Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA.

The site specificities of the type 1 DNA topoisomerases (topo 1) from rat liver and wheat germ were investigated. The nucleotide sequence at break sites on duplex SV40 DNA were determined for 245 wheat germ topo 1 sites and 223 rat liver topo 1 sites over a region of 1781 nucleotides. The enzymes from the two different sources show similar, but not identical patterns of DNA strand breakage. The sites occur frequently, but are not broken with equal probabilities. Major sites of breakage occur on the average every one to two turns of the helix, thus if sites of breakage accurately represent topo 1 sites of activity, the DNA sequence alone would appear to place few limits on the access of the enzyme to DNA. Sequences around the strongest sites for both enzymes show a bias in base composition for the four nucleotides immediately 5' to the break site (-4 to -1 positions), but no bias is observed 3' to the site of breakage. Consensus sequences for both enzymes were determined. Variations from the consensus sequence appear to affect the two enzymes differently and may account for the differences observed in the specificity of breakage.     

Protection of halogenated DNA from strand breakage and sister-chromatid exchange induced by the topoisomerase I inhibitor camptothecin

The fundamental nuclear enzyme DNA topoisomerase I (topo I), cleaves the double-stranded DNA molecule at preferred sequences within its recognition/binding sites. We have recently reported that when cells incorporate halogenated nucleosides analogues of thymidine into DNA, it interferes with normal chromosome segregation, as shown by an extraordinarily high yield of endoreduplication, and results in a protection against DNA breakage induced by the topo II poison m-AMSA [F. Cortés, N. Pastor, S. Mateos, I. Domínguez, The nature of DNA plays a role in chromosome segregation: endoreduplication in halogen-substituted chromosomes, DNA Repair 2 (2003) 719-726; G. Cantero, S. Mateos, N. Pastor; F. Cortés, Halogen substitution of DNA protects from poisoning of topoisomerase II that results in DNA double-strand breaks (DSBs), DNA Repair 5 (2006) 667-674]. In the present investigation, we have assessed whether the presence of halogenated nucleosides in DNA diminishes the frequency of interaction of topo I with DNA and thus the frequency with which the stabilisation of cleavage complexes by the topo I poison camptothecin (CPT) takes place, in such a way that it protects from chromosome breakage and sister-chromatid exchange. This protective effect is shown to parallel a loss in halogen-substituted cells of the otherwise CPT-increased catalytic activity bound to DNA.     

Niacin prevents DNA strand breakage by adenosine deaminase inhibitors

The adenosine deaminase inhibitors deoxycoformycin and erythro-9-(2-hydroxy-3 nonyl) adenine (EHNA) induce single-strand DNA breaks in cultured human lymphocytes. Deoxycoformycin produced a significant number of strand breaks (4-fold increase compared to controls) and EHNA induced strand breaks in a dose-dependent manner. Strand breaks stimulate repair by poly(ADP-ribosylation) which requires NAD+ as a cofactor. Niacin is a precursor of NAD+ and when preincubated with human lymphocytes prior to exposure to adenosine deaminase inhibitors, strand breakage was reduced significantly. The administration of niacin may represent an approach to decreasing the toxicity associated with these agents.     

DNA strand breakage after kidney storage

Ischemia and storage cause single-stranded DNA breaks. The breaks become evident after 30 min of warm storage or after only 4 hr of cold storage. The number of breaks increases with increasing ischemia time. The DNA damage was detected by alkaline sucrose gradient analysis of the DNA.     

Purification and characterization of wheat germ DNA topoisomerase I (nicking-closing enzyme)

Wheat germ contains an enzyme capable of removing supercoils from circular DNA. We have purified this enzyme using Polymin P fractionation, ammonium sulfate precipitation, and chromatography on Bio-Rex 70 and phenyl-Sepharose. Renaturation after electrophoresis on sodium dodecyl sulfate-polyacrylamide gels shows that topoisomerase activity is associated with a polypeptide with a Mr = about 111,000. The enzyme is similar to other eukaryotic type I DNA topoisomerases (nicking-closing enzymes) by the following criteria: it is capable of increasing or decreasing the topological linking number of covalently closed DNA substrate; it is capable of restoring an equilibrium distribution of linking numbers to DNA substrate with a single unique linking number; and it does not require magnesium ion or ATP for activity.     

DNA strand transfer reactions catalyzed by vaccinia topoisomerase I.

Vaccinia virus DNA topoisomerase I forms a 3'-phosphoryl intermediate with duplex DNAs containing the conserved binding/cleavage motif 5'CCCTT decreases. Covalently bound enzyme is capable of transferring the incised DNA strand to a heterologous DNA acceptor containing a 5'OH terminus. Both intramolecular and intermolecular religation reactions are catalyzed. Intramolecular strand transfer occurs to the noncleaved strand of the DNA duplex and results in formation of a hairpin loop. Intermolecular religation to an exogenous DNA strand is favored over hairpin formation and requires the potential for base pairing between the acceptor and the noncleaved strand of the donor complex. As few as 4 potential base pairs are sufficient to support intermolecular transfer. These results in vitro are consistent with the proposal that vaccinia topoisomerase can catalyze sequence-specific strand transfer during genetic recombination in vivo (Shuman, S. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 10104-10108.).     

Single strand DNA cleavage reaction of duplex DNA by Drosophila topoisomerase II

A unique reaction for type II DNA topoisomerase is its cleavage of a pair of DNA strands in concert. We show however, that in a reaction mixture containing a molar excess of EDTA over Mg2+, or when Mg2+ is substituted by Ca2+, Mn2+, or Co2+, the enzyme cleaves only one rather than both strands. These results suggest that the divalent cations may play an important role in coordinating the two subunits of DNA topoisomerase II during the strand cleavage reaction. The single strand and the double strand cleavage reactions are similar in the following aspects: both require the addition of a protein denaturant, can be reversed by low temperature or high salt, and a topoisomerase II molecule is attached covalently to the 5' phosphoryl end of each broken DNA strand. Furthermore, the single strand cleavage sites share a similar sequence preference with double strand cleavage sites. There is, however, a strand bias for the single strand cleavage reaction. We show also that under single strand cleavage conditions, topoisomerase II still possesses a low level of double strand passage activity: it can introduce topological knots into both covalently closed or nicked DNA rings, and change the linking number of a plasmid DNA by steps of two. The implication of this observation on the sequential cleavage of the two strands of the DNA duplex during the normal DNA double strand passage process catalyzed by type II DNA topoisomerases is discussed.     

Single strand breakage and repair in eukaryotic DNA as assayed by S1 nuclease.

A sensitive new approach for measuring the repair of single strand breaks in DNA induced by low doses of gamma irradiation was tested in cultured fibroblasts from Chinese hamster lung, human afflicted with ataxia telangiectasia or Fanconi's anemia and in normal cells of early and late passages. The assay is based on the increasing rate of strand separation of DNA duplexes in alkali for molecules with increasing numbers of single strand scissions. DNA strand separation is shown to follow the relation, in F = -(1/Mn - const) - tbeta where F is the proportion of double-stranded DNA, detected as S1 nuclease resistant, after alkaline denaturation time, t. Mn is the number-average molecular weight of DNA between single strand breaks. beta less than 1 is an empirically determined constant. The results suggest an increase in the number-average molecular weight between breaks, Mn, with increasing times for repair. The final level attained corresponds to the Mn of control DNA in unirradiated cells. As few as one break introduced into 109 daltons of single-stranded control cell DNA can be detected. The kinetics, requirements and sensitivities of this assay are described.     

Human DNA topoisomerase I: quantitative analysis of the effects of camptothecin analogs and the benzophenanthridine alkaloids nitidine and 6-ethoxydihydronitidine on DNA topoisomerase I-induced DNA strand breakage.

Human DNA topoisomerase I (topo I) has been purified from normal placenta and from a recombinant baculovirus expression system. A new radiolabeled plasmid DNA assay has been used to quantitate the activity of the purified enzymes and to compare the ability of several types of topo I-targeted drugs to induce topo I-mediated DNA strand breaks. The 100-kDa recombinant enzyme form isolated from the baculovirus expression system is able to relax 2564 ng of supercoiled M-13 mp19 plasmid per minute per nanogram of enzyme. The addition of camptothecin (1 microM) to the reaction lowers the rate to 1282 ng per minute per nanogram of enzyme. The 100-kDa topo I from human placenta is able to relax 1092 ng of supercoiled plasmid per minute per nanogram of enzyme and the 68-kDa topo I form from placenta is able to relax 2069 ng of supercoiled plasmid per minute per nanogram of enzyme. Camptothecin (1 microM) decreases the relaxation rate of the placental enzymes about 50%. In the presence of several different types of topo I-targeted drugs, both the recombinant and placental enzymes are induced to cleave plasmid DNA. Quantitative DNA cleavage assays with radioactive plasmid DNA and 9-aminocamptothecin, topotecan, SN-38, 10, 11-methylenedioxycamptothecin, 7-ethyl-10, 11-methylenedioxycamptothecin, 7-chloromethyl-10, 11-methylenedioxycamptothecin, nitidine, and 6-ethoxy-5, 6-dihydronitidine indicate that the order of potency in inducing topo I-mediated DNA breakage is methylenedioxycamptothecin analogs > SN-38 > 9-aminocamptothecin > topotecan and camptothecin > nitidine compounds. The order of potency correlates with the half-lives of the topo I-DNA drug complex determined with radiolabeled DNA in 0.45 M NaCl at 30 degrees C. The half-life of the complex formed with 7-chloromethyl-10,11-methylenedioxycamptothecin is greater than 90 min whereas the half-life of the topo I-DNA complex with 6-ethoxy-5, 6-dihydronitidine is less than 15 s. The other drugs tested were found to have drug complex half-lives which fall between these two extremes.     

Iron-mediated DNA damage: sensitive detection of DNA strand breakage catalyzed by iron.

Oxidative DNA damage is involved in mutagenesis, carcinogenesis, aging, radiation effects, and the action of several anticancer drugs. Accumulated evidence indicates that iron may play an important role in those processes. We studied the in vitro effect of low concentrations of Fe(II) alone or Fe(III) in the presence of reducing agents on supercoiled plasmid DNA. The assay, based on the relaxation and linearization of supercoiled DNA, is simple yet sensitive and quantitative. Iron mediated the production of single and double strand breaks in supercoiled DNA. Iron chelators, free radical scavengers, and enzymes of the oxygen reduction pathways modulated the DNA damage. Fe(III)-nitrilotriacetate (NTA) plus either H2O2, L-ascorbate, or L-cysteine produced single and double strand breaks as a function of reductant concentration. A combination of 0.1 microM Fe(III)-NTA and 100 microM L-ascorbate induced detectable DNA strand breaks after 30 min at 24 degrees C. Whereas superoxide dismutase was inhibitory only in systems containing H2O2 as reductant, catalase inhibited DNA breakage in all the iron-mediated systems studied. The effect of scavengers and enzymes indicates that H2O2 and .OH are involved in the DNA damaging process. These reactions may account for the toxicity and carcinogenicity associated with iron overload.     

DNA strand breakage by hydroxyphenyl radicals generated from mutagenic diazoquinone compounds.

The mutagenic diazoquinone compounds p-diazoquinone (p-DQ), o-diazoquinone (o-DQ) and 3-diazo-N-nitrosobamethan (D-BM) cleaved the phosphodiester bond of lambda DNA, phi X174 RFI DNA and M13mp8ss DNA. p-DQ also cleaved the phosphodiester bond of bis(p-nitrophenyl)phosphate. The breakage of the phosphodiester bond was inhibited by the antioxidant butyl hydroxyanisole (BHA), ethanol, the spin trapping agent DMPO, cysteine and 2-mercaptoethanol. While incubation of p-DQ and o-DQ alone gave p-hydroquinone and catechol, respectively, incubation of these compounds in the presence of BHA and ethanol gave phenol in large yields. Incubation of p-DQ and o-DQ with the spin trapping agents DMPO and PBN gave spin adducts assignable as p- and o-hydroxyphenyl adducts, respectively. The breakage of the phosphodiester bond of DNA by the diazoquinone compounds is suggested to be due to the hydroxyphenyl radicals generated during incubation.     

Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks.

The structure of replicating simian virus 40 minichromosomes, extracted from camptothecin-treated infected cells, was investigated by biochemical and electron microscopic methods. We found that camptothecin frequently induced breaks at replication forks close to the replicative growth points. Replication branches were disrupted at about equal frequencies at the leading and the lagging strand sides of the fork. Since camptothecin is known to be a specific inhibitor of type I DNA topoisomerase, we suggest that this enzyme is acting very near the replication forks. This conclusion was supported by experiments with aphidicolin, a drug that blocks replicative fork movement, but did not prevent the camptothecin-induced breakage of replication forks. The drug teniposide, an inhibitor of type II DNA topoisomerase, had only minor effects on the structure of these replicative intermediates.     

The basis for camptothecin enhancement of DNA breakage by eukaryotic topoisomerase I.

The eukaryotic topoisomerase I (topo I) is the target of the cytotoxic alkaloid camptothecin (CTT). In vitro, CTT enhances the breakage of DNA by topo I when the reaction is stopped with detergent. Although breakage at some sites is enhanced to a great extent while breakage at others is enhanced only minimally, CTT does not significantly change the breakage specificity of topo I in vitro. It has been suggested that CTT acts by slowing the reclosure step of the nicking-closing reaction. To test this hypothesis, we have measured the rate of reclosure for different break sites in the presence of CTT after adding 0.5 M NaCl to a standard low salt reaction. In support of the hypothesis, we find that topo I-mediated DNA breakage is enhanced the greatest at those sites where closure of the break is the slowest. These results suggest a mechanism for the toxicity of CTT in vivo.     

Mechanism of DNA strand breakage by piperidine at sites of N7-alkylguanines

The volatile, secondary amine piperidine is used in the Maxam-Gilbert chemical method of DNA sequencing to create strand breaks in DNA at sites of damaged bases. As such it is often used in generalized studies of DNA damage to identify 'alkali-labile lesions'. We confirm the mechanism proposed by Maxam and Gilbert (Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560) by which aqueous piperidine creates strand breaks at sites of N7-guanine alkylations: alkaline conditions catalyze rupture of the C8-N9 bond, forming a formamido-pyrimidine structure which is displaced from the ribose moiety by piperidine. In keeping with this mechanism, the tertiary amine, N-methylpiperidine, does not catalyze the formation of strand breaks in alkylated DNA. Our data confirm the prediction that high pH in and of itself will not create strand breaks at sites of N7-alkylguanines.     

Characterization of DNA strand breakage in vitro by the antitumor protein neocarzinostatin.

The antitumor protein antibiotic neocarzinostatin causes strand scission of DNA in vitro in the presence of a sulfhydryl compound. The breaks are single stranded in nature and bear 5'-phosphoryl termini. All four deoxymononucleotides are recoverable at the 5'-ends of the cleavage sites although a higher proportion of dGMP and TMP are consistently found. The lesions are not repairable with polynucleotide ligase from Escherichia coli. A quantitative assay was developed to determine the pH profile and time course of the reaction. Data from protection experiments with synthetic and natural DNAs indicate the requirement for thymidylic acid and deoxyadenylic acid in the DNA for cutting. In DNA-RNA hybrids, riboadenylic acid can substitute for deoxyadenylic acid, whereas ribouridylic acid cannot substitute for thymidylic acid. Release of thymine is detected, and the amount of release correlates well with the number of strand scissions.     

Mechanism of DNA strand breakage induced by photosensitized tetracycline-Cu(II) complex

Tetracyclines (TCs) in combination with Cu(II) ions exhibited significant DNA damaging potential vis a vis tetracyclines per se. Interaction of tetracyclines with DNA resulted in alkylation at N-7 and N-3 positions of adenine and guanine bases, and caused destabilization of DNA secondary structure. Significant release of acid-soluble nucleotides from tetracycline-modified DNA upon incubation with S(1) nuclease ascertained the formation of single stranded regions in the DNA. Also, the treatment of tetracycline-modified DNA with 0.1 and 0.5M NaOH resulted in 62 and 76% hydrolysis compared to untreated control. Comparative alkaline hydrolysis of DNA modified with tetracycline derivatives showed differential DNA damaging ability in the order as DOTC > DMTC > TC > OTC > CTC. Addition of Cu(II) invariably augmented the extent of tetracycline-induced DNA damage. The alkaline unwinding assay clearly demonstrated the formation of approximately six strand breaks per unit DNA at 1:10 DNA nucleotide/TC molar ratio in the presence of 0.1mM Cu(II) ions. At a similar Cu(II) concentration, a progressive transformation of covalently closed circular (CCC) (form-I) plasmid pBR322 DNA to forms-II and -III was noticed with increasing tetracycline concentrations. The results obtained with the free-radical quenchers viz. mannitol, thiourea, sodium benzoate and superoxide dismutase (SOD) suggested the involvement of reactive oxygen species in the DNA strand breakage. It is concluded that the tetracycline-Cu(II)-induced DNA damage occurs due to (i) significant binding of tetracycline and Cu(II) with DNA, (ii) methyl group transfer from tetracycline to the putative sites on nitrogenous bases, and (iii) metal ion catalyzed free-radical generation in close vicinity of DNA backbone upon tetracycline photosensitization. Albeit, the DNA alkylation and strand cleavage are repairable lesions, but any defect in the critical repair pathway may augment the damage accumulation and mutagenesis.