Radioprobing of DNA: distribution of DNA breaks produced by decay of 125I incorporated into a triplex-forming oligonucleotide correlates with geometry of the triplex.

The distribution of breaks produced in both strands of a DNA duplex by the decay of 125I carried by a triplex-forming DNA oligonucleotide was studied at single nucleotide resolution. The 125I atom was located in the C5 position of a single cytosine residue of an oligonucleotide designed to form a triple helix with the target sequence duplex. The majority of the breaks (90%) are located within 10 bp around the decay site. The addition of the free radical scavenger DMSO produces an insignificant effect on the yield and distribution of the breaks. These results suggest that the majority of these breaks are produced by the direct action of radiation and are not mediated by diffusible free radicals. The frequency of breaks in the purine strand was two times higher that in the pyrimidine strand. This asymmetry in the yield of breaks correlates with the geometry of this type of triplex; the C5 of the cytosine in the third strand is closer to the sugar-phosphate backbone of the purine strand. Moreover, study of molecular models shows that the yield of breaks at individual bases correlates with distance from the 125I decay site. We suggest the possible use of 125I decay as a probe for the structure of nucleic acids and nucleoprotein complexes.     

Determination and analysis of site-specific 125I decay-induced DNA double-strand break end-group structures

End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.     

Gene targeted DNA double-strand break induction by (125)I-labeled triplex-forming oligonucleotides is highly mutagenic following repair in human cells.

A parallel binding motif 16mer triplex-forming oligonucleotide (TFO) complementary to a polypurine-polypyrimidine target region near the 3'-end of the SupF gene of plasmid pSP189 was labeled with [5-(125)I]dCMP at position 15. Following triplex formation and decay accumulation, radiation-induced site-specific double-strand breaks (DSBs) were produced in the pSP189 SupF gene. Bulk damaged DNA and the isolated site-specific DSB-containing DNA were separately transfected into human WI38VA13 cells and allowed to repair prior to recovery and analysis of mutants. Bulk damaged DNA had a relatively low mutation frequency of 2.7 x 10(-3). In contrast, the isolated linear DNA containing site-specific DSBs had an unusually high mutation frequency of 7.9 x 10(-1). This was nearly 300-fold greater than that observed for the bulk damaged DNA mixture, and >1.5 x 10(4)-fold greater than background. The mutation spectra displayed a high proportion of deletion mutants targeted to the(125)I binding position within the SupF gene for both bulk damaged DNA and isolated linear DNA. Both spectra were characterized by complex mutations with mixtures of changes. However, mutations recovered from the linear site-specific DSB-containing DNA presented a much higher proportion of complex deletion mutations.     

Sequence-specific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides.

A triplex-forming oligonucleotide (TFO) complementary to the polypurine-polypyrimidine region of the nef gene of the Human Immunodeficiency Virus (HIV) was labeled with 125I at the C5 position of a single deoxycytosine residue. Labeled TFO was incubated with a plasmid containing a fragment of the nef gene. Decay of 125I was found to cause double-strand breaks (DSB) within the nef gene upon triplex formation in a sequence specific manner. No DSB were detected after incubation at ionic conditions preventing triplex formation or when TFO was labeled with 32P instead of 125I. Mapping DSB sites with single base resolution showed that they are distributed within 10 bp of a maximum located exactly opposite the position of the [125I] IdC in the TFO. We estimate that on average the amount of DSB produced per decay is close to one.     

Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides

Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.     

Strand breaks in plasmid DNA after positional changes of Auger electron-emitting iodine-125: direct compared to indirect effects.

To elucidate the nature and kinetics of DNA strand breaks caused by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA in the presence of the (.)OH scavenger dimethyl sulfoxide (DMSO) after the decay of (125)I (1) in proximity to DNA after minor-groove binding ((125)I-iodoHoechst 33342, (125)IH) and (2) at a distance from DNA ((125)I-iodoantipyrine, (125)IAP). DMSO is efficient at protecting supercoiled plasmid DNA from the decay of (125)I free in solution (dose modification factor, DMF = 59 +/- 4) and less effective when the (125)I decays occur close to DNA (DMF = 3.8 +/- 0.3). This difference is due mainly to the inability of DMSO to protect DNA from the double-strand breaks produced by groove-bound (125)I (DMF = 1.0 +/- 0.2). Additionally, the fragmentation of plasmid DNA beyond the production of single-strand and double-strand breaks that is seen after the decay of (125)IH and not (125)IAP (Kassis et al., Radiat. Res. 151, 167-176, 1999) cannot be modified by DMSO. These results demonstrate that the mechanisms underlying double-strand breaks caused by the decay of (125)IH differ in nature from those caused by the decay of (125)IAP.     

Plasmid DNA breakage by decay of DNA-associated Auger electron emitters: approaches to analysis of experimental data

Plasmid DNA is a popular substrate for the assay of DNA strand breakage by a variety of agents. The use of the plasmid assay relies on the assumption that individual damaging events occur at random, which allows the application of Poisson statistics. This assumption is not valid in the case of damage arising from decay of DNA-associated Auger electron emitters, since a single decay event can generate a few breaks in the same DNA strand, which is indistinguishable from a single break in the assay. The consequent analytical difficulties are overcome by considering relaxation events rather than single-strand breaks, and linearization events rather than double-strand breaks. A further consideration is that apart from damage at the site of DNA-associated decay, which is the principal interest of the analysis, some DNA damage also arises from the radiation field created by all decay events. These two components of damage are referred to as internal and external breakage, respectively, and they can be separated in the analysis since their contribution depends on the experimental conditions. The DNA-binding ligand Hoechst 33258 labeled with 125I was used in our experiments to study breakage in pBR322 plasmid DNA arising from the decay of this Auger electron emitter. The values obtained for the efficiency (per decay) of plasmid relaxation and linearization by the 125I-labeled ligand were 0.090 +/- 0.035 and 0.82 +/- 0.04, respectively. When dimethylsulfoxide was included as a radical scavenger, the efficiency values for relaxation and linearization were 0.15 +/- 0.02 and 0.65 +/- 0.05, respectively.     

Chemical and biological consequences of the radioactive decay of iodine-125 in plasmid DNA

Doubly labeled [U-14C, 5-125I]iododeoxycytidine (IdC) triphosphate was synthesized and incorporated enzymatically into defined positions of the plasmid pBR322. After storage under various conditions, the stable end products were analyzed using radio-GC, radio-HPLC, and electron microscopy. In addition, solutions of 14C-IdC-labeled DNA containing Na125I as an internal radiation source were studied to investigate the influence of internal radiolysis. Transmutation of the covalently bound 125I leads to complete destruction of the labeled nucleotide, giving rise to 14CO2 and 14CO as major products. Fragmentation of the pyrimidine base is independent of solvent and DNA configuration. Internal radiolysis caused by Na125I leads to only minor damage. Electron microscopy studies reveal that decay-induced double strand breaks (dsb) occur both at the site of decay and in areas as far as hundreds of base pairs apart from that site. Number and distribution of the breaks is strongly dependent on solvent and DNA configuration. A direct correlation exists between the extent of fragmentation of the nucleotide and the mean number of dsb.     

Repair of sequence-specific 125I-induced double-strand breaks by nonhomologous DNA end joining in mammalian cell-free extracts

In mammalian cells, nonhomologous DNA end joining (NHEJ) is considered the major pathway of double-strand break (DSB) repair. Rejoining of DSB produced by decay of (125)I positioned against a specific target site in plasmid DNA via a triplex-forming oligonucleotide (TFO) was investigated in cell-free extracts from Chinese hamster ovary cells. The efficiency and quality of NHEJ of the "complex" DSB induced by the (125)I-TFO was compared with that of "simple" DSB induced by restriction enzymes. We demonstrate that the extracts are indeed able to rejoin (125)I-TFO-induced DSB, although at approximately 10-fold decreased efficiency compared with restriction enzyme-induced DSB. The resulting spectrum of junctions is highly heterogeneous exhibiting deletions (1-30 bp), base pair substitutions, and insertions and reflects the heterogeneity of DSB induced by the (125)I-TFO within its target site. We show that NHEJ of (125)I-TFO-induced DSB is not a random process that solely depends on the position of the DSB but is driven by the availability of microhomology patches in the target sequence. The similarity of the junctions obtained with the ones found in vivo after (125)I-TFO-mediated radiodamage indicates that our in vitro system may be a useful tool to elucidate the mechanisms of ionizing radiation-induced mutagenesis and repair.     

Sequence specificity of 125I-labelled Hoechst 33258 in intact human cells

Using polyacrylamide/urea DNA sequencing gels, the DNA sequence selectivity of 125I-labelled Hoechst 33258 damage has been determined in intact human cells to the exact base-pair. This was accomplished using a novel procedure with human alpha RI-DNA as the target DNA sequence. In this procedure, after size fractionation, the alpha RI-DNA is selectively purified by hybridization to a single-stranded M13 clone containing an alpha RI-DNA insert. The sequence specificity of [125I]Hoechst 33258 was indistinguishable in intact cells from purified high molecular weight DNA; and this is surprising considering the more complex environment of DNA in the nucleus where DNA is bound to nucleosomes and other DNA binding proteins. The ligand preferentially binds to DNA sequences which have four or more consecutive A.T base-pairs. The extent of damage was measured with a densitometer and, relative to the damage hotspot at base-pair 94, the extent of damage was similar in both purified high molecular weight DNA and intact cells. [125I]Hoechst 33258 causes only double-strand breaks, since single-strand breaks or base damage were not detected. These experiments represent the first occasion that the sequence specificity of a DNA damaging agent, which causes only double-strand breaks, has been determined to the exact base-pair in intact cells.     

DNA breakage by decay of Auger electron emitters: experiments with 123I-iodoHoechst 33258 and plasmid DNA

The Auger electron-emitting isotope 123I is of interest in the context of potential exploitation of Auger electron emitters in radioimmunotherapy. The efficiency of induction of cytotoxic lesions by decay of DNA-associated 125I, the prototype Auger electron emitter, is well established, but its long half-life (60 days) is a limitation. However, the advantage of the much shorter half-life of 123I (13.2 h) might be outweighed by its "weaker" Auger electron cascade with an average of 8-11 Auger electrons, compared to about 15-21 electrons for 125I. Accordingly, the efficiency of DNA breakage for DNA-associated 123I was investigated by incubation of 123I-iodoHoechst 33258 with plasmid DNA. The efficiency of double-strand break induction by decay of 123I was 0.62 compared to 0.82 per decay of 125I in the same experimental system. In the presence of dimethylsulfoxide, the values were 0.54 and 0.65 for decay of 123I and 125I, respectively. The results also showed that at a very low ligand/plasmid molar ratio (<1), the majority of cleavage seemed to occur at a particular site on the plasmid molecule, indicating preferential binding of the 123I-ligand to a unique site or a cluster of neighboring sites.     

Site-specific strand breaks in RNA produced by (125)I radiodecay

Decay of ¹²⁵I produces a shower of low energy electrons (Auger electrons) that cause strand breaks in DNA in a distance-dependent manner with 90% of the breaks located within 10 bp from the decay site. We studied strand breaks in RNA molecules produced by decay of ¹²⁵I incorporated into complementary DNA oligonucleotides forming RNA/DNA duplexes with the target RNA. The frequencies and distribution of the breaks were unaffected by the presence of the free radical scavenger dimethyl sulfoxide (DMSO) or by freezing of the samples. Therefore, as was the case with DNA, most of the breaks in RNA were direct rather than caused by diffusible free radicals produced in water. The distribution of break frequencies at individual bases in RNA molecules is narrower, with a maximum shifted to the 3′-end with respect to the distribution of breaks in DNA molecules of the same sequence. This correlates with the distances from the radioiodine to the sugars of the corresponding bases in A-form (RNA/DNA duplex) and B-form (DNA/DNA duplex) DNA. Interestingly, when ¹²⁵I was located close to the end of the antisense DNA oligonucleotide, we observed breaks in RNA beyond the RNA/DNA duplex region. This was not the case for a control DNA/DNA hybrid of the same sequence. We assume that for the RNA there is an interaction between the RNA/DNA duplex region and the single-stranded RNA tail, and we propose a model for such an interaction. This report demonstrates that ¹²⁵I radioprobing of RNA could be a powerful method to study both local conformation and global folding of RNA molecules.     

Oligodeoxynucleotide-directed photo-induced cross-linking of HIV proviral DNA via triple-helix formation.

The HIV proviral genome contains two copies of a 16 bp homopurine.homopyrimidine sequence which overlaps the recognition and cleavage site of the Dra I restriction enzyme. Psoralen was attached to the 16-mer homopyrimidine oligonucleotide, d5'(TTTTCT-TTTCCCCCCT)3', which forms a triple helix with this HIV proviral sequence. Two plasmids, containing part of the HIV proviral DNA, with either one (pLTR) or two (pBT1) copies of the 16-bp homopurine.homopyrimidine sequence and either 4 or 14 Dra I cleavage sites, respectively, were used as substrates for the psoralen-oligonucleotide conjugate. Following UV irradiation the two strands of the DNA targeted sequence were cross-linked at the triplex-duplex junction. The psoralen-oligonucleotide conjugate selectively inhibited Dra I enzymatic cleavage at sites overlapping the two triple helix-forming sequences. A secondary triplex-forming site of 8 contiguous base pairs was observed on the pBT1 plasmid when binding of the 16 base-long oligonucleotide was allowed to take place at high oligonucleotide concentrations. Replacement of a stretch of six cytosines in the 16-mer oligomer by a stretch of six guanines increased binding to the primary sites and abolished binding to the secondary site under physiological conditions. These results demonstrate that oligonucleotides can be designed to selectively recognize and modify specific sequences in HIV proviral DNA.     

Mechanisms underlying production of double-strand breaks in plasmid DNA after decay of 125I-Hoechst

Previously, the kinetics of strand break production by (125)I-labeled m-iodo-p-ethoxyHoechst 33342 ((125)IEH) in supercoiled (SC) plasmid DNA had demonstrated that approximately 1 DSB is produced per (125)I decay both in the presence and absence of the hydroxyl radical scavenger DMSO. In these experiments, an (125)IEH:DNA molar ratio of 42:1 was used. We now hypothesize that this DSB yield (but not the SSB yield) may be an overestimate due to subsequent decays occurring in any of the 41 (125)IEH molecules still bound to nicked (N) DNA. To test our hypothesis, (125)IEH was incubated with SC pUC19 plasmids ((125)IEH:DNA ratio of approximately 3:1) and the SSB and DSB yields were quantified after the decay of (125)I. As predicted, the number of DSBs produced per (125)I decay is one-half that reported previously ( approximately 0.5 compared to approximately 1, +/- DMSO) whereas the number of SSBs ( approximately 3/(125)I decay) is similar to that obtained previously ( approximately 90% are generated by OH radicals). Direct visualization by atomic force microscopy confirms formation of L and N DNA after (125)IEH decays in SC DNA and supports the strand break yields reported. These findings indicate that although SSB production is independent of the number of (125)IEH bound to DNA, the DSB yield can be augmented erroneously by (125)I decays occurring in N DNA. Further analysis indicates that 17% of SSBs and 100% of DSBs take place within the plasmid molecule in which an (125)IEH molecule decays, whereas 83% of SSBs are formed in neighboring plasmid DNA molecules.     

Induction and repair of double- and single-strand DNA breaks in bacteriophage lambda superinfecting Escherichia coli

Induction and repair of double- and single-strand DNA breaks have been measured after decays of 125I and 3H incorporated into the DNA and after external irradiation with 4 MeV electrons. For the decay experiments, cells of wild type Escherichia coli K-12 were superinfected with bacteriophage lambda DNA labelled with 5'-(125I)iodo-2'-deoxyuridine or with (methyl-3H)thymidine and frozen in liquid nitrogen. Aliquots were thawed at intervals and lysed at neutral pH, and the phage DNA was assayed for double- and single-strand breakage by neutral sucrose gradient centrifugation. The gradients used allowed measurements of both kinds of breaks in the same gradient. Decays of 125I induced 0.39 single-strand breaks per double-strand break. No repair of either break type could be detected. Each 3H disintegration caused 0.20 single-strand breaks and very few double-strand breaks. The single-strand breaks were rapidly rejoined after the cells were thawed. For irradiation with 4 MeV electrons, cells of wild type E. coli K-12 were superinfected with phage lambda and suspended in growth medium. Irradiation induced 42 single-strand breaks per double-strand break. The rates of break induction were 6.75 x 10(-14) (double-strand breaks) and 2.82 x 10(-12) (single-strand breaks) per rad and per dalton. The single-strand breaks were rapidly repaired upon incubation whereas the double-strand breaks seemed to remain unrepaired. It is concluded that double-strand breaks in superinfecting bacteriophage lambda DNA are repaired to a very small extent, if at all.     

The stability of DNA triplexes inside cells as studied by iodine-125 radioprinting.

We studied the stability of a DNA triplex resulting from the binding of a 38 nt long purine motif triplex-forming oligonucleotide (TFO) to a covalently closed plasmid containing a target sequence from the human HPRT gene. Our in vitro experiments showed that the triplex formed at plasmid and TFO concentrations as low as 10(-9)M. Once formed, the triplex was remarkably stable and could withstand 10 min incubation at 65 degrees C. We next delivered these TFO-plasmid complexes into cultured human cells. To monitor the TFO-plasmid complexes inside cells we applied a new technique that we call 'radioprinting'. Because the TFO was(125)I labeled, we could quantitatively monitor the triplexes by measuring(125)I-induced DNA strand breaks in the target plasmid sequence. We found that the triplexes remain stable inside the cells for at least 48 h. Based on these findings we propose using TFO for indirect labeling of intact plasmid DNA. As a demonstration, we show that the intracellular distribution of a fluorescein-labeled TFO was different when it was liposome-delivered into cultured human cells alone or in a complex with the plasmid. In the latter case, the fluorescence was detected in nearly all the cells while detection of the plasmid by use of a marker gene (beta-galactosidase) revealed expression of the gene in only half of the cells.     

Molecular analysis of base damage clustering associated with a site-specific radiation-induced DNA double-strand break

Base damage flanking a radiation-induced DNA double-strand break (DSB) may contribute to DSB complexity and affect break repair. However, to date, an isolated radiation-induced DSB has not been assessed for such structures at the molecular level. In this study, an authentic site-specific radiation-induced DSB was produced in plasmid DNA by triplex forming oligonucleotide-targeted (125)I decay. A restriction fragment terminated by the DSB was isolated and probed for base damage with the E. coli DNA repair enzymes endonuclease III and formamidopyrimidine-DNA glycosylase. Our results demonstrate base damage clustering within 8 bases of the (125)I-targeted base in the DNA duplex. An increased yield of base damage (purine > pyrimidine) was observed for DSBs formed by irradiation in the absence of DMSO. An internal control fragment 1354 bp upstream from the targeted base was insensitive to enzymatic probing, indicating that the damage detected proximal to the DSB was produced by the (125)I decay that formed the DSB. Gas chromatography-mass spectrometry identified three types of damaged bases in the approximately 32-bp region proximal to the DSB. These base lesions were 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine. Finally, evidence is presented for base damage >24 bp upstream from the (125)I-decay site that may form via a charge migration mechanism.     

Auger electron-induced double-strand breaks depend on DNA topology

From a structural perspective, the factors controlling and the mechanisms underlying the toxic effects of ionizing radiation remain elusive. We have studied the consequences of superhelical/torsional stress on the magnitude and mechanism of DSBs induced by low-energy, short-range, high-LET Auger electrons emitted by (125)I, targeted to plasmid DNA by m-[(125)I]iodo-p-ethoxyHoechst 33342 ((125)IEH). DSB yields per (125)I decay for torsionally relaxed nicked (relaxed circular) and linear DNA (1.74+/-0.11 and 1.62+/-0.07, respectively) are approximately threefold higher than that for torsionally strained supercoiled DNA (0.52+/-0.02), despite the same affinity of all forms for (125)IEH. In the presence of DMSO, the DSB yield for the supercoiled form remains unchanged, whereas that for nicked and linear forms decreases to 1.05+/-0.07 and 0.76+/-0.03 per (125)I decay, respectively. DSBs in supercoiled DNA therefore result exclusively from direct mechanisms, and those in nicked and linear DNA, additionally, from hydroxyl radical-mediated indirect effects. Iodine-125 decays produce hydroxyl radicals along the tracks of Auger electrons in small isolated pockets around the decay site. We propose that relaxation of superhelical stress after radical attack could move a single-strand break lesion away from these pockets, thereby preventing further breaks in the complementary strand that could lead to DSBs.     

A method of calculating initial DNA strand breakage following the decay of incorporated 125I

Two sources of individual Auger electron spectra and an electron track code were used with a simple model of the DNA to successfully simulate the single-strand DNA breakage measured by Martin and Haseltine (1981). The conditions of the calculation were then extended to examine patterns of single-strand breaks in both strands of the DNA duplex to score double-strand breaks. The occurrences of five types of break were scored. The total number of double-strand breaks (dsb) per decay at the site of the decay was 0.90 and 0.65 for the different Auger electron spectra. It was shown that for mammalian cells an additional source of double-strand breaks from low LET radiation added approximately 0.17 dsb/decay to each, giving a final total of 1.07 and 0.85 dsb/decay for mammalian cells depending on the electron spectrum. Further is is shown that the energy deposition in the DNA from the iodine decay is very complex, with a broad range of energy depositions and products. Even for a particular energy deposited in the DNA different types of strand break are produced. These are identified and their probabilities calculated.     

High-throughput assays for DNA gyrase and other topoisomerases

We have developed high-throughput microtitre plate-based assays for DNA gyrase and other DNA topoisomerases. These assays exploit the fact that negatively supercoiled plasmids form intermolecular triplexes more efficiently than when they are relaxed. Two assays are presented, one using capture of a plasmid containing a single triplex-forming sequence by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by staining with a DNA-specific fluorescent dye. The other uses capture of a plasmid containing two triplex-forming sequences by an oligonucleotide tethered to the surface of a microtitre plate and subsequent detection by a second oligonucleotide that is radiolabelled. The assays are shown to be appropriate for assaying DNA supercoiling by Escherichia coli DNA gyrase and DNA relaxation by eukaryotic topoisomerases I and II, and E.coli topoisomerase IV. The assays are readily adaptable to other enzymes that change DNA supercoiling (e.g. restriction enzymes) and are suitable for use in a high-throughput format.