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PubMed1995

Interaction of an alkylating camptothecin derivative with a DNA base at topoisomerase I-DNA cleavage sites.

DNA topoisomerase I (top1) is a ubiquitous nuclear enzyme. It is specifically inhibited by camptothecin, a natural product derived from the bark of the tree Camptotheca acuminata. Camptothecin and several of its derivatives are presently in clinical trial and exhibit remarkable anticancer activity. The present study is a further investigation of the molecular interactions between the drug and the enzyme-DNA complex. We utilized an alkylating camptothecin derivative, 7-chloromethyl-10,11-methylenedioxycamptothecin (7-ClMe-MDO-CPT), and compared its activity against calf thymus top1 in a DNA oligonucleotide containing a single top1 cleavage site with the activity of its nonalkylating analog, 7-ethyl-10,11-methylenedioxycamptothecin (7-Et-MDO-CPT). In the presence of top1, 7-ClMe-MDO-CPT produced a DNA fragment that migrated more slowly than the top1-cleaved DNA fragment observed with 7-Et-MDO-CPT. Top1 was unable to religate this fragment in the presence of high NaCl concentration or proteinase K at 50 degrees C. This fragment was resistant to piperidine treatment and was also formed with an oligonucleotide containing a 7-deazaguanine at the 5' terminus of the top1-cleaved DNA (base + 1). It was however cleaved by formic acid treatment followed by piperidine. These observations are consistent with alkylation of the +1 base (adenine or guanine) by 7-ClMe-MDO-CPT in the presence of top1 covalent complexes and provide direct evidence that camptothecins inhibit top1 by binding at the enzyme-DNA interface.

Alkylation↗
PubMed1998

Substitution of 5-methylcytosines for cytosines enhances the stability of topoisomerase I-DNA complexes and modulates the sequence selectivity of camptothecin-induced DNA cleavage.

We have investigated the binding and cleavage of DNA by human topoisomerase I using a 160 bp restriction fragment containing either natural bases or 5-methylcytosine residues in place of cytosines. Experiments were performed in the presence and absence of the antitumour drug camptothecin which specifically inhibits topoisomerase I. Replacement of all cytosines with 5-methylcytosine residues (i) reinforces the enzyme-DNA interaction, (ii) enhances the stability of topoisomerase I-DNA complexes and (iii) modulates the sequence selectivity of camptothecin-induced DNA cleavage. The methyl group exposed in the major groove of the double helix is identified as a critical element for the interaction between topoisomerase I and DNA.

5-Methylcytosine↗
PubMedDate not supplied

Structural variety of copper(II)-peroxide adducts and its relevance to DNA cleavage.

The reactivity of copper (II) compounds with several tetradentate ligands towards some spin-trapping reagents was studied in the presence of hydrogen peroxide. The compounds used in this study are roughly divided into two groups based on the reactivity towards 2,2,6,6-tetramethyl-4-piperidinol (and also 2,2,6,6-tetramethyl-4-piperidone), which are trapping agents for singlet oxygen. 1O2(1deltag); The A-group compounds exhibited a high activity to form the corresponding nitrone radical, which was detected by ESR spectroscopy, but corresponding activity of the B-group compounds was very low. The A-group compounds defined as above exhibited high activity for cleavage of DNA (supercoiled) Form I) in the presence of hydrogen peroxide, yielding DNA Form II (relaxed circular) or Form III (linear duplex) under our experimental conditions ([Cu (II)] = 0.1 approximately 0.5 mM). On the other hand, the B-group compounds effected complete degradation of the DNA (double-strand scission) under the same experimental conditions, formation of Form II or Form III DNA was negligible. Two different DNA cleavage patterns observed for A- and B-group compounds were elucidated by the different structural property of the copper (II)-peroxide adducts, which is controlled by the interaction through both DNA and the peripheral group of the ligand system.

Copper↗
PubMed1987

Cell cycle stage dependent variations in drug-induced topoisomerase II mediated DNA cleavage and cytotoxicity.

The DNA cleavage produced by 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in mammalian cells is putatively mediated by topoisomerase II. We found that in synchronized HeLa cells the frequency of such cleavage was 4-15-fold greater in mitosis than in S while the DNA of G1 and G2 cells exhibited an intermediate susceptibility to cleavage. The hypersensitivity of mitotic DNA to m-AMSA-induced cleavage was acquired relatively abruptly in late G2 and was lost similarly abruptly in early G1. The susceptibility of mitotic cells to m-AMSA-induced DNA cleavage was not clearly paralleled by an increase in topoisomerase II activity (decatenation of kinetoplast DNA) in 350 mM NaCl extracts from mitotic cells compared to similar extracts from cells in G1, S, or G2. Furthermore, equal amounts of decatenating activity from cells in mitosis and S produced equal amounts of m-AMSA-induced cleavage of simian virus 40 (SV40) DNA; i.e., the interaction between m-AMSA and extractable enzyme was similar in mitosis and S. The DNA of mitotic cells was also hypersensitive to cleavage by 4'-demethylepipodophyllotoxin 4-(4,6-O-ethylidene-beta-D-glucopyranoside) (etoposide), a drug that produces topoisomerase II mediated DNA cleavage without binding to DNA. Thus, alterations in the drug-chromatin interaction during the cell cycle seem an unlikely explanation for results in whole cells. Cell cycle stage dependent fluctuations in m-AMSA-induced DNA cleavage may result from fluctuations in the structure of chromatin per se that occur during the cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Amsacrine↗
PubMed2000

Mechanism of DNA cleavage by the DNA/RNA-non-specific Anabaena sp. PCC 7120 endonuclease NucA and its inhibition by NuiA.

A structural model of the DNA/RNA non-specific endonuclease NucA from Anabaena sp. PCC7120 that has been obtained on the basis of the three-dimensional structure of the related Serratia nuclease, suggests that the overall architecture of the active site including amino acid residues H124, N155 and E163 (corresponding to H89, N119 and E127 in Serratia nuclease) is similar in both nucleases. Substitution of these residues by alanine leads to a large reduction in activity (<0.1 %), similarly as observed for Serratia nuclease demonstrating that both enzymes share a similar mechanism of catalysis with differences only in detail. NucA is inhibited by its specific polypeptide inhibitor with a K(i) value in the subpicomolar range, while the related Serratia nuclease at nanomolar concentrations is only inhibited at an approximately 1000-fold molar excess of NuiA. The artificial chromophoric substrate deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) is cleaved by NucA as well as by Serratia nuclease. Cleavage of this analogue by NucA, however, is not inhibited by NuiA, suggesting that small molecules gain access to the active site of NucA in the enzyme-inhibitor complex under conditions where cleavage of DNA substrates is completely inhibited. The active site residue E163 seems to be the main target amino acid for inhibition of NucA by NuiA, but R93, R122 and R167 (corresponding to K55, R87, R131 in Serratia nuclease) are also involved in the NucA/NuiA interaction. NuiA deletion mutants show that the structural integrity of the N and C-terminal region of the inhibitor is important for complex formation with NucA and inhibition of nuclease activity. Based on these results a mechanism of DNA cleavage by NucA and its inhibition by NuiA is proposed.

Amino Acid Sequence↗
PubMed2004

Ca(2+)-mediated site-specific DNA cleavage and suppression of promiscuous activity of KpnI restriction endonuclease.

The characteristic feature of type II restriction endonucleases (REases) is their exquisite sequence specificity and obligate Mg(2+) requirement for catalysis. Efficient cleavage of DNA only in the presence of Ca(2+) ions, comparable with that of Mg(2+), is previously not described. Most intriguingly, KpnI REase exhibits Ca(2+)-dependent specific DNA cleavage. Moreover, the enzyme is highly promiscuous in its cleavage pattern on plasmid DNAs in the presence of Mn(2+) or Mg(2+), with the complete suppression of promiscuous activity in the presence of Ca(2+). KpnI methyltransferase does not exhibit promiscuous activity unlike its cognate REase. The REase binds to oligonucleotides containing canonical and mapped noncanonical sites with comparable affinities. However, the extent of cleavage is varied depending on the metal ion and the sequence. The ability of the enzyme to be promiscuous or specific may reflect an evolutionary design. Based on the results, we suggest that the enzyme KpnI represents an REase evolving to attain higher sequence specificity from an ancient nonspecific nuclease.

Calcium↗
PubMed1993

A fast and accurate enzyme-linked immunosorbent assay for the determination of the DNA cleavage activity of restriction endonucleases.

We have developed an assay procedure to monitor the cleavage of DNA substrates by restriction endonucleases. This procedure uses DNA substrates that are labeled with biotin on one 5' end and with an antigenic group, e.g., fluorescein or digoxigenin, on the other 5' end. After incubation with the restriction enzyme, the reaction is stopped with EDTA and an aliquot is pipetted into the well of an avidin-coated microtiter plate. This immobilizes the unreacted substrate and the biotinylated cleavage product, whereas the other cleavage product labeled with the antigenic group is subsequently washed off. The unreacted substrate is detected by an enzyme-linked immunosorbent assay with an appropriate enzyme-linked antibody. To test our assay we have measured the steady-state rate constants for cleavage of DNA by EcoRI yielding a kcat of 8.6 min-1 and a Km of 150 nM, which are close to values measured with other assays. The advantage of this assay is that it is not only fast and accurate, but also very sensitive. It allows for many samples to be analyzed in parallel and lends itself to automation. Furthermore, this assay can be designed as a competitive assay, when two substrates carrying different antigenic groups are used. The usefulness of such competitive assay is demonstrated by determining the influence of sequence context on the rate of DNA cleavage by EcoRI.

Base Sequence↗
PubMed1993

Structural basis for the sequence selectivity of DNA cleavage by bleomycins.

DNA strand scission by the bleomycin analogs talisomycin S10b and a novel fluorescent mimic, fluoromycin, has been characterized and compared with that of bleomycin A2. Both analogs were found to have essentially the same sequence specificity for DNA strand scission as bleomycin A2. As observed with bleomycin A2, the preferred sites of DNA cleavage by talisomycin S10b and fluoromycin were 5'-GpT-3' and 5'-GpC-3'. These results suggest that the DNA sequence selectivity of bleomycins remains unaltered upon modification of the C-terminal domain.

Base Sequence↗
PubMed1992

Topoisomerase II-mediated DNA cleavage activity and irreversibility of cleavable complex formation induced by DNA intercalator with alkylating capability.

A group of chrysophanol and emodin derivatives with DNA-intercalating capability and with or without alkylating potential have been synthesized and shown to have antitumor activity in vitro. The topoisomerase II (Topo II)-mediated DNA cleavage activities induced by representative compounds 3-(2-chloroethylamino) methyl-1,8-dihydroxy-9,10-anthraquinone (SK-31690), 3-bis [(2-chloroethyl)amino]methyl-1,8-dihydroxy-9,10-anthraquinone (SK-31662), and 3-(2-hydroxyethylamino)methy-1,8-dihydroxy-9,10-anthraquinon e (SK-31694), and their cytotoxicities, have been investigated. All three compounds inhibited the kinetoplast DNA decatenation catalyzed by DNA Topo II. These compounds inhibited leukemia cell growth and stimulated, in a dose-dependent manner from 0.5 to 60 microM, the formation of Topo II-DNA cleavable complexes, when 3'-32P-labeled DNA was used. The mapping of Topo II-mediated DNA cleavage sites using HindIII-digested 3'-32P-labeled DNA showed that, at 10 microM, these compounds induced protein-linked DNA breaks that correlated with cytotoxicity, with respect to their maximal efficacy or the reciprocal concentration for the half-maximal effect. The reversibility study showed that the amounts of protein-linked DNA cleavage induced by 4'-(9-acridinylamino)methanesulfon-m-anisidide and VP-16 as well as SK-31694, which lacks alkylating potential, were markedly decreased during 30-sec exposure to 65 degrees or 0.5 M NaCl. In contrast, protein-linked DNA cleavages induced by SK-31662, which has two alkylating functionalities, and by SK-31690, which has one alkylating functionality in its structure, cannot be reversed during the 15-min exposure to 65 degrees or 0.5 M NaCl. These data suggest that Topo II is a major cellular target for cytotoxicity of these compounds. Furthermore, DNA intercalators with alkylating potential interact with Topo II-DNA cleavable complexes in an irreversible manner, with enhanced toxicity.

Alkylating Agents↗
PubMed1990

Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I.

Cleavage of a defined linear duplex DNA by vaccinia virus DNA topoisomerase I was found to occur nonrandomly and infrequently. Approximately 12 sites of strand scission were detected within the 5372 nucleotides of pUC19 DNA. These sites could be classified as having higher or lower affinity for topoisomerase based on the following criteria. Higher affinity sites were cleaved at low enzyme concentration, were less sensitive to competition, and were most refractory to religation promoted by salt, divalent cations, and elevated temperature. Cleavage at lower affinity sites required higher enzyme concentration and was more sensitive to competition and induced religation. Cleavage site selection correlated with a pentameric sequence motif (C/T)CCTT immediately preceding the site of strand scission. Noncovalent DNA binding by topoisomerase predominated over covalent adduct formation, as revealed by nitrocellulose filter-binding studies. The noncovalent binding affinity of vaccinia topoisomerase for particular subsegments of pUC19 DNA correlated with the strength and/or the number of DNA cleavage sites contained therein. Thus, cleavage site selection is likely to be dictated by specific noncovalent DNA-protein interactions. This was supported by the demonstration that a mutant vaccinia topoisomerase (containing a Tyr----Phe substitution at the active site) that was catalytically inert and did not form the covalent intermediate, nevertheless bound DNA with similar affinity and site selectivity as the wild-type enzyme. Noncovalent binding is therefore independent of competence in transesterification. It is construed that the vaccinia topoisomerase is considerably more stringent in its cleavage and binding specificity for duplex DNA than are the cellular type I enzymes.

Cloning, Molecular↗
PubMed2005

DNA cleavage in immunoglobulin somatic hypermutation depends on de novo protein synthesis but not on uracil DNA glycosylase.

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step of Ig somatic hypermutation (SHM). However, its molecular mechanism is controversial. The RNA editing hypothesis postulates that AID deaminates cytosine in an unknown mRNA to generate a new mRNA encoding SHM endonuclease. On the other hand, the DNA deamination hypothesis explains DNA cleavage by cytosine deamination in DNA, followed by uracil removal by uracil DNA glycosylase (UNG). By using the protein synthesis inhibitor cycloheximide, we showed that SHM requires de novo protein synthesis in accord with predictions by the RNA editing hypothesis. In addition, we found that cycloheximide but not Ugi (the specific inhibitor of UNG) inhibited AID-dependent DNA cleavage in the Ig gene during SHM, by using histone H2AX focus formation as a marker of DNA cleavage. The results indicate the following order of events: AID expression, protein synthesis, DNA cleavage, and SHM. The requirement of protein synthesis but not of UNG for the DNA cleavage step of SHM forces us to reconsider the DNA deamination hypothesis and strengthens the RNA editing hypothesis.

Animals↗
PubMed1997

Identification of a novel 97 kDa endonuclease capable of internucleosomal DNA cleavage.

The two steps of DNA digestion seen in apoptotic cells were recreated in nuclei isolated from 5123tc rat hepatoma cells. The initial DNA cleavage, into high molecular weight fragments (300-50 kb), was stimulated by magnesium ions alone, whereas the second step required both calcium and magnesium ions and produced the ladder of oligonucleosomes. Endonucleolytic activities involved in both steps of DNA cleavage could be separated under appropriate conditions since the magnesium-modulated activity was tightly bound to the chromatin whereas the calcium/magnesium-dependent internucleosomal cleaving activity was easily extractable with a low ionic strength buffer. This calcium/ magnesium-dependent activity was attributed to a novel 97 kDa endonuclease which was also activated by manganese and cobalt and inhibited by millimolar concentrations of zinc, consistent with the properties ascribed to the apoptotic endonuclease. Furthermore, this activity became resistant to extraction with a low salt buffer in nuclei of apoptotic cells. Isoelectrofocusing revealed that the p97 protein existed in multiple forms of different isoelectric points (pI range 4.6-5.0), indicative of its postranslational modification. The p97 enzyme was present constitutively in a variety of cultured cells and rat tissues. It was active over a broad range of pH (6-9), but it was inactivated by reducing agents. In vitro, it displayed both endo- and exonucleolytic activities, and it was capable of both single- and double-stranded DNA cleavage. Rabbit polyclonal anti-p97 antibodies were generated and used to further distinguish this protein from other known cellular nucleases, namely, DNases I and II.

Animals↗
PubMed1999

A new method for determining the stereochemistry of DNA cleavage reactions: application to the SfiI and HpaII restriction endonucleases and to the MuA transposase.

A new method was developed for tracking the stereochemical path of enzymatic cleavage of DNA. DNA with a phosphorothioate of known chirality at the scissile bond is cleaved by the enzyme in H218O. The cleavage produces a DNA molecule with the 5'-[16O,18O, S]-thiophosphoryl group, whose chirality depends on whether the cleavage reaction proceeds by a single-step hydrolysis mechanism or by a two-step mechanism involving a protein-DNA covalent intermediate. To determine this chirality, the cleaved DNA is joined to an oligonucleotide by DNA ligase. Given the strict stereochemistry of the DNA ligase reaction, determined here, the original chirality of the phosphorothioate dictates whether the 18O is retained or lost in the ligation product, which can be determined by mass spectrometry. This method has advantages over previous methods in that it is not restricted to particular DNA sequences, requires substantially less material, and avoids purification of the products at intermediate stages in the procedure. The method was validated by confirming that DNA cleavage by the EcoRI restriction endonuclease causes inversion of configuration at the scissile phosphate. It was then applied to the reactions of the SfiI and HpaII endonucleases and the MuA transposase. In all three cases, DNA cleavage proceeded with inversion of configuration, indicating direct hydrolysis of the phosphodiester bond by water as opposed to a reaction involving a covalent enzyme-DNA intermediate.

5' Untranslated Regions↗
PubMed2005

Bacterial cell killing mediated by topoisomerase I DNA cleavage activity.

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or E. coli topoisomerase I can result in a mutant enzyme that has the SOS-inducing and cell-killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents.

Amino Acid Motifs↗
PubMed1990

Stabilization of the topoisomerase II-DNA cleavage complex by antineoplastic drugs: inhibition of enzyme-mediated DNA religation by 4'-(9-acridinylamino)methanesulfon-m-anisidide.

In order to elucidate the mechanism by which the intercalative antineoplastic drug 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) stabilizes the covalent topoisomerase II-DNA cleavage complex, the effect of the drug on the DNA cleavage/religation reaction of the type II enzyme from Drosophila melanogaster was examined. At a concentration of 60 microM, m-AMSA enhanced topoisomerase II mediated double-stranded DNA breakage approximately 5-fold. Drug-induced stabilization of the enzyme-DNA cleavage complex was readily reversed by the addition of EDTA or salt. When a DNA religation assay was utilized, m-AMSA was found to inhibit the topoisomerase II mediated rejoining of cleaved DNA approximately 3.5-fold. This result is similar to that previously reported for the effects of etoposide on the activity of the Drosophila enzyme [Osheroff, N. (1989) Biochemistry 28, 6157-6160]. Thus, it appears that structurally disparate classes of topoisomerase II targeted antineoplastic drugs stabilize the enzyme's DNA cleavage complex primarily by interfering with the ability of topoisomerase II to religate DNA.

Aminacrine↗
PubMed2002

Site-specific DNA cleavage by Chlorella virus topoisomerase II.

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.

Animals↗
PubMed1990

Nucleosome positioning as a critical determinant for the DNA cleavage sites of mammalian DNA topoisomerase II in reconstituted simian virus 40 chromatin.

We have assessed the ability of nucleosomes to influence the formation of mammalian topoisomerase II-DNA complexes by mapping the sites of cleavage induced by four unrelated topoisomerase II inhibitors in naked versus nucleosome-reconstituted SV40 DNA. DNA fragments were reconstituted with histone octamers from HeLa cells by the histone exchange method. Nucleosome positions were determined by comparing micrococcal nuclease cleavage patterns of nucleosome-reconstituted and naked DNA. Three types of DNA regions were defined: 1) regions with fixed nucleosome positioning; 2) regions lacking regular nucleosome phasing; and 3) a region around the replication origin (from position 5100 to 600) with no detectable nucleosomes. Topoisomerase II cleavage sites were suppressed in nucleosomes and persisted or were enhanced in linker DNA and in the nucleosome-free region around the replication origin. Incubation of reconstituted chromatin with topoisomerase II protected nucleosome-free regions from micrococcal nuclease cleavage without changing the overall micrococcal nuclease cleavage pattern. Thus, the present results indicate that topoisomerase II binds preferentially to nucleosome-free DNA and that the presence of nucleosomes at preferred DNA sequences influences drug-induced DNA breaks by topoisomerase II inhibitors.

Animals↗
PubMed1997

Actinomycin D amplifies site-specific DNA cleavage induced by neocarzinostatin.

Neocarzinostatin (NCS) is an antineoplastic antibiotic causing DNA cleavage. Actinomycin D (ActD) is an antineoplastic antibiotic, which does not cleave DNA strands. We examined the mechanism of ActD-mediated amplification of NCS-induced DNA cleavage using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene. NCS plus glutathione caused DNA cleavage at thymine and adenine residues in the absence of ActD. The addition of ActD enhanced the DNA cleavage at the sites at which the lesions on opposite strands are staggered 2 bases in a 3' direction, particularly at the 5'TCT-3'.3'-AGA-5', 5'-TGT-3'.3'-ACA-5' and 5'-ACT-3'.3'-TGA-5' sequences, suggesting that ActD amplified double-stranded DNA cleavage. The mechanism of ActD-mediated amplification of NCS-induced DNA cleavage can be explained by assuming that binding of ActD to DNA changes the DNA conformation to allow NCS to bind to DNA at the specific sequences.

Adenine↗