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1.
Intrachromosomal recombination between repeated elements can result in deletion (DEL recombination) events. We investigated the inducibility of such intrachromosomal recombination events at different stages of the cell cycle and the nature of the primary DNA lesions capable of initiating these events. Two genetic systems were constructed in Saccharomyces cerevisiae that select for DEL recombination events between duplicated alleles of CDC28 and TUB2. We determined effects of double-strand breaks (DSBs) and single-strand breaks (SSBs) between the duplicated alleles on DEL recombination when induced in dividing cells or cells arrested in G1 or G2. Site-specific DSBs and SSBs were produced by overexpression of the I-Sce I endonuclease and the gene II protein (gIIp), respectively. I-Sce I-induced DSBs caused an increase in DEL recombination frequencies in both dividing and cell-cycle-arrested cells, indicating that G1- and G2-arrested cells are capable of completing DSB repair. In contrast, gIIp-induced SSBs caused an increase in DEL recombination frequency only in dividing cells. To further examine these phenomena we used both gamma-irradiation, inducing DSBs as its most relevant lesion, and UV, inducing other forms of DNA damage. UV irradiation did not increase DEL recombination frequencies in G1 or G2, whereas gamma-rays increased DEL recombination frequencies in both phases. Both forms of radiation, however, induced DEL recombination in dividing cells. The results suggest that DSBs but not SSBs induce DEL recombination, probably via the single-strand annealing pathway. Further, DSBs in dividing cells may result from the replication of a UV or SSB-damaged template. Alternatively, UV induced events may occur by replication slippage after DNA polymerase pausing in front of the damage. 相似文献
2.
A system is described for mapping oxidative DNA damage (sites sensitive to formamidopyrimidine-DNA glycosylase and single-strand breaks) at nucleotide resolution in the nuclear and mitochondrial DNA of Saccharomyces cerevisiae. Our 3' end labelling method is sensitive and was first developed using the well-studied inducer of oxidative DNA damage, methylene blue (MB) plus light. We treated yeast DNA in vitro with this so as to maximise levels of damage for assay development. Unfortunately, MB does not remain in yeast cells and yeast DNA repair mutants sensitive to active oxygen species are not sensitive to this agent, thus for in vivo experiments we turned to a polycyclic aromatic, RO 19-8022 (RO). This resulted in oxidative DNA damage when light was applied to yeast cells in its presence. The spectra of enzyme-sensitive sites and single-strand breaks induced by MB in vitro or by RO plus light in vivo or in vitro were examined in two yeast reporter genes: the nuclear MFA2 and the mitochondrial OLI1. The experiments revealed that most of the enzyme-sensitive sites and single-strand breaks induced by MB or RO plus light are at the same positions in these sequences, and that these are guanines. 相似文献
3.
DNA damage by reactive oxygen species results in a spectrum of DNA lesions including single-strand breaks (ssb) and double-strand breaks (dsb). However, most damage is not lethal, and the location and nature of the DNA damage, in addition to total number of breaks, are likely to be critical in determining ultimate survival. Generally associated only with ionizing radiation, multiply damaged sites (i.e., complex lesions and clusters of complex lesions in DNA) are more likely to be lethal because they are less easily repaired. We examined five drugs known to cause DNA adducts, strand breaks, and reactive oxygen species for their ability to produce complex lesions: 4-nitroquinoline-1-oxide (4NQO), H2O2, doxorubicin, Tirapazamine, and etoposide. As indicators of lesion complexity we compared 1) the ratio of ssb to dsb, 2) the rate of rejoining of single-strand breaks, 3) the relative lethality of the breaks (number of breaks per mean lethal dose), and 4) the ability to produce complex lesions. Tirapazamine, etoposide, and doxorubicin gave dsb/ssb ratios similar to that for X-rays, whereas 4NQO and H2O2 showed dsb/ssb ratios of 200 and 3250, respectively. The number of dsb per LD50 varied from 2.5 to 500 for different drugs. There was no apparent relation between ssb rejoining half-time (3.5-85 min) and relative lethality or lesion complexity. A modified (nonionic detergent) filter elution method confirmed that tirapazamine, like ionizing radiation, produced multiple dsb within single chromatin domains. These data indicate that complex lesions can be produced by a number of different chemicals and suggest that the damage that results in killing by these drugs may be related to production of multiply damaged sites in DNA. 相似文献
4.
S Kamesaki H Kamesaki TJ Jorgensen A Tanizawa Y Pommier J Cossman 《Canadian Metallurgical Quarterly》1993,53(18):4251-4256
Previous studies have shown that bcl-2 overexpression can inhibit apoptosis induced by DNA-damaging agents widely used in cancer chemotherapy, including X-irradiation, alkylating agents (hydroperoxycyclophosphamide, etc.), and topoisomerase II inhibitors (etoposide, etc.). However, little is known about the mechanism by which bcl-2 overexpression inhibits apoptosis triggered by these agents. In this study, we examined whether bcl-2 overexpression could have effects on etoposide-induced DNA damage and its repair. For these experiments, we developed CH31 clones (mouse B-cells) stably transfected with human bcl-2 sense plasmids and compared these clones with a parental CH31 clone or CH31 clones with antisense plasmids. Overexpression of bcl-2 protein inhibited etoposide-induced apoptosis and cytotoxicity. However, there was no or little difference in the production and repair of DNA-protein cross-links, DNA single-strand breaks, and double-strand beaks among a parental CH31 clone and CH31 clones with human bcl-2 sense or antisense plasmids. These findings indicate that (a) apoptosis or cytotoxicity induced by etoposide can be separated into early events (formation of double-strand breaks, DNA single-strand breaks, and double-strand breaks) and later events (secondary DNA fragmentation or cell death) and (b) bcl-2 inhibits apoptosis and cytotoxicity induced by etoposide at some steps between these events. 相似文献
5.
As judged by alkaline elution, exposure of Walker cells to either 3-amino-1,2,4-benzotriazine-1,4-dioxide (SR 4233) or nitromin results in a dose-dependent increase in DNA damage due to single-strand breaks. With nitromin or SR 4233 there was little difference in the extent of DNA single-strand breaks between Walker cells incubated either hypoxically or aerobically. In contrast, there was a 24-fold enhancement in the differential hypoxic/aerobic response to SR 4233 in clonogenic studies. Following incubation of cells with nitrogen mustard, DNA cross-linking is observed. Bioreduction of nitromin would be expected to yield nitrogen mustard as the putative reactive metabolite. However, only DNA strand-breaks could be detected in Walker cells incubated with nitromin, suggesting that reduction of this pro-drug to nitrogen mustard was not a major activation pathway. In cells incubated under aerobic conditions, SR 4233 induces oxidative DNA damage, as indicated by the formation of 8-hydroxydeoxyguanosine, suggesting the involvement of futile redox cycling. In rats dosed with SR 4233 in vivo, significantly higher levels of 8-hydroxydeoxyguanosine could be detected in liver, compared to vehicle-dosed controls. 相似文献
6.
The chronic administration of estradiol by subcutaneous (s.c.) implantation into male Syrian hamsters induces kidney tumors. Free radicals generated by redox cycling between catecholestrogens and their quinones have been proposed to damage DNA and to thus mediate renal hormone-induced carcinogenesis. As part of an examination of this postulate, we assayed by a filter elution technique DNA single-strand breaks in livers and kidneys of male hamsters treated with estrogen by single intraperitoneal (i.p.) injection, by s.c. implant or by continuous infusion and compared values to those in untreated controls. The DNAs of hamster liver and kidney were not affected by one i.p. injection of 5, 15 or 150 mg/kg estradiol. However, treatment of hamsters with one 25 mg estradiol implant/animal for 2 weeks elevated by 10% the levels of DNA single-strand breaks in kidney, but only to a minor extent in liver, which is not a target of estrogen-induced carcinogenesis. An infusion of 250 micrograms/day/animal of estradiol or 4-hydroxyestradiol for one week by osmotic pumps into hamsters resulted in a comparable increase of single-strand breaks in kidney DNA, whereas 2-hydroxyestradiol under these conditions had a negligible effect. It is concluded that the induction of DNA single-strand breakage by either estradiol or 4-hydroxyestradiol in hamster kidney supports a mechanism of estrogen-induced carcinogenesis by free radical generation via redox cycling between 4-hydroxyestradiol and its corresponding quinone. 相似文献
7.
We improved alkaline sucrose density gradient sedimentation to detect very long single-strand DNA at the megabase level (from less than 1 to about 4 Mb). Hitherto, these have not sedimented correctly due to some artifacts. One artifact was aggregation of sticky DNA and proteins formed in the gradient. Then, in some gradients, biphasic distribution was observed, the major peak of which was reasonable as a result of random scission by X-rays, but the minor, fast-sedimenting population was another artifact resulting from incomplete denaturation of the DNA. We mainly reduced the centrifugal force and used a solution for cell lysis with a high concentration of salt. By means of this procedure, DNA single-strand breaks induced by relatively low doses of X-rays and subsequent repair processes can be measured in human fibroblasts. The protocol is also applicable to the study of DNA damage accompanied by strand scission, such as by UV or dimethyl sulfate as well as their repair. The technique is sensitive enough to detect even single-strand breaks induced by 0.1 J/m2 UV and sufficiently reproducible that breaks induced by increasing UV dosages were dose dependent. Thus, this technique was proven to be very sensitive, reliable and simple to perform. Therefore, this improvement will be extremely useful to investigators studying DNA repair. 相似文献
8.
Mammalian cells are hypersensitive to very low doses of X-rays (< 0.2 Gy), a response which is followed by increased radioresistance up to 1 Gy. Increased radioresistance is postulated to be a response to DNA damage, possibly single-strand breaks, and it appears to be a characteristic of low linear energy transfer (LET) radiation. Here we demonstrate a correspondence between the extent of the increased radioresistance and linear energy transfer of 250 kVp X-rays and plateau and Bragg peak negative pi-mesons. The results support our hypothesis since the size of the increased radioresistant response appears to correspond to the number of radiation induced single-strand breaks. Furthermore, since survival prior to the increased radioresistant response (< 0.2 Gy) was LET-independent, these data support the notion that the increased radioresistant response may dictate the overall survival response to higher doses. However, while these data provide further circumstantial evidence for the involvement of DNA strand breaks in the triggering of increased radioresistance, more direct conclusions cannot be made. The data are not accurate enough to detect structure in the single-strand break profiles, the production of single-strand breaks being apparently linear with dose. 相似文献
9.
Increasing evidence indicates that nitric oxide (NO) may play a role in immune-mediated injury to beta-cells. One site for the action of this agent is the mitochondrion. Although the exact targets for damage within this organelle have yet to be fully elucidated, a potential location for injury is mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by NO. Both exogenous NO generation (spermine/NO adduct [sper/NO]) and endogenous production of NO (IL-1beta) were studied. To study the effects of exogenously produced NO, neonatal rat islet cells in monolayers were exposed to varying doses of sper/NO for 30 min. Total cellular DNA was isolated and treated with alkali to produce strand breaks at abasic sites resulting from exposure to NO. Damage to mtDNA was evaluated using a quantitative Southern blot technique. The results showed that sper/NO caused dose-dependent damage to mtDNA. Additionally, mtDNA was found to be more sensitive to injury generated by either source than a similarly sized fragment of nuclear DNA. To evaluate the effects of endogenously produced NO, beta-cell cultures were treated with IL-1beta for 18 h. Other cultures were treated with IL-1beta and an inhibitor of the inducible form of nitric oxide synthase, aminoguanidine. DNA was evaluated as described for the sper/NO studies. IL-1beta caused appreciable damage to mtDNA, and this damage was reduced in mtDNA from cultures treated with IL-1beta and aminoguanidine. These studies show that mtDNA is a sensitive target for NO generated both endogenously and exogenously and that this DNA is more vulnerable to NO-induced damage than nuclear DNA. 相似文献
10.
DNA of plasmid pBR322 irradiated with laser at a wavelength of 193 mm was treated with an extract containing proteins from E.coli K12 AB1157 (wild-type). The enzymes were found to produce single- and double-strand DNA breaks, which was interpreted as a transformation of a portion of cyclobutane pyrimidine dimers and (6-4) photoproducts into nonrepairable single-strand DNA breaks. The products resulted from ionization of DNA, in particular, single-strand breaks, transform to double-strand breaks. A comparison of these data with the data on survival of plasmid upon transformation of E.coli K12 AB1157 enables one to assess the biological significance of single- and double-strand breaks. The inactivation of the plasmid (in AB1157) is mainly determined by the number of directly formed laser-induced single-strand breaks, whereas the contribution of enzymatically produced single- and double-strand breaks is insignificant. 相似文献
11.
An endonuclease (AP-endonuclease II) that specifically attacks double stranded or single stranded depurinated DNA, resulting in single-strand nicks, has been purified 320-fold from Micrococcus luteus. The enzyme is not stimulated by 0.002 M MgCl2, it induces 3'OH-5'PO4 breaks on the 5' side of apurinic sites, it has no activity towards UV-irradiated DNA and has a molecular weight of about 30 000. In cooperation with DNA-polymerase from M. luteus and T4 DNA ligase, AP-endonuclease II has been shown capable of carrying out complete excision repair of depurinated DNA in vitro. 相似文献
12.
Recent evidence suggests that DNA damage of various origins is not randomly distributed in the genome but appears to be clustered in unidentified hypersensitive regions of the chromatin. A model was proposed that stipulates that unpaired DNA stretches, such as those found in scaffold- (or matrix)-associated regions (SARs) under torsional strain, are candidate regions of hypersensitivity to DNA damage in vivo. In this study, we assessed in vitro the relative susceptibility of supercoiled plasmids containing a SAR or chromatin loop DNA segment to DNA damage induced by acid-catalyzed depurination or FeIII-bleomycin. Single-strand specific S1 nuclease was used in combination with 3'-end-labeling to detect single-strand breaks or gaps, after cleavage of abasic sites or removal of 3'-phosphoglycolates by Escherichia coli endonuclease IV. The optimal conditions of DNA cleavage specificity by S1 nuclease were determined. Using these conditions, the DNA cleavage patterns obtained showed (i) a preferential localization of S1 hypersensitive sites in the SAR DNA as compared with plasmid or chromatin loop DNA and (ii) a strikingly similar localization of DNA damage with the two clastogenic treatments. 相似文献
13.
M Gupta S Fan Q Zhan KW Kohn PM O'Connor Y Pommier 《Canadian Metallurgical Quarterly》1997,3(9):1653-1660
Camptothecin (CPT) derivatives are topoisomerase I (top1) inhibitors recently introduced as clinical agents. To explore the role of p53 in CPT-induced cytotoxicity, we examined CPT effects in two isogenic pairs of human cancer cell lines, MCF-7 breast carcinoma and HCT116 colon carcinoma cells, in which p53 function had been disrupted by transfection with the human papillomavirus type-16 E6 gene. Clonogenic survival assays showed that both MCF-7/E6 and HCT116/E6 cells were more sensitive to CPT. No differences in top1 protein levels and activity analyzed by a novel in vitro oligonucleotide assay were observed in the E6 transfectants. Also, CPT showed comparable top1 cleavable complex formation in vivo, as determined by DNA single-strand breaks and DNA protein cross-links. These results suggest that p53 can protect against CPT-induced cytotoxicity and that this protection is mediated downstream of CPT-induced DNA damage. Flow cytometry analyses showed that CPT can induce G1 arrest in cells with normal p53. This G1 arrest was markedly reduced in the p53-deficient cells. These results demonstrate a critical role of p53 as a G1 checkpoint regulator after CPT-induced DNA damage and suggest a rationale for the selectivity of CPT toward tumors with p53 mutations. 相似文献
14.
We have studied the role of DNA polymerase III, encoded in S. cerevisiae by the CDC2 gene, in the repair of yeast nuclear DNA. It was found that the repair of MMS-induced single-strand breaks is defective in the DNA polymerase III temperature-sensitive mutant cdc2-1 at the restrictive temperature (37 degrees C), but is not affected at the permissive temperature (23 degrees C). Under conditions where only a small number of lesions was introduced into DNA (80% survival), the repair of MMS-induced damage could also be observed in the mutant at the restrictive temperature, although with low efficiency. When the quantity of lesions increased (50% survival or less), the repair of single-strand breaks was blocked. At the same time we observed a high rate of reversion in the meth, his and trp loci of the cdc2-1 mutant under restrictive conditions. The results presented suggest that DNA polymerase III is involved in the repair of MMS-induced lesions in yeast DNA and that the cdc2-1 mutation affects the proofreading activity of this polymerase. 相似文献
15.
C Pfister G Vallancien J Bougaran-André P Grise 《Canadian Metallurgical Quarterly》1997,32(4):448-454
We describe a childhood mitochondrial disorder in which the clinical symptoms began and remained confined to the gastrointestinal (GI) system during the first 4 y. Seizures heralded the onset of progressive encephalopathy at age 7. Peripheral neuropathy, retinitis pigmentosa, and neural deafness developed subsequently. Laboratory investigations disclosed elevated levels of plasma lactate, and a muscle biopsy revealed ragged red fibers lacking cytochrome c oxidase activity and diminished levels of respiratory chain enzyme complexes. Molecular genetic tests failed to show any of the previously reported pathogenic mitochondrial DNA (mtDNA) mutations. We therefore screened the whole mitochondrial genome by coupling restriction digestions with single-strand conformational polymorphism (SSCP) patterns. We identified a unique SSCP in the segment that encompassed the tRNA(Lys) gene, and direct sequencing of this segment revealed a G-->A transition at an evolutionarily conserved nucleotide at mtDNA position 8313. This G8313A transition was heteroplasmic in muscle and fibroblasts of the patient, but was absent in the white blood cells and platelets from his maternal relatives. This report illustrates how GI symptoms can be the initial manifestation in a mitochondrial disorder and suggests that mitochondrial dysfunction should be considered in differentials of unexplained chronic GI symptoms, especially when lactic acidosis or other unrelated clinical signs or symptoms are present. 相似文献
16.
Radiation-induction and rejoining of single-strand breaks (SSBs) in the DNA of synchronized HeLa S3 cells were investigated by alkaline sucrose density gradients. We could not find any significant differences in the extent of SSBs induced in cellular DNA and in the extent of their rejoining throughout the cell cycle, including mitosis. The cyclic variation curve of the content of non-protein sylfhydryls (NPSH) during the cell cycle is similar to that of X-ray survivals except in mitosis, although there was no close correlation between the content of apparent total sulfhydryls (APSH) and X-ray survivals. Radiation-induced mutants resistant to 8-azaguanine (8AG) occurred in higher frequency in the radio-sensitive G1-S boundary phase than in the radio-resistant G1, S and early G2 phases. Further, the pre-irradiation treatment with 50 mM cysteamine prevented reproductive death and induction of 8AG-resistant mutants by X-rays throughout the cell cycle. These findings seem to indicate that there is a close correlation between the extent of lethal radiation damage to the cells and their mutability, and that sulfhydryls may play an important role as a factor governing cellular radio-sensitivity. 相似文献
17.
J LaDou 《Canadian Metallurgical Quarterly》1998,53(4):247-248
A modified technique of alkaline filter elution was used to evaluate single-strand breaks (SSB) as a measure of DNA lesions in lymphocytes of male Wistar rats exposed to 60 or 80 vol% oxygen, over 23 h/day and for 6 or 5 days, respectively. An acceleration of elution was observed with samples from experiments at both increased levels of oxygen. With proteinase K treatment, a twofold increase in elution rates after exposure to 60 vol% oxygen and a threefold increase at 80 vol% oxygen were indicative of increased occurrence of SSB. This is explained by an involvement of reactive oxygen species in DNA damage under elevated oxygen levels. 相似文献
18.
R Wiger JA Holme JK Hongslo G Brunborg K Haug V Rodilla E Dybing EJ S?derlund 《Canadian Metallurgical Quarterly》1998,14(4):267-282
We investigated 1,2-dibromo-3-chloropropane (DBCP)-induced DNA damage, cell cycle alterations and cell death in two cell lines, the human leukemia HL-60 and the pig kidney LLCPK1, both of which are derived from potential target sites for DBCP-induced toxicity. DBCP (30-300 micromol/L) caused a concentration-dependent increase in the levels of DNA single-strand breaks in both cell lines as well as in cultured human renal proximal tubular cells. After extended DBCP exposure in LLCPK1 cells (100 micromol/L, 30 h), the level of DNA breaks returned almost to control values. Incubation for 48 h showed a clear reduction of growth with DBCP concentrations as low as 10 micromol/L. Flow cytometric analysis showed that DBCP (1-10 micromol/L) exposure for 24 h caused an accumulation of LLCPK1 cells in the G2/M-phase. In HL-60 cells the accumulation in G2/M-phase was less marked, and at higher concentrations the cells accumulated in S-phase. Flow cytometric studies of HL-60 and LLCPK1 cells exposed to 100-500 micromol/L DBCP showed increased number of apoptotic cells/bodies with a lower DNA content than that of the G1 cells. Microscopic studies revealed that there were increased numbers of cells with nuclear condensation and fragmentation, indicating that apoptosis was the dominant mode of death in these cell lines, following exposure to DBCP. The characteristic ladder pattern of apoptotic cells was observed when DNA from DBCP-treated HL-60 cells and LLCPK1 cells was electrophoresed in agarose. The finding that DBCP can cause an accumulation of cells in G2/M-phase and induce apoptosis in vitro may be of importance for the development of DBCP-induced toxicity in vivo. 相似文献
19.
ML Smith XY Hua DL Marsden D Liu NG Kennaway KY Ngo RH Haas 《Canadian Metallurgical Quarterly》1997,82(9):2826-2831
A 6-yr-old boy presented with muscle weakness, lactic acidemia, and insulin-dependent diabetes mellitus (IDDM). Using PCR and restriction enzyme analysis, he was found to have the classical A3248G mitochondrial DNA (mtDNA) mutation frequently associated with mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS). The mutation was confirmed by sequencing muscle mtDNA. The mutation in mtDNA from muscle, lymphoblasts, and blood was clearly demonstrable by standard methods using ethidium bromide staining. His mother also had IDDM, but no A3243G mutation could be detected in her blood or transformed lymphoblasts using the same PCR technique. When PCR was carried out in the presence of [32P]deoxycytidine triphosphate, subsequent autoradiography detected the presence of the mutation at low levels in mtDNA from the mother's lymphoblasts and blood. Study of the mother's muscle showed a mitochondrial myopathy, despite the fact that she was asymptomatic. We emphasize that the increased sensitivity of radiolabeled PCR may be necessary to detect small percentages of heteroplasmic A3243G mtDNA mutation in blood from diabetic subjects. Otherwise the incidence of mtDNA mutations in both IDDM and non-insulin dependent diabetes may be underestimated. 相似文献
20.
To elucidate the kinetics of the induction of DNA strand breaks by low-energy Auger electron emitters, we compared the yields of DNA breaks in supercoiled pUC19 DNA after the decay of 125I (1) in proximity to DNA after minor-groove binding (125I-iodoHoechst 33342, 125IH) and (2) at a distance from DNA (125I-iodoantipyrine, 125IAP). Iodine-125 bound to the minor groove in DNA or free in solution is equally effective per decay in producing single-strand breaks (SSBs), while 125I bound to the minor groove is 6.7-fold more efficient than 125I free in solution in producing double-strand breaks (DSBs) (1.08 +/- 0.13 compared to 0.16 +/- 0.01 DSB/decay). Consequently, SSB to DSB ratios for 125IAP and gamma radiation (20.7 +/- 2.9 and 43.8 +/- 1.5, respectively) are greater than that for 125IH (2.9 +/- 0.4). Finally, the decay of 125IH leads to fragmentation of plasmid DNA beyond SSBs and DSBs. 相似文献