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.     

DNA-incorporated 125I induces more than one double-strand break per decay in mammalian cells

The Auger-electron emitter 125I releases cascades of 20 electrons per decay that deposit a great amount of local energy, and for DNA-incorporated 125I, approximately one DNA double-strand break (DSB) is produced close to the decay site. To investigate the potential of 125I to induce additional DSBs within adjacent chromatin structures in mammalian cells, we applied DNA fragment-size analysis based on pulsed-field gel electrophoresis (PFGE) of hamster V79-379A cells exposed to DNA-incorporated 125IdU. After accumulation of decays at -70 degrees C in the presence of 10% DMSO, there was a non-random distribution of DNA fragments with an excess of fragments <0.5 Mbp and the measured yield was 1.6 DSBs/decay. However, since these experiments were performed under high scavenging conditions (DMSO) that reduce indirect effects, the yield in cells exposed to 125IdU under physiological conditions would most likely be even higher. In contrast, using a conventional low-resolution assay without measurement of smaller DNA fragments, the yield was close to one DSB/decay. We conclude that a large fraction of the DSBs induced by DNA-incorporated 125I are nonrandomly distributed and that significantly more than one DSB/decay is induced in an intact cell. Thus, in addition to DSBs produced close to the decay site, DSBs may also be induced within neighboring chromatin fibers, releasing smaller DNA fragments that are not detected by conventional DSB assays.     

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.     

Measurement of double-strand breaks in Chinese hamster cell DNA by neutral filter elution: calibration by 125I decay

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.     

Influence of chromatin structure on induction of double-strand breaks in mammalian cells irradiated with DNA-incorporated 125I

In this study the induction of double-strand breaks (DSBs) was investigated in Chinese hamster V79-379A cells irradiated with the Auger-electron emitter (125)I incorporated into DNA. The role of chromatin organization was studied by pulse-labeling synchronized cells with (125)IdU before decay accumulation in early or late S phase. Pulsed-field gel electrophoresis and fragment-size analysis were used to quantify the distribution of DNA fragments in irradiated intact cells and naked DNA as well as in DNA from asynchronously labeled cultures in a different scavenging environment. The results show that in intact cells, after accumulation of decays at -70 degrees C in the presence of 10% DMSO, almost four times more DSBs were induced in late S phase compared with early S phase and the fragment distribution was clearly non-random with an excess of fragments <0.2 Mbp. The DSB yield was 0.6 DSB/cell and decay for cells irradiated in early S phase and 2.3 DSBs/cell and decay for cells irradiated in late S phase. When similar experiments were performed on naked genomic DNA or intact cells irradiated with gamma rays, the difference in yield was not as prominent. These data imply a role of chromatin organization in the induction of DSBs by DNA-incorporated (125)I. In summary, the results presented here suggest that the yield of DSBs as well as the fragment distribution induced by (125)IdU decay may vary significantly depending on the chromatin organization during S phase and the labeling procedure used.     

The number of superhelical turns in native virion SV40 DNA and minicol DNA determined by the band counting method.

By a method of overlapping the results obtained after agarose gel electrophoresis under two different sets of conditions, it has become possible to determine the number of superhelical turns in a given DNA by counting the bands present after partially relaxing the DNA (Keller and Wendel, 1974) with highly purified nicking-closing (N-C) enzyme from LA9 mouse cell nuclei. Because native supercoiled DNA is heterogeneous with respect to superhelix density, an average number of superhelical turns was determined. Virion SV40 DNA contains 26 +/- 0.5 superhelical turns, and native Minicol DNA contains 19 +/- 0.5 superhelical turns. The above are values at 0.2 M NaCl and at 37 degrees C, the condition under which the enzymatic relaxations were performed. The superhelix densities determined by the band counting method have been compared with superhelix densities determined by buoyant equilibrium in PDl-CsCl gradients. The Gray, Upholt, and Vinograd (1971) calculation procedure has been used for evaluating the superhelix densities by the latter method with the new statement, however, that relaxed DNA has zero superhelical turns. Comparison of the superhelix densities obtained by both methods permits a calculation of an unwinding angle for ethidium. The mean value from experiments with SV40 DNA is 23 +/- 3 degree. The average number of superhelical turns in SV40, 26, combined with the value, 21, obtained by both Griffith (1975) and Germond et al. (1975) for the average number of nucleosomes per SV40 genome, yields an average of 1.25 superhelical turns per 1/21 of the SV40 genome. If the regions of internucleosomal DNA are fully relaxed, 1.25 correesponds to the average number of superhelical turns with a nucleosome. When analyzed under identical conditions, the limit product generated by ligating a nicked circular substrate in the presence of 0.001 M Mg2+ at 37 degrees C (ligation conditions) is slightly more positively supercoiled than the limit product obtained when the N-C reaction is performed in 0.2 M NaCl at 37 degrees C. The difference in superhelix density as measured in gels between the two sets of limit products for both Minicol and SV40 DNAs is 0.0059 +/- 0.0005. This result indicates that the DNA duplex is overwound in the ligation solvent relative to its state in 0.2 M NaCl.     

Nuclease TT1 from Thermus thermophilus HB8 has an endonuclease activity preferential to circular DNAs.

Homogeneously purified nuclease TT1 from Thermus thermophilus HB8 is known as an exonuclease to produce 5'-mononucleotides. Besides the exonuclease activity, nuclease TT1 also possesses endonuclease activity preferential to superhelical (form I) and single-stranded circular DNA. Although the rate of cleavage is slower than that of form I, covalently closed circular DNA (form I') is also cleaved. Form I DNA was nicked to yield relaxed circles (form II) first, and was then nicked at the opposite site to yield unit length linear DNA (form III) which was subsequently hydrolyzed to 5'-mononucleotides exonucleolytically. Both endo- and exo-nuclease activities co-migrate on polyacrylamide gels. The general properties of the endonuclease activity are very similar to those of the exonuclease activity. The temperature optimum for endonuclease activity was 85 degrees C. The pH-optimum was in pH-range from 7.5-9.1. The enzyme was active over a wide range of Mg2+ concentrations (2.5-125 mM), and was inhibited by EDTA. A linear substrate such as (dT)8 was a competitive inhibitor for this endonuclease activity.     

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.     

Strand break induction by photoabsorption in DNA-bound molecules

Dried samples of a DNA-chloroterpyridine platinum complex were irradiated with monochromatic X rays tuned to the photoabsorption resonance of the L(III) inner shell of the platinum atom. The number of single- and double-strand breaks (SSBs and DSBs) triggered by the Auger effect in supercoiled DNA plasmids was measured by the production of circular nicked and linear forms. To probe the specific contribution of the L(III) inner-shell excitation of the platinum atom, photon wavelengths were tuned on the resonance energy (on peak) and below (off peak). The quantum yields of the resonance radiation were typically found to be 11 for the SSBs and 1 for the DSBs. The DSB-to-SSB ratio increased by 20% when switching from off-resonance to on-resonance irradiation.     

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.     

Enhanced DNA damage induced by secondary electron emission from a tantalum surface exposed to soft x rays

Both thick and thin films of pGEMR-3Zf- plasmid DNA deposited on a tantalum foil were exposed to soft X rays (effective energy of 14.8 keV) for various times in air under a relative humidity of 45% (Gamma approximately 6, where Gamma is the number of water molecules per nucleotide) and 84% (Gamma approximately 21), respectively. For a thick film, the DNA damage was induced chiefly by X-ray photons. For a thin film of DNA, X-ray-induced secondary electrons emitted from the tantalum result in a substantial increase in DNA damages. Different forms of plasmid DNA were separated and quantified by agarose gel electrophoresis and laser scanning. The exposure curves for the formation of nicked circular (single-strand break, SSB), linear (double-strand break, DSB), and interduplex crosslink forms 1 and 2 were obtained for both thick and thin films of DNA. The secondary electron enhancement factor for SSBs, DSBs and crosslinks of the thin film of DNA were derived to be 3.8 +/- 0.5, 2.9 +/- 0.7 and 7 +/- 3 at Gamma approximately 6 and 6.0 +/- 0.8, 7 +/- 1 and 3.9 +/- 0.9 at Gamma approximately 21, respectively. This study provides a molecular basis for understanding the enhanced biological effects at interfaces during diagnostic X-ray examination and radiotherapy.     

Separation of closed circular DNA from linear DNA by electrophoresis in two dimensions in agarose gels

A method that provides an easy, rapid, and reproducible way for separating closed circular DNA species from linear DNA and nicked circles is described. The method is based on the difference in mobility of form I (supercoiled) DNA and form II (nicked circles), and the differential mobility of relaxed circular DNA in the presence and absence of ethidium bromide (EtdBr). It can be used for detection or for purification of plasmid, episomal, or viral DNA from the bulk of cellular DNA, or from other DNA mixtures.     

Repair of radiation-induced DNA double-strand breaks is dependent upon radiation quality and the structural complexity of double-strand breaks

Mammalian cells primarily repair DSBs by nonhomologous end joining (NHEJ). To assess the ability of human cells to mediate end joining of complex DSBs such as those produced by chemicals, oxidative events, or high- and low-LET radiation, we employed an in vitro double-strand break repair assay using plasmid DNA linearized by these various agents. We found that human HeLa cell extracts support end joining of complex DSBs and form multimeric plasmid products from substrates produced by the radiomimetic drug bleomycin, 60Co gamma rays, and the effects of 125I decay in DNA. End joining was found to be dependent on the type of DSB-damaging agent, and it decreased as the cytotoxicity of the DSB-inducing agent increased. In addition to the inhibitory effects of DSB end-group structures on repair, NHEJ was found to be strongly inhibited by lesions proximal to DSB ends. The initial repair rate for complex non-ligatable bleomycin-induced DSBs was sixfold less than that of similarly configured (blunt-ended) but less complex (ligatable) restriction enzyme-induced DSBs. Repair of DSBs produced by gamma rays was 15-fold less efficient than repair of restriction enzyme-induced DSBs. Repair of the DSBs produced by 125I was near the lower limit of detection in our assay and was at least twofold lower than that of gamma-ray-induced DSBs. In addition, DSB ends produced by 125I were shown to be blocked by 3'-nucleotide fragments: the removal of these by E. coli endonuclease IV permitted ligation.     

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.     

Purification and properties of DNA endonucleases associated with Friend leukemia virus.

An endonuclease associated with the core of Friend leukemia virus (FLV) has been purified more than 10(3)-fold by ion exchange chromatography and gel filtration. Its molecular weight was determined by gel filtration to be about 40,000. Divalent cations were required for the endonuclease to function and KCl concentrations above 50 mM inhibited the enzyme activity. In the presence of Mg++ the purified enzyme nicked preferentially supercoiled circular DNA duplexes and in most of these molecules only one single-stranded nick was introduced per strand. The regions into which the nick could be introduced appeared to be randomly distributed on the circular molecule. When Mn++ was substituted for Mg++ the number of nicks introduced into DNA by the purified enzyme was greatly increased, and both relaxed circular and linear DNA duplexes were nicked as well as supercoiled circular DNA duplexes. Prior to its purification, however, in the presence of Mn++ the endonuclease activity in the virus extract was able to differentiate between circular and linear DNA duplexes, since both supercoiled and relaxed circular duplexes were nicked much more readily than linear duplexes. Single-stranded DNA functioned poorly as a substrate for the purified enzyme.     

Iodine-125 decay in a synthetic oligodeoxynucleotide. II. The role of auger electron irradiation compared to charge neutralization in DNA breakage

Lobachevsky, P. N. and Martin, R. F. Iodine-125 Decay in a Synthetic Oligodeoxynucleotide. II. The Role of Auger Electron Irradiation Compared to Charge Neutralization in DNA Breakage. The dramatic chemical and biological effects of the decay of DNA-incorporated (125)I stem from two consequences of the Auger electron cascades associated with the decay of the isotope: high local deposition of radiation energy from short-range Auger electrons, and neutralization of the multiply charged tellurium atom. We have analyzed the extensive data reported in the companion paper (Radiat. Res. 153, 000-000, 2000), in which DNA breakage was measured after (125)I decay in a 41-bp oligoDNA. The experimental data collected under scavenging conditions (2 M dimethylsulfoxide) were deconvoluted into two components denoted as radiation and nonradiation, the former being attributed to energy deposition by Auger electrons. The contribution of the components was estimated by adopting various assumptions, the principal one being that DNA breakage due to the radiation mechanism is dependent on the distance between the decaying (125)I atom and the cleaved deoxyribosyl unit, while the nonradiation mechanism, associated with neutralization of the multiply charged tellurium atom, contributes equally at corresponding nucleotides starting from the (125)I-incorporating nucleotide. Comparison of the experimental data sets collected under scavenging and nonscavenging (without dimethylsulfoxide) conditions was used to estimate the radiation-scavengeable component. Our analysis showed that the nonradiation component plays the major role in causing breakage within 4-5 nucleotides from the site of (125)I incorporation and produces about 50% of all single-stranded breaks. This overall result is consistent with the relative amounts of energy associated with Auger electrons and the charged tellurium atom. However, the nonradiation component accounts for almost four times more breaks in the top strand, to which the (125)I is bound covalently, than in the bottom strand, thus suggesting an important role of covalent bonds in the energy transfer from the charged tellurium atom. The radiation component dominates at the distances beyond 8-9 nucleotides, and 36% of the radiation-induced breaks are scavengeable.     

Monte Carlo simulation of strand-break induction on plasmid DNA in aqueous solution by monoenergetic electrons

The radiation-induced process of strand breaks on pBR322 plasmid DNA in aqueous solution for different energy electrons was studied by Monte Carlo simulation. Assumptions of induction mechanisms of single- and double-strand breaks (SSBs and DSBs) used in the simulation are that SSB is induced by OH or H reaction with DNA and that DSB is induced by two SSBs on the opposite strands within 10 bp. Dose-response relationships of SSBs and DSBs were demonstrated for monoenergetic electrons of 100 eV, 10 keV, 1 keV and 1 MeV, and the yields of SSB and DSB were calculated. The dose-response relationships of SSBs and DSBs can be fitted by linear and linear-quadratic functions, respectively. The ratio of quadratic to linear components of DSB induction changes due to the electron energy. A high contribution of the linear component is observed for 1 keV electrons in the dose range below 160 Gy. The yields of SSBs and DSBs for all examined electron energies lie well within the experimental data when the probability of strand-break induction by OH and H is assumed to be around 0.1-0.2. The yield of SSBs has a minimum at 1 keV, while the yield of DSBs has a maximum at 1 keV in the examined energies. The strand breaks are formed most densely for 1 keV electrons.     

Early melting of supercoiled DNA.

Denaturing gradient gel electrophoresis (formamide with urea) has been used to study the melting of supercoiled DNA. A linear gradient of denaturant concentration proportional to a 25 degrees C linear increase of temperature (Teff) from the left to the right edge of the gel was created perpendicular to DNA migration. The mobility of supercoiled DNA molecules was shown to drop to the level of relaxed molecules a long way (5-30 degrees C) before linear DNA began to melt. The further increase of Teff, including the melting range for linear molecules, caused no appreciable changes in the mobility of relaxed molecules. The transition curves are S-shaped for all the topoisomers, and an increase of superhelicity shifts the transition towards lower Teff values. The analysis of the results indicates that the observed relaxation of superhelical molecules is due to denatured region forming in them, their size increasing with the topoisomer number.