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.     

Variation in radiation-induced formation of DNA double-strand breaks as a function of chromatin structure.

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.     

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.     

Chromatin organization contributes to non-randomly distributed double-strand breaks after exposure to high-LET radiation

The influence of higher-order chromatin structure on the non-random distribution of DNA double-strand breaks induced by high-LET radiation was investigated. Five different chromatin structures (intact cells, condensed and decondensed chromatin, nucleoids and naked genomic DNA) from GM5758 cells or K562 cells were irradiated with (137)Cs gamma-ray photons and 125 keV/microm nitrogen ions (16-25 MeV/nucleon). DNA was purified with a modified lysis procedure to avoid release of heat-labile sites, and fragment size distributions and double-strand break yields were analyzed by different pulsed-field gel electrophoresis protocols. Whereas double-strand breaks in photon-irradiated cells were randomly distributed, irradiation of intact K562 cells with high-LET nitrogen ions produced an excess of non-randomly distributed DNA fragments 10 kb-1 Mbp in size. Complete removal of proteins eliminated this non-random component. There was a gradual increase in the yield of double-strand breaks for each chromatin decondensation step, and compared to intact cells, the yields for naked DNA (in buffer without scavengers) increased 83 and 25 times after photon and nitrogen-ion irradiation, respectively. The corresponding relative biological effectiveness decreased from 1.6-1.8 for intact cells to 0.49 for the naked DNA. We conclude that the organization of DNA into chromatin fiber and higher-order structures is responsible for the majority of non-randomly distributed double-strand breaks induced by high-LET radiation. However, our data suggest a complex interaction between track structure and chromatin organization over several levels.     

A model relating cell survival to DNA fragment loss and unrepaired double-strand breaks

The model of radiation action that is presented relates the surviving fraction of irradiated cells to unrepaired DNA double-strand breaks (DSBs). The following assumptions are made in the model: (i) A DNA fragment created by the induced DSBs may move out of its chromosome (become lost), and the probability of that process depends on the fragment size. (ii) An irradiated cell will lose its proliferative capacity if it has an unrepaired DSB (including DNA fragments) at certain points in the cell cycle. Mathematical expressions of the model yield the dose and time dependencies of the surviving fraction, the number of unrepaired DSBs, and the number of prematurely condensed chromosome fragments. Radiobiological phenomena described include effects of low dose rate, delayed plating, hypertonic solution, araA, and high-LET radiation. The calculated dose dependence of the residual number of unrepaired DSBs for ataxia telangiectasia and normal fibroblast cells is very close to the experimentally obtained [M. N. Cornforth and J. S. Bedford, Radiat. Res. 111, 385-405 (1987)] total number of chromosomal aberrations. This leads to the conclusion that each unrepaired DSB becomes a chromosomal aberration. Analysis in terms of the model shows that the radiosensitivity of various cell lines is predominantly due to the different amounts of time available for DSB repair in these cells.     

Relative biological effectiveness of the alpha-particle emitter (211)At for double-strand break induction in human fibroblasts

The purpose of this study was to quantify and to determine the distribution of DNA double-strand breaks (DSBs) in human cells irradiated in vitro and to evaluate the relative biological effectiveness (RBE) of the alpha-particle emitter (211)At for DSB induction. The influence of the irradiation temperature on the induction of DSBs was also investigated. Human fibroblasts were irradiated as intact cells with alpha particles from (211)At, (60)Co gamma rays and X rays. The numbers and distributions of DSBs were determined by pulsed-field gel electrophoresis with fragment analysis for separation of DNA fragments in sizes 10 kbp-5.7 Mbp. A non-random distribution was found for DSB induction after irradiation with alpha particles from (211)At, while irradiation with low-LET radiation led to more random distributions. The RBEs for DSB induction were 2.1 and 3.1 for (60)Co gamma rays and X rays as the reference radiation, respectively. In the experiments studying temperature effects, nuclear monolayers were irradiated with (211)At alpha particles or (60)Co gamma rays at 2 degrees C or 37 degrees C and intact cells were irradiated with (211)At alpha particles at the same temperatures. The dose-modifying factor (DMF(temp)) for irradiation of nuclear monolayers at 37 degrees C compared with 2 degrees C was 1.7 for (211)At alpha particles and 1.6 for (60)Co gamma rays. No temperature effect was observed for intact cells irradiated with (211)At. In conclusion, irradiation with alpha particles from (211)At induced two to three times more DSB than gamma rays and X rays.     

Spatial distribution of radiation-induced double-strand breaks in plasmid DNA as resolved by atomic force microscopy

Atomic force microscopy (AFM) has been used to directly visualize, size and compare the DNA fragments resulting from exposure to low- and high-LET radiation. Double-stranded pUC-19 plasmid ("naked") DNA samples were irradiated by electron-beam or reactor neutron fluxes with doses ranging from 0.9 to 10 kGy. AFM scanning in the tapping mode was used to image and measure the DNA fragment lengths (ranging from a few bp up to 2864 bp long). Double-strand break (DSB) distributions resulting from high-LET neutron and lower-LET electron irradiation revealed a distinct difference between the effects of these two types of radiation: Low-LET radiation-induced DSBs are distributed more uniformly along the DNA, whereas a much larger proportion of neutron-induced DSBs are distributed locally and densely. Furthermore, comparisons with predictions of a random DSB model of radiation damage show that neutron-induced DSBs deviate more from the model than do electron-induced DSBs. In summary, our high-resolution AFM measurements of radiation-induced DNA fragment-length distributions reveal an increased number of very short fragments and hence clustering of DSBs induced by the high-LET neutron radiation compared with low-LET electron radiation and a random DSB model prediction.     

Induction and rejoining of DNA double-strand breaks in normal human skin fibroblasts after exposure to radiation of different linear energy transfer: possible roles of track structure and chromatin organization

DNA double-strand breaks are nonrandomly induced by high-LET radiation. Differences in the induction and rejoining of DSBs after irradiation with ions having different LET were detected by fragment analysis. The data obtained indicate that the track structure of the traversing particle and its interaction with the different chromatin structures of the cellular DNA influence the yield as well as the distribution of the induced damage. The induction and rejoining of clustered DSBs induced by the same nitrogen ion fluence at LETs of 80-225 keV/microm were investigated by a detailed analysis of the DNA fragmentation patterns in normal human fibroblasts. The DSBs in the cells were allowed to rejoin during incubations for 0-20 h. Two separate pulsed-field gel electrophoresis protocols were used, optimized for separation of fragments in the size ranges 1-6 Mbp and 5 kbp-1.5 Mbp. A strong influence of LET on the level of DSB induction was evident. The DSB yield increased from 4.5 +/- 0.2 to 10.0 +/- 0.3 DSBs per particle traversal through the cell nucleus when LET increased from 80 to 225 keV/microm. Further, the size distribution of the DNA fragments showed a significant dependence on radiation quality, with an excess of fragments at 50-200 kbp and around 1 Mbp. Differences in repair kinetics were also evident, with slower rejoining for increasing LET, and the initial nonrandom fragment distributions were still present after 1 h of repair.     

Efficient rejoining of radiation-induced DNA double-strand breaks in centromeric DNA of human cells

Although major efforts in elucidating different DNA double-strand break (DSB) repair pathways and their contribution to accurate repair or misrepair have been made, little is known about the influence of chromatin structure on the fidelity of DSB repair. Here, the repair of ionizing radiation-induced DSBs was investigated in heterochromatic centromeric regions of human cells in comparison with other genomic locations. A hybridization assay was applied that allows the quantification of correct DSB rejoining events in specific genomic regions by measuring reconstitution of large restriction fragments. We show for two primary fibroblast lines (MRC-5 and 180BR) and an epithelial tumor cell line that restriction fragment reconstitution is considerably more efficient in the centromere than in average genomic locations. Importantly, however, DNA ligase IV-deficient 180BR cells show, compared with repair-proficient MRC-5 cells, impaired restriction fragment reconstitution both in average DNA and in the centromere. Thus, the efficient repair of DSBs in centromeric DNA is dependent on functional non-homologous end joining. It is proposed that the condensed chromatin state in the centromere limits the mobility of break ends and leads to enhanced restriction fragment reconstitution by increasing the probability for rejoining correct break ends.     

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.     

Relative biological effectiveness of accumulated 125IdU and 125I-estrogen decays in estrogen receptor-expressing MCF-7 human breast cancer cells

The therapeutic potential for delivering a cytotoxic dose of radiation (using the decay of Auger-electron emitters) to the cell nucleus of cancer cells that express estrogen receptors (ERs) by radiolabeled estrogen was investigated in the ER-expressing human breast cancer cell line, MCF-7. The radiolabeled estrogen/ER complex irradiates the cell nucleus by binding specific DNA sequences called estrogen response elements (EREs). Cell clonogenicity and induction of DNA double-strand breaks (DSBs) by gamma radiation or accumulation of (125)I-iododeoxyuridine ((125)IdU) or E-17alpha[(125)I]iodovinyl-11betamethoxyestradiol ((125)IVME2) decays were determined. MCF-7 cells were efficiently killed by accumulation of (125)IdU (D(0) = 30 decays per cell) and (125)IVME2 decays (D(0) = 28 decays per cell). DNA DSBs were induced by the accumulation of (125)IdU (approximately 3750 decays per cell required to reduce the mean value of the elution profile to 50%) or (125)IVME2 decays (approximately 465 decays per cell required to reduce the mean value to 50%). For survival of MCF-7 cells after gamma irradiation, the D(0) was 1 Gy, and approximately 65 Gy was required to reduce the mean value to 50% for induction of DSBs. The RBE values for cell killing and induction of DSBs by (125)IVME2 relative to gamma radiation were 4.8 and 18.8, respectively. The RBE values for cell killing and induction of DSBs by (125)IdU relative to gamma radiation were 4.5 and 2.3, respectively. Cell killing in a manner similar to that induced by high-LET radiation and the high RBE for induction of DSBs by (125)IVME2 in the ER-expressing MCF-7 cells provide a biological rationale for the use of Auger electron-emitting radionuclides covalently bound to estrogen to deliver a cytotoxic dose of radiation to ER-positive cancers.     

DNA repair in the context of chromatin: new molecular insights by the nanoscale detection of DNA repair complexes using transmission electron microscopy

The recognition and repair of DNA double-strand breaks (DSBs) occurs in the context of highly structured chromatin. Here, we established a transmission electron microscopy (TEM) approach to localize gold-labeled DSB repair components in different chromatin environments within the intact nuclear architecture of cells in irradiated mouse tissues. The ultra-high resolution of TEM offers the intriguing possibility of detecting core components of the DNA repair machinery at the single-molecule level and visualizing their molecular interactions with specific histone modifications. By labeling phosphorylated Ku70, which binds directly to broken DNA ends in preparation for rejoining, this TEM approach can monitor formation and repair of actual DSBs in euchromatic versus heterochromatic regions. While DNA lesions in euchromatin are detected and rejoined without any delay, DNA packaging in heterochromatin appears to retard DSB processing, leading to slower repair kinetics. Of significance, the assembly of γH2AX, MDC1, and 53BP1 occurs exclusively at DSBs in heterochromatic (characterized by H3K9me3), but not euchromatic domains, suggesting involvement in localized chromatin decondensation (which increases heterochromatic DNA accessibility). Collectively, this TEM approach provides fascinating insights into the dynamic events of the DSB repair process that depend decisively upon the actual chromatin structure around the break.     

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.     

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.     

Pulsed-field gel electrophoresis of chromosomal DNA of Saccharomyces pastorianus after exposure to x-rays (30–50 keV) and neutrons (14 MeV)

Chromosomes of budding yeast Saccharomyces pastorianus were used to determine the extent of DNA double-strand breaks (DSBs) induced by x-rays (30-50 keV) and 14 MeV neutrons. The yeast chromosomes were separated by pulsed-field gel electrophoresis (PFGE) and the proportion of unbroken molecules corresponding to the largest chromosome no. IV (1500 kbp) was used to calculate the DSB frequency assuming a random distribution of hits. To determine the protective contribution of the cell environment, chromosomes embedded in agarose plugs as well as intact yeast cells, were irradiated under conditions completely inhibiting DNA repair. Following irradiation, the intact cells were also embedded in agarose plugs and the chromosomes isolated to perform PFGE. All radiation experiments resulted in a linear dose-effect curve for DSBs. For both radiation qualities, the yield of DSBs for exposed isolated chromosomes exceeded that for intact yeast cells by a factor of 13. The relative biological effectiveness (RBE) of 14 MeV neutrons in the induction of DNA DSBs was about 2.5. This figure was found to be identical for the in vivo and in vitro exposure of yeast chromosomes (neutrons 36.7 and 2.8, x-rays 14.5 and 1.1 x 10(-8) DSB x Bp-1 Gy-1 for isolated DNA and intact cells, respectively).     

Comparative radiation tolerance based on the induction of DNA double-strand breaks in tobacco BY-2 cells and CHO-K1 cells irradiated with gamma rays

Higher plants are generally more tolerant to ionizing radiation than mammals. To explore the radiation tolerance of higher plants, the induction of DNA double-strand breaks (DSBs) by gamma rays was investigated in tobacco BY-2 cells and compared with that in Chinese hamster ovary (CHO)-K1 cells as a reference. This is the first examination of radiation-induced DSBs in a higher plant cell. The resulting DNA fragments were separated by pulsed-field gel electrophoresis and stained with SYBR Green I. The initial yield of DSBs was then quantified from the fraction of DNA fragments shorter than 1.6 Mbp based on the assumption of random distribution of DSBs. The DSB yield in tobacco BY-2 cells (2.0 +/- 0.1 DSBs Gbp(-1) Gy(-1)) was only one-third of that in CHO-K1 cells. Furthermore, the calculated number of DSBs per diploid cell irradiated with gamma rays at the mean lethal dose was five times greater in BY-2 cells (263 +/- 13) than in CHO-K1 cells. These results suggest that the radiation tolerance of BY-2 cells appears to be due not only to a lower induction of DNA damage but also to a more efficient repair of the induced DNA damage.     

Simulation of DNA damage after proton irradiation

The biophysical radiation track simulation model PARTRAC was improved by implementing new interaction cross sections for protons in water. Computer-simulated tracks of energy deposition events from protons and their secondary electrons were superimposed on a higher-order DNA target model describing the spatial coordinates of the whole genome inside a human cell. Induction of DNA double-strand breaks was simulated for proton irradiation with LET values between 1.6 and 70 keV/microm and various reference radiation qualities. The yield of DSBs after proton irradiation was found to rise continuously with increasing LET up to about 20 DSBs per Gbp and Gy, corresponding to an RBE up to 2.2. About half of this increase resulted from a higher yield of DSB clusters associated with small fragments below 10 kbp. Exclusion of experimentally unresolved multiple DSBs reduced the maximum DSB yield by 30% and shifted it to an LET of about 40 keV/microm. Simulated fragment size distributions deviated significantly from random breakage distributions over the whole size range after irradiation with protons with an LET above 10 keV/microm. Determination of DSB yields using equations derived for random breakage resulted in an underestimation by up to 20%. The inclusion of background fragments had only a minor influence on the distribution of the DNA fragments induced by radiation. Despite limited numerical agreement, the simulations reproduced the trends in proton-induced DNA DSBs and fragment induction found in recent experiments.     

Clusters of DNA double-strand breaks induced by different doses of nitrogen ions for various LETs: experimental measurements and theoretical analyses

The yields and clustering of DNA double-strand breaks (DSBs) were investigated in normal human skin fibroblasts exposed to gamma rays or to a wide range of doses of nitrogen ions with various linear energy transfers (LETs). Data obtained by pulsed-field gel electrophoresis on the dose and LET dependence of DNA fragmentation were analyzed with the randomly located clusters (RLC) formalism. The formalism considers stochastic clustering of DSBs along a chromosome due to chromatin structure, particle track structure, and multitrack action. The relative biological effectiveness (RBE) for the total DSB yield did not depend strongly on LET, but particles with higher LET produced higher fractions of small DNA fragments, corresponding in the formalism to an increase in the average number of DSBs per DSB cluster. The results are consistent with the idea that DSB clustering along chromosomes is what leads to large RBEs of high-LET radiations for major biological end points. At a given dose, large fragments are less affected by the variability in LET than small fragments, suggesting that the two free ends in large fragments are often produced by two different tracks. The formalism successfully described an extra increase in small DNA fragments as dose increases and a related decrease in large fragments, mainly due to interlacing of DSB clusters produced along a chromosome by different tracks, since interlacing cuts larger DNA fragments into smaller ones.     

Enhanced fidelity for rejoining radiation-induced DNA double-strand breaks in the G2 phase of Chinese hamster ovary cells

The influence of cell cycle phase on the fidelity of DNA double-strand break (DSB) repair is largely unknown. We investigated the rejoining of correct and incorrect DSB ends in synchronized populations of Chinese hamster ovary cells irradiated with 80 Gy X-rays. A specialized pulsed-field gel electrophoresis assay based on quantitative Southern hybridization of individual large restriction fragments was employed to measure correct DSB rejoining by monitoring restriction fragment reconstitution. Total DSB repair, representing both correct and incorrect rejoining, was analyzed using conventional pulsed-field gel electrophoresis. We present evidence that restriction fragment reconstitution is more efficient in G₂ than in G₁, suggesting that DSB rejoining in G₂ proceeds with higher fidelity. DNA-dependent protein kinase-deficient V3 and xrs-6 cells show impaired restriction fragment reconstitution in G₁ and G₂ compared with wild-type AA8 and K1 cells, demonstrating that the enhanced fidelity of DSB rejoining in G₂ occurs by non- homologous end joining. Additionally, homologous recombination-deficient irs1SF and wild-type cells show identical DSB rejoining in G₁ and G₂. We propose that structural characteristics of G₂ phase chromatin, such as the cohesion of sister chromatids in replicated chromatin, limit the mobility of radiation-induced break ends and enhance the fidelity of DSB rejoining.