Repair of potentially lethal damage by introduction of T4 DNA ligase in eucaryotic cells.

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.     

Repair of Potentially Lethal Damage by Introduction of T4 DNA Ligase in Eucaryotic Cells

Summary

The bacterial enzyme PvuII, which generates blunt-ended DNA double-strand breaks, and T4 DNA ligase, which seals adjacent DNA fragments in coupling to ATP cleavage, were introduced in mouse C3H10T1/2 fibroblasts using osmolytic shock of pinocytic vesicles. Cells were then assayed for their clonogenic ability. In agreement with previous studies by others, we find that the PvuII restriction endonuclease simulates ionizing radiation effects by causing a dose-dependent loss of reproductive capacity. Here we show that the concomitant treatment with DNA ligase considerably increases cell survival. Survival curves were shown to be dependent on the ligase enzyme dose and on ATP concentration in the hypertonic medium. We conclude that T4 DNA ligase is able to repair some of the potentially lethal damage produced by restriction endonucleases in eucaryotic cells.     

Transient DNA Double-Strand Breakage in 4-Hydroperoxyifosfamide-Treated Mammalian Cells in Vitro Does Not Interact with the Rejoining of Radiation-Induced Double-Strand Breaks

BACKGROUND: 4-Hydroxyifosfamide is the primary metabolite in vivo of the bifunctional alkylating cytostatic ifosfamide. DNA interstrand cross-linking induced by bifunctional alkylators may be repaired through an intermediate with unligated repair patches on both strands which should uncover analytically as DNA double-strand breaks and allow to measure the rejoining kinetic of this repair intermediate. Additionally, the combined effects of drug and radiation treatment on rejoining of double-strand breaks was investigated with two different mammalian cell lines. MATERIAL AND METHODS: V79 (rodent fibroblasts) and Widr (human colon carcinoma) cells were treated for 2 hours with 4-hydroperoxyifosfamide which rapidly decays to 4-hydoxyifosfamide in aqueous solution or were exposed in combination with ionizing radiation followed by incubation for repair with or without the drug. DNA double-strand breakage was measured by pulsed-field electrophoresis. RESULTS: The 2 hours 4-hydroperoxyifosfamide treatment (30 micrograms/ml) resulted in a pronounced DNA fragmentation that, 2-4 hours after drug removal, declined with an estimated half-live of about 4 hours for both cell lines. When the cells were additionally irradiated with 10 Gy given in the middle of drug exposure, the residual fragmentation after 12 or 24 hours incubation for repair was only marginally increased, roughly corresponding to the respective value after radiation, alone. A continuous drug exposure of 6 hours (at 10 micrograms/ml) resulted in a fragmentation that was independent of a preirradiation with a high dose of 30 Gy, immediately before drug addition. CONCLUSIONS: The present data support the idea that unligated/unrejoined double-stranded DNA ends are generated during the repair of lesions from bifunctional alkylators. The rate of subsequent rejoining is in the order of magnitude of the slow rejoining of radiation-induced double-strand breaks. Processing of double-stranded DNA damage from either 4-hydroperoxyifosfamid or radiation exposure is apparently unaffected in combined treatments.     

Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells.

Chinese hamster ovary cells were exposed to various heat treatments followed by X-irradiation, and the induction and repair of DNA strand breaks was studied using the alkaline unwinding technique. Heat treatments alone were found to cause DNA strand breakage only for temperatures greater than or equal to 43 degrees C, whereas the number of radiation-induced strand breaks was unaffected by additional heating. Strand break repair was studied for irradiated cells preheated at temperatures ranging from 42 degrees C to 45 degrees C. The total repair curve could be separated into three phases, a fast (t = 0-15 min), an intermediate (t = 15-120 min) and a slow (t greater than or equal to 120 min) phase. All phases were altered when cells were heated either prior to or after irradiation. The fast and the intermediate phase could be well interpreted by the assumption that irradiation leads to both primary and secondary single-strand breaks, the latter being generated by enzymatic incision at sites of damaged bases. For irradiation alone, the ratio of all secondary strand breaks to all primary breaks was fsec = 1.5 +/- 0.5. This ratio was not altered by preceding heat treatments (mean fsec = 1.7 +/- 0.2). The main effect of heating on the repair kinetics of single-strand breaks was an increase in the repair half-time of primary and secondary breaks (maximum increase by a factor of 3.4), whereas the generation of secondary breaks was only slightly retarded (factor 1.3). The slow repair phase, which is assumed to represent the repair of DNA double-strand breaks, was best described by a single exponential component. The half-time of this component was found to increase from tau slow = 170 +/- 70 min for non-heated cells to tau slow = 345 +/- 80 min for cells heated at 45 degrees C for 20 min, indicating that heat inhibited the repair of double-strand breaks. For irradiation alone, the initial fraction of the slow component was fslow = 0.065 +/- 0.004. This fraction was enhanced by additional heating, with a maximum increase by a factor of 2.7 for cells heated at 45 degrees C for 20 min. This elevation cannot be the result of an enhanced induction of double-strand breaks, but must be associated with an additional formation of slowly repaired strand breaks during repair incubation. These additional strand breaks must arise from strand breaks which in non-heated cells are repaired during the fast or intermediate phase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role for non-homologous end-joining in the repair of UVA-induced DNA damage

PURPOSE: The biological significance of long-wavelength ultraviolet (UV) light, UVA, is increasingly realized, but the precise nature of the cellular damage responsible for the effects of this radiation is still not clear. It has been reported that UVA can induce double-strand breaks in DNA, but the biological significance of these is not known. We have therefore examined the UVA sensitivity of a cell line deficient in non-homologous end-joining, the major pathway for the repair of DNA double-strand breaks in mammalian cells in order to determine the biological importance of UVA-induced DSB. MATERIALS AND METHODS: Xrs-6, a Chinese hamster ovary cell line mutant for XRCC5 (Ku80) was compared with its parental CHO-K1 cell line for its sensitivity to UVA radiation (365 nm) using both a clonogenic assay and the micronucleus assay. RESULTS: Xrs-6 cells were sensitive to the cytotoxic effects of UVA. This resulted in the formation of chromosome damage, as measured by the micronucleus assay, which this cell line was unable to repair. CONCLUSIONS: Owing to the nature of the repair defect in these cells, these results imply that DNA double-strand breaks are produced in cells following UVA irradiation, that the non-homologous end-joining repair pathway is involved in their repair and that they are produced with sufficient frequency to have biological significance.     

Role for non-homologous end-joining in the repair of UVA-induced DNA damage

Purpose : The biological significance of long-wavelength ultraviolet (UV) light, UVA, is increasingly realized, but the precise nature of the cellular damage responsible for the effects of this radiation is still not clear. It has been reported that UVA can induce double-strand breaks in DNA, but the biological significance of these is not known. We have therefore examined the UVA sensitivity of a cell line deficient in non-homologous end-joining, the major pathway for the repair of DNA double-strand breaks in mammalian cells in order to determine the biological importance of UVA-induced DSB. Materials and methods : Xrs-6, a Chinese hamster ovary cell line mutant for XRCC5 (Ku80) was compared with its parental CHO-K1 cell line for its sensitivity to UVA radiation (365 nm) using both a clonogenic assay and the micronucleus assay. Results : Xrs-6 cells were sensitive to the cytotoxic effects of UVA. This resulted in the formation of chromosome damage, as measured by the micronucleus assay, which this cell line was unable to repair. Conclusions : Owing to the nature of the repair defect in these cells, these results imply that DNA double-strand breaks are produced in cells following UVA irradiation, that the non-homologous end-joining repair pathway is involved in their repair and that they are produced with sufficient frequency to have biological significance.     

Repair of DNA strand breaks at hyperthermic temperatures in Chinese hamster ovary cells.

The repair of DNA double-strand breaks was measured by pH 7.2 filter elution in cells incubated at 25-45 degrees C either before or after X-irradiation. Exposure to 45 degrees C for 15 minutes immediately prior to X-irradiation significantly increased both the half-time for DNA double-strand break closure and the number of DNA double-strand breaks remaining in nuclear DNA 180 minutes after irradiation. Exposure to temperatures between 41 and 45 degrees C immediately after X-irradiation accelerated DNA double-strand break closure and resulted in no increase in the number of DNA double-strand breaks remaining in the cell's genome 180 minutes after irradiation. The results indicate either that the radiosensitization produced by the administration of hyperthermic temperatures before and after irradiation result from two characteristically different molecular mechanisms, or that neither the rate of DNA strand break closure nor the number of DNA strand breaks remaining in nuclear DNA after irradiation accurately predict hyperthermic radiosensitization. These conclusions assume that no DNA strand breaks are below the resolution of this DNA damage assay and that a comparison between cytotoxicity and DNA repair after exposure to high radiation doses is valid.     

Repair of DNA single- and double-strand breaks in proliferating and quiescent murine tumor cells

We evaluated the relationship between the repair of DNA single- and double-strand breaks and cellular radiosensitivity in proliferating vs. quiescent cells of the mouse mammary tumor lines 66 and 67 in vitro, using the technique of filter elution at pH 12.2, pH 7.2 and pH 9.6. In these lines, quiescent (Q; unfed plateau-phase) cells are more radiosensitive than are proliferating (P) cells. At doses of 4-6 Gy, both 66 and 67 Q cells repair single-strand breaks (ssb) with kinetics similar to those of P cells. However, repair of ssb was slightly retarded in Q cells at a higher dose (10 Gy) than at the lower doses. In contrast, repair of ssb in P cells was dose-independent, at least for doses up to 10 Gy. The rate of repair of DNA double-strand breaks (dsb), measured at pH 7.2, was dose-independent in P and Q cells of both lines. The repair kinetics were biphasic, with an initial half-time less than 15 min, and the early phase was similar in all cell groups. The half-time for repair in the slow phase ranged from about 2 to greater than 20 h. The fraction of damage repaired by the slow phase was relatively high in all cell groups (40-70 per cent). In line 66, P cells repaired a higher percentage of dsb by 2 h postirradiation than did Q cells. The opposite was observed in line 67: Q cells repaired more dsb in 2 h than did P cells. The survival of 66 St4 cells (Q cultures which have been refed with complete medium and incubated 4 h) was significantly greater than that of 66 Q; nevertheless St4 cells repaired both ssb and dsb at rates similar to those of Q cells. Therefore, survival does not necessarily correlate with the rates of either ssb or dsb repair among these cell lines in different growth states.     

DNA Double-Strand Break Induction and Repair in Irradiated Lymphoblastoid, Fibroblast Cell Lines and White Blood Cells from ATM, NBS and Radiosensitive Patients

BACKGROUND AND PURPOSE: DNA double-strand breaks (dsbs) in lymphoblastoid cell lines (LCLs), fibroblasts and white blood cells from healthy donors, cancer patients with and without late effects of grade 3-4 (RTOG) as well as donors with known radiosensitivity syndromes were examined with the aim to detect dsb repair ability as a marker for radiosensitivity. MATERIAL AND METHODS: LCLs from six healthy donors, seven patients with a heterozygous or homozygous genotype for ataxia-telangiectasia (ATM) and Nijmegen breakage syndrome (NBS), two patients with a late toxicity of grade 3-4 (RTOG), and one cell line with a ligase IV-/- status and its parental cell line were examined. Furthermore, fibroblasts from patients with ATM, NBS, two healthy control individuals, and leukocytes from 16 healthy and 22 cancer patients including seven patients with clinical hypersensitivity grade 3 (RTOG) were examined. Cells were irradiated in vitro with 0-150 Gy. Initial damage as well as remaining damage after 8 and 24 h were measured using constant field gel electrophoresis. RESULTS: In contrast to cells derived from patients homozygous for NBS, impaired dsb repair ability could be detected both in fibroblast and lymphoblastoid cells from ATM and ligase IV-/- patients. The dsb repair ability of all 38 leukocyte cell lines (patients with grade 3-4 late effects and controls) was similar, whereas the initial damage among healthy donors was less. CONCLUSION: Despite showing a clinically elevated radiosensitivity after irradiation, the DNA repair of the patients with clinical hypersensitivity grade 3 (RTOG) appeared to be normal. Other mechanisms such as mutations, altered cell cycle or defective apoptosis could play a critical role toward determining radiosensitivity.     

Genetic changes induced in human cells in Space Shuttle experiment (STS-95)

BACKGROUND: Results of past space experiments suggest that the biological effect of space radiation could be enhanced under microgravity. To assess the radiation risk for humans during long-term spaceflight, it is very important to clarify whether human cells exhibit a synergistic effect of radiation and microgravity. HYPOTHESIS: If significant synergism occurs in human cells, genetic changes induced during spaceflight may be detected by using human tumor HCT-116 cells which are hypermutable due to a defect in the DNA mismatch repair system. METHODS: Cultured HCT-116 cells were loaded on the Space Shuttle Discovery (STS-95) and grown during the 9-d mission. After landing, many single-cell clones were isolated, microsatellite repetitive sequences in each clone were amplified by PCR, and mutations in the microsatellite loci were detected as changes in the length of PCR fragments. Mutation frequencies of ouabain-resistant phenotype were also analyzed. RESULTS: The frequencies of microsatellite mutations as well as ouabain-resistant mutations in the flight sample were similar to those of the ground control samples. Some cells were treated in space with bleomycin which mimics the action of radiation, but the frequencies of microsatellite mutations were not significantly different between the flight and the ground control samples. CONCLUSION: Under the present flight conditions, neither space radiation (about 20 mSv during this mission) nor microgravity caused excess mutations in human cells.     

Increased sensitivity to sparsely ionizing radiation due to excessive base excision in clustered DNA damage sites in Escherichia coli

PURPOSE: In order to clarify the cellular processing and repair mechanisms for radiation-induced clustered DNA damage, we examined the correlation between the levels of DNA glycosylases and the sensitivity to ionizing radiation in Escherichia coli. MATERIALS AND METHODS: The lethal effects of gamma-rays, X-rays, alpha-particles and H2O2 were determined in E. coli with different levels of DNA glycosylases. The formation of double-strand breaks by post-irradiation treatment with DNA glycosylase was assayed with gamma-irradiated plasmid DNA in vitro. RESULTS: An E. coli mutM nth nei triple mutant was less sensitive to the lethal effect of sparsely ionizing radiation (gamma-rays and X-rays) than the wild-type strain. Overproduction of MutM (8-oxoguanine-DNA glycosylase), Nth (endonuclease III) and Nei (endonulease VIII) increased the sensitivity to gamma-rays, whereas it did not affect the sensitivity to alpha-particles. Increased sensitivity to gamma-rays also occurred in E. coli overproducing human 8-oxoguanine-DNA glycosylase (hOgg1). Treatment of gamma-irradiated plasmid DNA with purified MutM converted the covalently closed circular to the linear form of the DNA. On the other hand, overproduction of MutM conferred resistance to H2O2 on the E. coli mutM nth nei mutant. CONCLUSIONS: The levels of DNA glycosylases affect the sensitivity of E. coli to gamma-rays and X-rays. Excessive excision by DNA glycosylases converts nearly opposite base damage in clustered DNA damage to double-strand breaks, which are potentially lethal.     

Radiation-induced DNA damage and repair in quiescent and proliferating human tumor cells in vitro

The purpose of this study was to examine radiation-induced DNA strand breakage and repair in quiescent and proliferating human tumor cells in vitro and determine their relationship to radiation sensitivity and potentially lethal damage repair (PLDR). Using centrifugal elutriation we have isolated from fed plateau-phase cultures of HEp-3 human squamous carcinoma cells, relatively pure populations of quiescent and proliferating cells. This was confirmed by both [3H]-thymidine labelling and acridine orange (AO) staining with flow cytometry. Quiescent cells were more sensitive to ionizing radiation (Do = 0.97 Gy) than were proliferating cells (Do = 1.28 Gy). However, quiescent cells showed higher repair of potentially lethal damage (PLDR) than did proliferating cells. Repair of single-strand breaks (ssb) and double-strand breaks (dsb) as measured by filter elution did not differ significantly between quiescent and proliferating cells. For both populations, ssb and dsb repair kinetics and final damage remaining were the same, suggesting that repair of DNA strand breaks is not entirely responsible for the difference in radiation sensitivity between quiescent and proliferating cells.     

UV-C radiation induced conformational relaxation of pMTa4 DNA in Escherichia coli may be the cause of single strand breaks

PURPOSE: The biological consequences of initial physicochemical events following exposure of DNA to germicidal (254 nm) ultraviolet C (UV-C) radiation are not fully understood despite progress that has been made. In particular the cause of UV-C induced single strand breaks is not known. This question has been addressed in the present investigation. MATERIALS AND METHODS: A plasmid construct, pMTa4, was exposed to UV-C in vitro as well as in vivo after transforming the plasmid into a repair proficient wild type and repair deficient, recF, mutant of E. coli. Following UV exposure in vivo, the plasmid was isolated under repair non-permissive and permissive conditions. The plasmid isolate and the pure super-coiled closed circular (CC) topological form of the plasmid were analyzed by agarose gel electrophoresis. The dependence of UV-C induced damage and conformational changes on the dose of radiation as well as on the duration of post-irradiation repair incubations was observed. The influence of UV-C on hyperchromic change and intercalation of ethidium bromide into plasmid DNA were also recorded. RESULTS: UV-C exposure of pMTa4 DNA in vitro and in vivo induced dose dependent, but sparsely placed, single strand breaks (SSB). While the wild type (AB1157) E. coli was able to repair SSB nearly completely under repair permissive condition, the recF (JC9239) mutant failed to do so. A dose-dependent relaxation of super-structure of CC form of pMTa4 DNA concomitant with enhanced ethidium bromide intercalation into the plasmid DNA was observed. CONCLUSION: It is proposed that the conformational relaxation generated negative super-coiling strain on the DNA backbone of CC form of plasmid as well as exposed chemical bonds for hydrolytic cleavage. This might be the cause of the production of sparsely placed single strand breaks in pMTa4 upon exposure to low doses of UV-C.     

Efficiency of radiation-induced base lesion excision and the order of enzymatic treatment

Purpose: To clarify whether initial base excision repair processes at clustered DNA damage sites comprising multiple base lesions affect subsequent excision processes via the formation of additional strand breaks by glycosylase and apurinic/apyrimidinic (AP) endonuclease base excision enzymes.

Materials and methods: Plasmid DNA (pUC18) as a model DNA molecule was exposed to high-linear-energy-transfer (LET) ionizing radiation (He^2+?or C^6+?ions) or low-LET ionizing radiation (X-rays) under various conditions to produce varied radical-scavenging effects. pUC18 was then treated sequentially or simultaneously with two bacterial base excision enzymes (glycosylases), namely, endonuclease III and formamidopyrimidine-DNA glycosylase, which convert pyrimidine (or abasic [AP] site) and purine (or AP site) lesions to single-strand breaks (SSB), respectively. Yields of additional SSB or double-strand breaks (DSB) as digestion products were examined after changing the order of enzymatic treatment.

Results: There were few differences among the enzymatic treatments, indicating that treatment order did not affect the final yields of additional SSB or DSB formed by glycosylase activity. This suggests that of the total damage, the fraction of clustered damage sites with a persistent base lesion dependent on the order of glycosylase treatment was insignificant if present.

Conclusion: Base lesion clusters induced by high- or low-LET radiation appear three or more base pairs apart, and are promptly converted to a DSB by glycosylase, regardless of the order of enzymatic treatment.     

Kinetics of chromosome lesion repair in synchronized quiescent and proliferating CHO cells

The induction of chromosome aberrations by X-rays was investigated in synchronized quiescent and proliferating CHO-K1 cells. Kinetics of chromosome lesion repair was studied using the method of fractionated irradiation. In both cell types the time-course of repair during fractionation intervals followed first-order kinetics. Comparison with kinetic data reported on DNA double-strand break repair supports the hypothesis that DNA double-strand breaks are the lesions underlying chromosome aberration formation. Quiescent CHO cells showed higher aberration yields than proliferating cells, and chromosome lesion repair was faster in quiescent cells. This correlation can be interpreted in terms of a higher degree of repair synchronism during pairwise lesion interaction. The effect of delayed plating on aberration induction was studied in quiescent cells. The time-course of repair active during the delayed plating interval followed first-order kinetics. The kinetics observed in delayed plating experiments is slower than the repair kinetics observed in fractionation experiments, suggesting the involvement of two different processes.     

Radiation-induced DNA double-strand breaks and rejoining in malignant glioma cells.

PURPOSE: This study was performed to standardize experimental conditions for the quantification by pulsed-field gel electrophoresis (PFGE) of radiation-induced DNA double-strand breaks (dsb) and rejoining in a human malignant brain tumour xenograft model. MATERIALS AND METHODS: Because no correlation was found between DNA dsb induction or rejoining and clinical radiation response for six fresh glioblastoma (GBM) specimens, assay conditions were examined in detail. SF-767 human GBM cells were implanted into the flanks of athymic mice. Resulting tumours were irradiated in vivo, dissociated mechanically or using an enzyme cocktail, and assayed for DNA dsb induction and repair. In other experiments, excised tumour portions were irradiated and allowed to repair either as chunks (>50 mm3 pieces), as minced tumour (approximately 1 mm3 pieces), or as single-cell suspensions. Finally, the effect of holding excised tumours in vitro for times of up to 72 h before irradiation and assay for DNA dsb and cell survival was studied. RESULTS AND CONCLUSIONS: The method of tumour dissociation had no effect on results; however, both the configuration of specimens during irradiation and the in vitro maintenance time markedly affected the experimental outcome. Chunks irradiated in vitro had DNA dsb results that were very similar to those obtained when tumours were irradiated in situ, while minced pieces or single-cell suspensions resulted in steeper dose-response curves. When tumour chunks were maintained at 4 degrees C in medium, DNA dsb induction was not affected for 24 h and DNA dsb rejoining remained constant for 48 h but then decreased. Cell survival, however, decreased continually during the 72 h in vitro maintenance time.     

Inhibition of repair of X-ray-induced DNA double-strand breaks in human lymphocytes exposed to sodium butyrate

Purpose : Sodium butyrate is known to inhibit histone deacetylase enzymes and to enhance the frequencies of X-ray-induced dicentrics and rings in human lymphocytes. In this study an investigation was made of the mechanisms underlying this enhancement by assessing the effect of sodium butyrate on the extent of X-ray-induced DNA damage and its repair in human peripheral blood lymphocytes. Methods and materials : Unstimulated G 0 lymphocytes were pre-treated for 24h with sodium butyrate at a final concentration of 5 mM, irradiated with different doses of X-rays and then analysed for different endpoints either immediately or after different repair periods. The frequencies of DNA strand breaks were determined biochemically using nucleoid sedimentation, alkaline elution and immunochemical analysis as well as cytogenetically using the premature chromosome condensation (PCC) technique. Results : The results show that sodium butyrate pre-treatment does not lead to a significant increase of DNA double- or single-strand breaks nor to an increase of alkali labile base damage in G 0 lymphocytes. Moreover, sodium butyrate treatment had no effect on the initial frequency of chromosome breaks. However, PCC analysis clearly showed that the presence of sodium butyrate post-irradiation severely inhibited DNA double-strand break (DSB) repair, which most likely accounts for the increase in X-ray-induced chromosome aberrations. Conclusions : Sodium butyrate treatment leading to changes in histone acetylation and increased accessibility of chromatin had no effect on the initial levels of X-ray-induced DNA damage. However, sodium butyrate may affect either the chromatin configuration or the enzymatic activities that play a key role in the repair of DSB.     

CHEF electrophoresis, a sensitive technique for the determination of DNA double-strand breaks

It has been demonstrated that clamped homogeneous electrical field (CHEF) electrophoresis is a suitable method for the determination of DNA double-strand breaks in Chinese hamster ovary (CHO) cells. It allows the separation of DNA molecules up to 10 Mbp. The fraction of DNA fragments of this size is correlated with the number of radiation induced double-strand breaks. The resolution limit of the technique is equivalent to the effect of about 1 Gy (gamma-rays). Double-strand break repair was monitored after irradiation with Co-60 gamma rays and the repair time constant determined to t1/2 = 30-35 min. In combination with the detection of DNA by fluorescence, CHEF electrophoresis provides an easy and sensitive method for the determination of double-strand break repair which does not require the radioactive labelling of cells.     

Comparison of DNA damage and repair following radiation challenge in buccal cells and lymphocytes using single-cell gel electrophoresis

PURPOSE: To develop a reproducible single-cell gel electrophoresis assay for DNA damage and repair in buccal mucosa and sublingual exfoliated cells. MATERIALS AND METHODS: Buccal mucosa and sublingual cells and lymphocytes from six individuals (three males, three females, aged 34-45 years) were challenged with increasing doses of gamma-rays. DNA strand breaks and DNA repair were measured using the single-cell gel electrophoresis assay. RESULTS: Baseline DNA strand breaks were significantly greater in buccal mucosa and sublingual cells compared with lymphocytes. Buccal mucosa and sublingual cells did not differ from each other with respect to induction of DNA strand breaks by 2 or 4 Gy gamma-rays. However, they showed a smaller increase in gamma-ray-induced DNA strand breaks compared with lymphocytes (32-53% less than lymphocytes; ANOVA p<0.0001). Unlike lymphocytes, which repaired 83% of DNA strand breaks, buccal mucosa and sublingual cells exhibited only a minimal capacity for DNA repair (approximately 0-14% of the level in lymphocytes). CONCLUSIONS: Buccal mucosa and sublingual cells exhibit an apparent resistance to the expression of radiation-induced DNA strand breaks in vitro and an apparent lack of DNA strand break repair in the single-cell gel electrophoresis assay.     

泛素连接酶CUL4A-DDB1复合体参与DNA损伤修复的研究进展

泛素化修饰在DNA损伤信号中发挥重要功能,包括细胞周期监控、DNA修复、细胞衰老和程序性死亡的调控。CUL4A-DDB1泛素连接酶通过DCAFs靶向调控特异性的底物,启动DNA切除修复机制对受损DNA进行修复。近期的研究表明CUL4A-DDB1泛素连接酶协助DNA修复因子与受损DNA的识别,来维持基因组的稳定性和正确性。