Induction and repair of DNA strand breaks in Bacteroides fragilis

Alkaline sucrose gradient sedimentation was used to establish whether strand breakage and repair take place in the DNA of UV-irradiated Bacteroides fragilis during the removal of pyrimidine dimers. A B. fragilis wild-type strain and two of its repair mutants, a mitomycin C sensitive mutant (MTC25) having wild-type levels of UV survival, and a UV-sensitive, mitomycin C sensitive mutant (UVS9), were investigated. Under anaerobic conditions, far-UV irradiation induced metabolically regulated strand breakage and resynthesis in the wild-type strain, but this was markedly reduced in both the MTC25 and UVS9 mutants. Approximately half of the strand breaks generated by the various strains were rejoined during further holding in buffer. Under replicating conditions, complete repair of strand breaks in the wild type was observed. Caffeine treatment under anaerobic conditions caused direct DNA strand breakage in B. fragilis cells but did not inhibit UV-induced breakage or repair.     

The rate of strand separation in alkali of DNA of irradiated Mammalian cells

When mammalian cells are treated with alkali of pH at about 12, the cells are lysed and the released DNA starts to uncoil. This process of DNA strand separation is accelerated if the cells have been exposed to ionizing radiation, and the effect is clearly detectable in the dose range 10-100 rads. The rate of strand separation is also influenced by temperature and ionic strength of the alkaline solution. The kinetics of DNA strand separation in alkali is studied for three conditions in terms of ionic strength and temperature, chosen in such a way that the effect of irradiation may conveniently be studied in the dose range 10 rads to 20 krads. The accelerating effect of ionizing radiation on DNA strand separation is probably due to DNA strand breakage and the technique described is thus a sensitive method of studying such damage to DNA. A model for the strand-separation process, based on the assumption that strand breakage causes the accelerating effect, is proposed and found to describe the experimental data adequately.     

Impaired DNA double strand break repair in cells from Nijmegen breakage syndrome patients

Nijmegen breakage syndrome, caused by mutations in the NBS1 gene, is an autosomal recessive chromosomal instability disorder characterized by cancer predisposition. Cells isolated from Nijmegen breakage syndrome patients display increased levels of spontaneous chromosome aberrations and sensitivity to ionizing radiation. Here, we have investigated DNA double strand break repair pathways of homologous recombination, including single strand annealing, and non-homologous end-joining in Nijmegen breakage syndrome patient cells. We used recently developed GFP-YFP-based plasmid substrates to measure the efficiency of DNA double strand break repair. Both single strand annealing and non-homologous end-joining processes were markedly impaired in NBS1-deficient cells, and repair proficiency was restored upon re-introduction of full length NBS1 cDNA. Despite the observed defects in the repair efficiency, no apparent differences in homologous recombination or non-homologous end-joining effector proteins RAD51, KU70, KU86, or DNA-PK(CS) were observed. Furthermore, comparative analysis of junction sequences of plasmids recovered from NBS1-deficient and NBS1-complemented cells revealed increased dependence on microhomology-mediated end-joining DNA repair process in NBS1-complemented cells.     

Gamma radiation-induced single strand breaks in DNA and their repair in spheroplasts and nuclei of light-grown and dark-grown Euglena cells

Exposure of light-grown and dark-grown Euglena cells to gamma radiation causes single strand breaks in nuclear DNA as assessed by sedimentation analysis in alkaline sucrose density gradients. The number of radiation-induced single strand breaks in nuclear DNA of light-grown cells is found to be less than that in dark-grown cells. Post-irradiation incubation of both types of cells in 0 . 1 M phosphate buffer, pH 7 . 0 at 25 degrees C for 1 hour results in restitution of the strand breaks in DNA. Light-grown cells (cells with chloroplasts) are able to rejoin all the single strand breaks in DNA produced by gamma irradiation at D50 and D5 doses. On the other hand, dark-grown cells (cells devoid of chloroplasts) are unable to rejoin all the strand breaks caused by irradiation at either of the doses. The rate of DNA repair in dark-grown cells is also much slower than that in light-grown cells. Radiation-induced single strand breaks in DNA and their repair in nuclei from both types of cells is found to be similar to that observed in the spheroplasts. It is suggested that some factor(s) elaborated by chloroplasts may contribute towards the efficiency of nuclear DNA repair in Euglena cells.     

The mechanism of bilirubin-photosensitized DNA strand breakage in human cells exposed to phototherapy light

Exposure of normal human fibroblasts to visible light (420–490 nm) in the presence of exogenously added 1–100 μg/ml bilirubin enhanced the level of DNA strand breakage compared with cells irradiated in the absence of added bilirubin. Treatment of cells in the dark with an irradiated bilirubin solution also induced DNA strand breaks. However, strand breakage was not detected in cells treated with an irradiated bilirubin solution that had been incubated with catalase (H₂O₂: H₂O₂ oxidoreductase EC 1.11.1.6). Examination of irradiated bilirubin solutions demonstrated the presence of hydrogen peroxide although, apparently, not at concentrations sufficient to account for the level of DNA strand breakage detected. Hence, irradiation of bilirubin results in the generation of hydrogen peroxide and possibly other peroxides that can cause DNA damage.     

衰老过程中大白鼠脾细胞的DNA单链断裂与重接能力的变化

 DNA被紫外线损伤后,由DNA切除修复酶切除嘧啶二聚体,随之以另一条正常的DNA链为模板修复合成DNA片段,最后由DNA连接酶将新合成的DNA片与原有的DNA链连接。本文用荧光法测定DNA修复过程中DNA单链的断裂及重接能力与衰老的关系。结果表明,不同年龄大鼠脾细胞均具有修复DNA单链断裂的能力,DNA单链断裂重接的能力与年龄有相关性,断乳鼠及青年鼠的脾细胞当保温至30min时,即开始了DNA链的重接,保温90min后则恢复到原有水平;而老年鼠脾细胞保温至90min时才开始DNA链的重接,保温150min,尚未恢复到原有水平。还发现,断乳鼠及老年鼠脾细胞的单链DNA含量高于青年鼠。     

Possible involvement of DNA strand breaks in regulation of cell differentiation

The present review summarizes data on the accumulation of DNA strand breaks in differentiating cells. Large 50 Kbp free DNA fragments were observed by several research teams in non-apoptotic insect, mammal and plant cells. A more intensive DNA breakage was observed during maturation of spermatides, embryo development, and differentiation of myotubes, epidermal cells, lymphocytes and neutrophils. In general, accumulation of DNA strand breaks in differentiating cells cannot be attributed to decrease of the DNA repair efficiency. Poly(ADP)ribose synthesis often follows the DNA breakage in differentiating cells. We hypothesize that DNA fragmentation is an epigenetic tool for regulation of the differentiation process. Scarce data on localization of the differentiation-associated DNA strand breaks indicate their preferred accumulation in specific DNA sequences including the nuclear matrix attachment sites and repeats. Recent data on non-apoptotic functions of caspases provide more evidence for possible existence of a DNA breakage mechanism in differentiating cells resembling the initial stage of apoptosis. Excision of methylated cytosine and recombination are other possible explanations of the phenomenon. Elucidation of mechanisms of differentiation-induced DNA strand breaks appears to possess considerable research potential.     

Estimates of DNA strand breakage in bottlenose dolphin (Tursiops truncatus) leukocytes measured with the Comet and DNA diffusion assays

The analysis of DNA damage by mean of Comet or single cell gel electrophoresis (SCGE) assay has been commonly used to assess genotoxic impact in aquatic animals being able to detect exposure to low concentrations of contaminants in a wide range of species. The aims of this work were 1) to evaluate the usefulness of the Comet to detect DNA strand breakage in dolphin leukocytes, 2) to use the DNA diffusion assay to determine the amount of DNA strand breakage associated with apoptosis or necrosis, and 3) to determine the proportion of DNA strand breakage that was unrelated to apoptosis and necrosis. Significant intra-individual variation was observed in all of the estimates of DNA damage. DNA strand breakage was overestimated because a considerable amount (~29%) of the DNA damage was derived from apoptosis and necrosis. The remaining DNA damage in dolphin leukocytes was caused by factors unrelated to apoptosis and necrosis. These results indicate that the DNA diffusion assay is a complementary tool that can be used together with the Comet assay to assess DNA damage in bottlenose dolphins.     

Raman spectroscopic study of DNA after photosensitive damage caused by hypocrellins A and B

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Replication forks stalled at ultraviolet lesions are rescued via RecA and RuvABC protein-catalyzed disintegration in Escherichia coli

Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.     

Different effects of tert-butylhydroperoxide-induced peroxynitrite-dependent and -independent DNA single-strand breakage on PC12 cell poly(ADP-ribose) polymerase activity.

The short-chain lipid hydroperoxide analogue tert-butylhydroperoxide induces peroxynitrite-dependent and -independent DNA single strand breakage in PC12 cells. U937 cells that do not express constitutive nitric oxide synthase respond to tert-butylhydroperoxide treatment with peroxynitrite-independent DNA cleavage. Under experimental conditions leading to equivalent strand break frequencies, the analysis of poly(ADP-ribose) polymerase activity showed an increase in PC12 cells but not in U937 cells. The enhanced poly(ADP-ribose) polymerase activity observed in PC12 cells was paralleled by a significant decline in NAD+ content and both events were prevented by treatments suppressing formation of peroxynitrite. Although DNA breaks were rejoined at similar rates in the two cell lines, an inhibitor of poly(ADP-ribose) polymerase delayed DNA repair in PC12 cells but had hardly any effect in U937 cells. The results obtained using the latter cell type were confirmed with an additional cell line (Chinese hamster ovary cells) that does not express nitric oxide synthase. Collectively, our data suggest that tert-butylhydroperoxide-induced peroxynitrite-independent DNA strand scission is far less effective than the DNA cleavage generated by endogenous peroxynitrite in stimulating the activity of poly(ADP-ribose) polymerase.     

Visualization of DNA loops in nucleoids from HeLa cells: assays for DNA damage and repair

An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0-7.5 micrograms/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50-50 micrograms/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1-20 Gy of 137Cs gamma-rays, the ability to rewind the DNA supercoils was inhibited in a dose-dependent manner, presumably because of the presence of radiation-induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post-irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of gamma-irradiation, such that no difference from control cells could be detected. The half-time for repair of the radiation-induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.     

Analysis of metal-induced DNA lesions and DNA-repair replication in mammalian cells

The potency of several metal compounds in causing lesions in DNA either directly or by exposure of intact cultured cells has been examined using the neutral conditions of nucleoid gradient sedimentation. HgCl2 was clearly the most potent inducer of single-strand breakage when added to isolated nucleoids or when nucleoids were prepared from cells treated with this compound. CaCrO4 , however, caused DNA-strand breaks in nucleoids isolated from cells treated with this agent but did not induce DNA strand breaks when added directly to nucleoids. Although less potent than HgCl2, NiCl2 also caused significant single strand breakage in isolated nucleoids or in nucleoids prepared from cells treated with this metal. Since strand breakage of DNA in intact cells may occur secondary to activation of DNA-dependent nucleases during repair replication, CsCl gradient density sedimentation was utilized to examine whether repair processes were induced by exposure of cells to NiCl2, HgCl2 and CaCrO4 . CaCrO4 and NiCl2 induced substantial DNA-repair activity at concentrations and exposure times where DNA lesions could not be detected whereas HgCl2 induced a 10-fold lower level of DNA-repair activity compared to CaCrO4 at optimal concentrations which again were below the concentrations of this metal that produced measurable DNA lesions. Both the induction of DNA-repair activity and DNA-strand breakage by these metals was concentration- and time-dependent. These results demonstrate some unique aspects of the interaction of HgCl2, NiCl2 and CaCrO4 with the DNA of intact cells and point to the possible important correlation of induction of DNA repair to carcinogenesis since nickel and chromate have clearly been implicated as carcinogens and induce considerable repair whereas HgCl2 is not considered a carcinogen and induces the least DNA repair despite its potency in producing DNA lesions.     

Interactions between lac repressor protein and site-specific bromodeoxyuridine-substituted operator DNA. Ultraviolet footprinting and protein-DNA cross-link formation

Specific contacts between the lac repressor and operator have been explored using 5-bromodeoxyuridine-substituted DNA. Substitution of BrdU for single thymidine positions in a synthetic 40-base pair operator provides substrate for ultraviolet irradiation; upon irradiation, strand scission occurs at the BrdU residues. When bound, lac repressor protein provides protection against UV-induced breakage depending on the nature of the sites and type of interaction. We have confirmed 13 unique sites of inducer-sensitive protection along the operator sequence using this method compared to complete substitution with BrdU; differences were observed at two positions for singly substituted versus completely substituted DNAs (Ogata, R., and Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4973-4976). The ability of these photosensitive DNAs to form short range cross-links to bound protein has been used to determine the efficiency with which cross-linked protein-DNA complexes are generated at each individual site of BrdU substitution. Five sites of high efficiency cross-linking to the repressor protein have been identified. At one site, cross-linking without protection from strand scission was observed; this result suggests an unusual mechanism of strand scission and/or cross-linking at this site. Comparison of the UV protection results and the cross-linking data show that these processes provide complementary tools for identifying and analyzing individual protein-DNA contacts.     

PARP-1 inhibition induces a late increase in the level of reactive oxygen species in cells after ionizing radiation

Poly(ADP-ribose) polymerase 1 (PARP1), an enzyme activated by DNA strand breaks, synthesizes polymers of poly(ADP-ribose) (PAR) that modify chromatin and other proteins and play a role in DNA repair. Inhibition of PARP1 activity is considered a potentially important strategy in clinical practice, especially to sensitize tumor cells to chemo- and radio-therapy. Here we examined the influence of inhibition of PARP1 on formation of reactive oxygen species (ROS) and on DNA repair in cells exposed to ionizing radiation (IR). K562 (human myelogenous leukaemia) cells were grown and exposed to 4 or 12Gy of ionizing radiation in presence or absence of the PARP inhibitor NU1025 (100μM). Intracellular ROS were assayed using the probe 2,7-dichlorofluorescein with detection by flow cytometry and the rejoining of DNA strand breaks were followed by alkaline single cell gel electrophoresis (comet) assays. In untreated cells a significant increase in PAR formation occurred during the first 5min after IR, followed by a gradual decrease up to 30min. Addition of a PARP inhibitor arrested the production of PAR almost completely and decreased the rate of rejoining of DNA strand breaks significantly; however, 3h after irradiation we observed no difference in the amount of DNA strand breaks between PARP inhibitor-treated and untreated cells. Twelve to 48h after irradiation, an increase of ROS concentration was observed in irradiated cells and ROS levels in PARP inhibitor-treated cells were significantly higher than in cells without inhibitor. Irradiated cells grown in the presence or absence of PARP inhibitor did not differ in the frequencies of apoptotic and necrotic cells or in the activity of caspases at 24, 48 and 72h after irradiation. Poly(ADP-ribosylation) and inhibition of PARP1 appeared to modulate DNA strand break rejoining and influence the concentration of ROS in irradiated cells.     

Inhibition and enhancement of phleomycin-induced DNA breakdown by aromatic tricyclic compounds.

Cationic aromatic tricyclic compounds including triphenylmethane dyes, phenazines, phenoxazines, acridines, phenothiazines, phenanthridinium compounds, anthracenes and xanthene dyes, which amplify cell killing in phleomycin-treated Escherichia coli B cells also modified phleomycin-induced breakdown of DNA to acid-soluble fragments. A plot of DNA breakdown as a function of concentration was bell-shaped for each of the active compounds, i.e. as the concentration increased, DNA breakdown was enhanced initially, but above a certain concentration, the proportion of DNA degraded declined, often to zero. One of the compounds, acriflavine, when tested also inhibited DNA breakdown following ultraviolet irradiation. A study, by sedimentation methods, of DNA single-strand breakage in phleomycin-treated E. coli cells, using 3 representative compounds, Crystal Violet, 3,6-diaminoacridine and Methylene Blue, revealed a consistent increase in DNA strand breaks as concentration of compound increased. In similar experiments with ethidium bromide the breakage yield/concentration curve exhibited a maximum. In general, however, it seems that the inhibition of DNA-breakdown observed at higher concentrations of these amplifying compounds is not explicable by an effect on the primary breakage event, but is due to suppression of exonucleolytic activity in the cells.     

Incisions in ultraviolet irradiated circular bacteriophage lambda DNA molecules in excision proficient and deficient lysogens of E. coli

Summary The breakage of closed covalent DNA molecules in lysogenic host cells after ultravilet irradiation was investigated. In repair proficient host cells incisions are introduced immediately following irradiation. A steady-state of strand interruptions is observed within 20–50 seconds after irradiation, where the number of broken molecules is dose dependent for doses up to 600 ergs/mm². No ultraviolet promoted strand breaks were observed in uvrA or uvrB lysogens, in accordance with previous results obtained by Shimada, Ogawa & Tomizawa [Molec. gen. Genet. 101, 245 (1968)]. In contrast, uvrC mutants have the ability to form breaks in superinfecting DNA molecules after ultraviolet irradiation. The ultraviolet specific endonucleolytic activity observed in uvrC host cells differs from that observed in uvr ⁺ host cells in that, (i) the first break is introduced at least 15 times slower, (ii) for doses below 300 ergs/mm² the number of strand breaks is higher, (iii) the dose dependence terminates at a lower dose. The possible function of the uvrC gene product in the repair is discussed.     

Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.     

不同离子辐照对离体质粒DNA损伤与转化活性的影响

利用 30Kev的N+ 、Ar+ 离子辐照离体质粒DNA ,分析了不同离子对DNA单双链断裂及转化活性的影响。结果表明 :N+ 、Ar+ 离子辐照均可引起质粒DNA单双链断裂和转化活性的变化 ,且随着辐照剂量的增加 ,单双链断裂频率增加 ,转化活性下降。Ar+对离体质粒DNA比N+ 具有更强的单双链断裂效应 ,且从 9× 10 15Ar+ cm2 剂量开始 ,质粒可完全丧失转化活性。质粒转化活性的大小与DNA单双链断裂频率呈正相关     

Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C. It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure. We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P. furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli. At 100 degrees C, DNA in P. furiosus cells is about 20 times more resistant to thermal breakage than that in E. coli cells, and six times fewer breaks were found in P. furiosus DNA after exposure to 110 degrees C for 30 min than in E. coli DNA at 95 degrees C. Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.