Misrejoining of DNA double-strand breaks in primary and transformed human and rodent cells: a comparison between the HPRT region and other genomic locations.

Many studies of radiation response and mutagenesis have been carried out with transformed human or rodent cell lines. To study whether the transfer of results between different cellular systems is justified with regard to the repair of radiation-induced DNA double-strand breaks (DSBs), two assays that measure the joining of correct DSB ends and total rejoining in specific regions of the genome were applied to primary and cancer-derived human cells and a Chinese hamster cell line. The experimental procedure involves Southern hybridization of pulsed-field gel electrophoresis blots and quantitative analysis of specific restriction fragments detected by a single-copy probe. The yield of X-ray-induced DSBs was comparable in all cell lines analyzed, amounting to about 1 x 10(-2) breaks/Mbp/Gy. For joining correct DSB ends following an 80 Gy X-ray exposure all cell lines showed similar kinetics and the same final level of correctly rejoined breaks of about 50%. Analysis of all rejoining events revealed a considerable fraction of unrejoined DSBs (15-20%) after 24 h repair incubation in the tumor cell line, 5-10% unrejoined breaks in CHO cells and complete DSB rejoining in primary human fibroblasts. To study intragenomic heterogeneity of DSB repair, we analyzed the joining of correct and incorrect break ends in regions of different gene density and activity in human cells. A comparison of the region Xq26 spanning the hypoxanthine guanine phosphoribosyl transferase locus with the region 21q21 revealed identical characteristics for the induction and repair of DSBs, suggesting that there are no large variations between Giemsa-light and Giemsa-dark chromosomal bands.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

Age-dependent decline in rejoining of X-ray-induced DNA double-strand breaks in normal human lymphocytes

Unstimulated human peripheral blood lymphocytes (HPBL), separated by density centrifugation from anticoagulated whole blood, were X-irradiated (30 Gy) on ice and incubated in medium at 37 degrees C for repair times of 15, 30, and 120 min. Blood donors were 18 normotensive, non-smoking Caucasians aged 23-78, free from overt pathology and not taking any medications. Neutral filter elution was used to assay DNA double-strand break (DSB) induction and completeness of DSB rejoining (plus rejoining of any X-ray-induced alkali-labile sites converted to DSBs in vitro at pH 9.6). After 30 or 120 min repair incubation, the percentage of DSBs rejoined by cells from older donors (aged 66-78 years) was less than half the percentage of DSBs rejoined by cells from younger donors (aged 23-39 and 42-57). When data from the 3 age groups were pooled, the age-related decline in percent DSBs rejoined was significant for repair times 30 min (r = -0.63, p less than 0.005) and 120 min (r = -0.64, p less than 0.005) but not for 15 min (r = -0.04). These age-related declines were observed even though DNA from older donors sustained fewer strand breaks as demonstrated by the negative correlation between donor age and DSB induction (r = -0.65, p less than 0.005). These results suggest that the efficacy of X-ray-induced DSB repair diminishes with in vivo age in unstimulated HPBL.     

DNA double-strand break repair in eukaryotic cell lines having radically different radiosensitivities

TN-368 lepidopteran insect cells are on the order of 100 times more resistant to the lethal effects of ionizing radiation than cultured mammalian cells. DNA double-strand breaks (DSB) are believed by many to be the critical molecular lesion leading to cell death. We have therefore compared the rejoining of DSB in TN-368 and V79 Chinese hamster cells. Cells were irradiated on ice with 137Cs gamma rays at a dose rate of 2.5 Gy/min, incubated for various periods of time, and assayed for DNA DSB using the method of neutral elution. The kinetics of DSB rejoining following a dose of 90.2 Gy is similar for both cell lines with 50% of the rejoining completed in about 12 min. Approximately 83 and 87% of the DSB are rejoined in the TN-368 and V79 cells, respectively, by 1 h postirradiation. However, no further rejoining occurs in the TN-368 cells through at least 6 h postirradiation, whereas approximately 92% of the DSB are rejoined in the V79 cells by 2 h postirradiation. Other studies (from 22.6 to 226 Gy) demonstrate that the amount of rejoining of DSB varies inversely with dose for both cell lines, but this relationship is not as pronounced for the TN-368 cells. In general, these findings do not support the hypothesis that unrejoined DNA DSB represent the critical molecular lesion responsible for cell death.     

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

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.     

Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (L?brich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.     

Different oxygen enhancement ratios for induced and unrejoined DNA double-strand breaks in eukaryotic cells

DNA double-strand breaks (DSBs) are 2.9 times more frequently induced in yeast cells exposed to sparsely ionizing 30-MeV electrons under oxic compared to anoxic conditions. The rejoining of DSBs induced under anoxic conditions was investigated under conditions allowing repair of potentially lethal damage and compared to the rejoining of DSBs induced in oxic cells. In contrast to the biphasic rejoining kinetics of DSBs induced in oxic cells, the rejoining kinetics of DSBs induced in anoxic cells is complicated by the formation of secondary DSBs. These arise during postirradiation incubation of cells, presumably as a consequence of repair processes acting on radiation-induced lesions other than DSBs. These secondary DSBs may at least partially explain the finding that a greater fraction of unrejoinable DSBs is present in cells irradiated under anoxic compared to oxic conditions. As a consequence, the oxygen enhancement ratio of the yield of the remaining DSBs is decreasing in the course of DSB rejoining.     

Combined use of Monte Carlo DNA damage simulations and deterministic repair models to examine putative mechanisms of cell killing

A kinetic repair-misrepair-fixation (RMF) model is developed to better link double-strand break (DSB) induction to reproductive cell death. Formulas linking linear-quadratic (LQ) model radiosensitivity parameters to DSB induction and repair explicitly account for the contribution to cell killing of unrejoinable DSBs, misrepaired and fixed DSBs, and exchanges formed through intra- and intertrack DSB interactions. Information from Monte Carlo simulations is used to determine the initial yields and complexity of DSBs formed by low- and high-LET radiations. Our analysis of published survival data for human kidney cells suggests that intratrack DSB interactions are negligible for low-LET radiations but increase rapidly with increasing LET. The analysis suggests that no class of DSB is intrinsically unrejoinable or that DSB reparability is not strictly determined by the number of lesions forming the DSB. For radiations with LET >110 keV/mum, the model predicts that the relative cell killing efficiency, per unit absorbed dose, should continue to increase, whereas data from published experiments indicate a reduced cell killing efficiency. This observation suggests that the Monte Carlo simulation overestimates the DSB yield beyond 110 keV/microm or that other biological phenomena not included in the model, such as proximity effects, are important. For 200-250 kVp X rays ( approximately 1.9 keV/microm), only about 1% of the one-track killing is attributed to intratrack binary misrepair interactions. The analysis indicates that the remaining 99% of the lethal damage is due to other types of one-track damage, including possible unrepairable, misrepaired and fixed damage. Compared to the analysis of the X-ray results, 48% of the one-track lethal damage caused by 5.1 MeV alpha particles (approximately 88 keV/microm) is due to intratrack DSB interactions while the remainder is due to other forms of one-track damage.     

Evidence to support the existence of efficient DNA double-strand break rejoining in a radiosensitive mutant of V79-4 following irradiation with 250 kVp X-rays or neutrons

The repair of ionising-radiation-induced DNA double-strand break type damage was measured by Kohn neutral elution in an X-ray-sensitive mutant of V79-4, irs1. This was done in order to investigate further the likelihood that irs1 carries a defect which leads to error-prone repair of DNA damage, and not simply a reduced ability to rejoin DNA double-strand breaks. The mutant displayed an equal increase in sensitivity to the lethal effects of neutrons, as compared to X-rays. Both irs1 and V79-4 showed an increased sensitivity to the killing effects of neutrons of around 2 at 10% survival. irs1 also showed an exponential survival after either X-rays or neutrons. The induction of DNA double-strand breaks was measured in both cell lines over a dose range of 10-40 Gy using Kohn neutral filter elution. Induction of breaks by X-rays in irs1 seemed to increase slightly with dose, relative to induction in V79-4, so that at 40 Gy 1.5 times more DNA double-strand breaks were measured in irs1 cells than in V79-4. Neutron irradiation resulted in a more similar level of induction in either strain after 10-40 Gy. This difference in induction of damage may be due to a different cell-cycle composition in either cell line. The rejoining of X-ray induced double-strand breaks showed a very similar pattern (on a percentage rejoined basis) in both cell lines, although from the induction data at 40 Gy, the dose at which rejoining was measured, fewer breaks were rejoined in V79-4 but also fewer breaks remained unsealed. Neutron-induced breaks, however, were rejoined more efficiently in irs1 again on a percentage basis, but also in absolute terms since similar induction was seen after 40 Gy. This data, together with the differences seen in the rejoining of X-ray compared to neutron induced breaks, may indirectly support the proposal that irs1 is a misrepair mutant.     

DNA double-strand break misrejoining after exposure of primary human fibroblasts to CK characteristic X rays, 29 kVp X rays and 60Co gamma rays

The efficiency of ionizing photon radiation for inducing mutations, chromosome aberrations, neoplastic cell transformation, and cell killing depends on the photon energy. We investigated the induction and rejoining of DNA double-strand breaks (DSBs) as possible contributors for the varying efficiencies of different photon energies. A specialized pulsed-field gel electrophoresis assay based on Southern hybridization of single Mbp genomic restriction fragments was employed to assess DSB induction and rejoining by quantifying the restriction fragment band. Unrejoined and misrejoined DSBs were determined in dose fractionation protocols using doses per fraction of 2.2 and 4.4 Gy for CK characteristic X rays, 4 and 8 Gy for 29 kVp X rays, and 5, 10 and 20 Gy for 60Co gamma rays. DSB induction by CK characteristic X rays was about twofold higher than for 60Co gamma rays, whereas 29 kVp X rays showed only marginally elevated levels of induced DSBs compared with 60Co gamma rays (a factor of 1.15). Compared with these modest variations in DSB induction, the variations in the levels of unrejoined and misrejoined DSBs were more significant. Our results suggest that differences in the fidelity of DSB rejoining together with the different efficiencies for induction of DSBs can explain the varying biological effectiveness of different photon energies.     

Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae

The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately rejoined by at least two discrete pathways, homologous recombination and nonhomologous end-joining (NHEJ). The NHEJ pathway is essential for repair of specific classes of DSB termini in cells of the budding yeast Saccharomyces cerevisiae. Endonuclease-induced DSBs retaining complementary single-stranded DNA overhangs are repaired efficiently by end-joining. In contrast, damaged DSB ends (e.g., termini produced by ionizing radiation) are poor substrates for this pathway. NHEJ repair involves the functions of at least 10 genes, including YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2. Most or all of these genes are required for efficient recombination-independent recircularization of linearized plasmids and for rejoining of EcoRI endonuclease-induced chromosomal DSBs in vivo. Several NHEJ mutants also display aberrant processing and rejoining of DSBs that are generated by HO endonuclease or formed spontaneously in dicentric plasmids. In addition, all NHEJ genes except DNL4 and LIF1 are required for stabilization of telomeric repeat sequences. Each of the proteins involved in NHEJ appears to bind, directly or through protein associations, with the ends of linear DNA. Enzymatic and/or structural roles in the rejoining of DSB termini have been postulated for several proteins within the group. Most yeast NHEJ genes have homologues in human cells and many biochemical activities and protein:protein interactions have been conserved in higher eucaryotes. Similarities and differences between NHEJ repair in yeast and mammalian cells are discussed.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Induction and rejoining of DNA double-strand breaks in bladder tumor cells

The induction and rejoining of radiation-induced double-strand breaks (DSBs) in cells of six bladder tumor cell lines (T24, UM-UC-3, TCC-SUP, RT112, J82, HT1376) were measured using the neutral comet assay. Radiation dose-response curves (0-60 Gy) showed damage (measured as mean tail moment) for five of the cell lines in the same rank order as cell survival (measured over 0-10 Gy), with the least damage in the most radioresistant cell line. Damage induction correlated well with clonogenic survival at high doses (SF10) for all six cell lines. At the clinically relevant dose of 2 Gy, correlation was good for four cell lines but poor for two (TCC-SUP and T24). The rejoining process had a fast and slow component for all cell lines. The rate of these two components of DNA repair did not correlate with cell survival. However, the time taken to reduce the amount of DNA damage to preirradiated control levels correlated positively with cell survival at 10 Gy but not 2 Gy; radioresistant cells rejoined the induced DSBs to preirradiation control levels more quickly than the radiosensitive cells. Although the results show good correlation between SF10 and DSBs for all six cell lines, the lack of correlation with SF2 for TCC-SUP and T24 cells would suggest that a predictive test should be carried out at the clinically relevant dose. At present the neutral comet assay cannot achieve this.     

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.     

Facilitated detection of chromosome break and repair at low levels of ionizing radiation by addition of wortmannin to G1-type PCC fusion incubation

We have measured rejoining kinetics of chromosome breaks using a modified cell fusion-based premature chromosome condensation (PCC) technique in confluent cultures of normal human fibroblasts irradiated at low doses of X-rays. In order to enhance the sensitivity of the fusion-based PCC assay, we added a DNA double strand break (DSB) repair inhibitor wortmannin during the incubation period for PCC/fusion process resulting in a significantly higher yield of G1-type chromosome breaks. The initial number of chromosome breaks (without repair) gave a linear dose response with about 10 breaks per cell per Gy which is about two times higher than the value with the conventional G1-type PCC method. The chromosome rejoining kinetics at 0.5 and 2.0 Gy X-rays reveal a bi-phasic curve with both a fast and a slow component. The fast component (0-30 min) is nearly identical for both doses, but the slow component for 2 Gy kinetics is much slower than that for 0.5 Gy, indicating that the process occurring during this period may be crucial for the ultimate fate of irradiated cells. The chromosome rejoining kinetics obtained here is similar to that of other methods of detecting DNA DSB repair such as the gammaH2AX assay. Our chromosome repair assay is useful for evaluating the accuracy of other assays measuring DNA DSB repair at doses equal or less than 0.5 Gy of ionizing radiation.