugs to the DNA gyrase-DNA complex.

The primary target for the quinolone group of antibacterial agents is DNA gyrase. One model for the interaction of quinolone drugs with gyrase and DNA suggests that the drugs bind to the single-stranded regions revealed following DNA cleavage by the enzyme. We have tested this hypothesis by using mutants which have the active-site tyrosine in the gyrase A subunit altered to phenylalanine or serine. We have found that proteins bearing these mutations are still able to bind drug, suggesting that DNA cleavage is not a prerequisite for drug binding. We have also found that the blocking of transcription by RNA polymerase in vitro by the gyrase-quinolone complex on DNA does not occur when the active-site tyrosine is mutated to serine; i.e., polymerase blocking requires DNA cleavage.

Anti-Infective Agents↗

PubMed1996

Drug-specific sites of topoisomerase II DNA cleavage in Drosophila chromatin: heterogeneous localization and reversibility.

DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells.

Amsacrine↗

PubMed2004

De novo protein synthesis is required for activation-induced cytidine deaminase-dependent DNA cleavage in immunoglobulin class switch recombination.

Activation-induced cytidine deaminase is required for the DNA cleavage step of Ig class switch recombination (CSR). However, its molecular mechanism is controversial. RNA-editing hypothesis postulates that activation-induced cytidine deaminase deaminates cytosine in an unknown mRNA to generate a new mRNA encoding an endonuclease for CSR and thus predicts that DNA cleavage depends on de novo protein synthesis. On the other hand, DNA deamination hypothesis proposes that DNA cleavage is initiated by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase. By using the chromatin immunoprecipitation assay to detect gamma-H2AX focus formation as a marker for DNA cleavage, we found that cycloheximide inhibited DNA cleavage in the Ig heavy-chain locus during CSR. Requirement of protein synthesis in the DNA cleavage step of CSR strengthens the RNA-editing hypothesis.

Animals↗

PubMed2001

Effect of phosphorothioate substitutions on DNA cleavage by Escherichia coli DNA topoisomerase I.

To evaluate the structural influence of the DNA phosphate backbone on the activity of Escherichia coli DNA topoisomerase I, modified forms of oligonucleotide dA(7) were synthesized with a chiral phosphorothioate replacing the non-bridging oxygens at each position along the backbone. A deoxy-iodo-uracil replaced the 5'-base to crosslink the oligonucleotides by ultraviolet (UV) and assess binding affinity. At the scissile phosphate there was little effect on the cleavage rate. At the +1 phosphate, the rectus phosphorus (Rp)-thio-substitution reduced the rate of cleavage by a factor of 10. At the +3 and -2 positions from the scissile bond, the Rp-isomer was cleaved at a faster rate than the sinister phosphorus (Sp)-isomer. The results demonstrate the importance of backbone contacts between DNA substrate and E. coli topoisomerase I.

Binding Sites↗

PubMedDate not supplied

Acridine conjugates of 3-clip-phen: influence of the linker on the synthesis and the DNA cleavage activity of their copper complexes.

To increase the DNA cleavage activity and the cell delivery of the bis(phenanthroline) DNA cleaver "3-Clip-Phen", conjugates between 3-Clip-Phen and the intercalators acridine and 6-chloro-2-methoxyacridine, through amino acid linkers of various length, were prepared. After complexation with CuCl(2), the ability of these conjugates to cleave phiX 174 DNA in the presence of a reductant and air was compared. The results indicated that (i) the coupling of 3-Clip-Phen to an acridine derivative increased the DNA cleavage efficiency of the copper complexes, (ii) the acridine derivatives were more active than 6-chloro-2-methoxyacridine derivatives, (iii) the linker length influenced cleavage efficiency, the highest DNA cleavage activity being obtained for an aminocaproic spacer.

Acridines↗

PubMed1985

Inhibition of epipodophyllotoxin cytotoxicity by interference with topoisomerase-mediated DNA cleavage.

This laboratory and others previously proposed that the antitumor effects of the epipodophyllotoxin compounds are based on their abilities to stimulate DNA cleavage by a DNA topoisomerase. To explore this relationship further, we studied the intercalating agent ethidium bromide and found that it blocked epipodophyllotoxin-induced DNA cleavage by DNA topoisomerase II in vitro as well as in vivo. Using an in vitro assay consisting of purified calf thymus DNA topoisomerase II, end-labeled DNA, and the epipodophyllotoxin teniposide, we found that ethidium bromide markedly interfered with the enzyme-mediated DNA cleavage. Furthermore, ethidium bromide also blocked the formation of DNA single- and double-strand breaks in mouse L1210 cells when exposed to the epipodophyllotoxin etoposide. This effect cannot be explained by alterations in drug accumulation since steady-state drug concentrations were unchanged, and the effect was also observed in isolated nuclei. In addition to its effects on epipodophyllotoxin-mediated DNA breakage, ethidium bromide also potently inhibited the cytotoxic effects of etoposide but only when present during drug treatment. Thus, we believe that ethidium bromide may be a useful tool to investigate drug-induced perturbations of topoisomerase activity and their relationship to antitumor effect. Our data strongly support the hypothesis that the antitumor activity of epipodophyllotoxins is based on the ability to stimulate the formation of a cleavable complex between DNA topoisomerase and DNA.

Animals↗

PubMed1998

Intercomparison of apoptosis morphology with active DNA cleavage on single cells in vitro and on testis tumours.

Apoptosis morphology (DNA condensation) and endonucleolytical DNA cleavage (TdT assay) were measured simultaneously on double fluorescence labelled cells employing confocal laser scanning and conventional immunofluorescence microscopy. In vitro experiments on irradiated HL-60 cells revealed a high correspondence of non-apoptotic (normal) cells without detectable DNA cleavage, versus apoptotic cells and apoptotic bodies showing DNA cleavage. Experiments performed on histological slides of testis tumours reflected a heterogeneous picture: non-apoptotic (normal) cells, apoptotic cells, and apoptotic bodies appeared either with or without detectable DNA cleavage. These data allowed the characterization and quantitation of the grade of disturbance/heterogeneity of the apoptosis programme in vivo. Furthermore, the measured apoptotic index (AI) based on apoptosis morphology was lower than the AI assessed by DNA cleavage, in contrast to published work. Taken together, these methods represent a new approach and might be suitable for improved correlation with clinical parameters. In addition, the data presented confirm frequently published doubts regarding the ability of the TdT assay to detect apoptosis as defined by morphological criteria in tumours.

Apoptosis↗

PubMed2004

Hindering the strand passage reaction of human topoisomerase IIalpha without disturbing DNA cleavage, ATP hydrolysis, or the operation of the N-terminal clamp.

DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIalpha enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIalpha enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.

Adenosine Triphosphate↗

PubMed1999

Oxidation of ochratoxin A by an Fe-porphyrin system: model for enzymatic activation and DNA cleavage.

Ochratoxin A (OTA, 1) is a fungal toxin that facilitates single-strand DNA cleavage, DNA adduction, and lipid peroxidation when metabolically activated. To model the enzymatic activation of OTA, we have employed the water-soluble iron(III) meso-tetrakis(4-sulfonatophenyl)porphyrin (FeTPPS) oxidation system. In its presence, OTA has been found to facilitate single-strand cleavage of supercoiled plasmid DNA through production of reactive oxygen species (ROS) (i.e., the hydroxyl radical, HO(*)). The reaction of OTA with the FeTPPS oxidation system also generated three hydroxylated products (chlorine atom still attached), which was taken as evidence for production of the known hydroxylated metabolites (2-4) of OTA. This result suggested that the FeTPPS system served as a reasonable model for the enzymatic activation of OTA. When the reaction of OTA with FeTPPS was carried out in the presence of excess hydrogen peroxide (H(2)O(2)) and sodium ascorbate, a hydroquinone species (OTHQ, 5) was detected in which an OH group has replaced the chlorine atom of OTA. The production of OTHQ (5) was dependent on the presence of the reducing agent, sodium ascorbate, which suggested that the oxidation catalyst furnished the quinone derivative OTQ (6) that was subsequently reduced to OTHQ (5) by ascorbate. Utilizing a synthetic sample of OTHQ (5), the hydroquinone was found to undergo autoxidation with a t(1/2) of 11.1 h at pH 7.4, and to possess a pK(a) value of 8.03 for the phenolic oxygen ortho to the carbonyl groups. Our findings imply that the hydroquinone (OTHQ) and quinone (OTQ) metabolites of OTA have the ability to cause alkylation/redox damage and have allowed us to propose a viable pathway for oxidative damage by OTA.

Ascorbic Acid↗