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PubMed1979

DNA damage and DNA repair in cultured human cells exposed to chromate.

DNA damage and DNA repair have been observed in cultured human skin fibroblasts exposed to potassium chromate but not to a chromic glycine complex. DNA repair synthesis (unscheduled incorporation of [3H]thymidine (TdR)) was measured in cells during or following exposure to chromate and was significant for chromate concentrations above 10(-6) M. Maximal DNA repair was observed at about 10(-4) M chromate. DNA repair capacity was found to be saturated at this concentration. Chromate was stable for at least 8 h in culture medium and produced approximately a linear increase in repair with duration of exposure. DNA damage as determined by alkaline sucrose gradient sedimentation was detected after treatment for 1.5 h with 5 . 10(-4) M chromate. Exposure to 10(-7) M chromate solution for 7 days inhibited colony formation while acute (1 h) treatment was toxic at 5 . 10(-6) M. The chromic glycine complex was toxic above 10(-3) M for a 1-week exposure but was not observably toxic after a 1-h treatment. These results indicate that chromate and not chromic compounds may be the carcinogenic form for man. The nature of the ultimate carcinogen is discussed. These findings illustrate the utility of the DNA repair technique to study the effects on human cells of inorganic carcinogens and mutagens.

Adult↗
PubMed1979

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells.

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.

Animals↗
PubMed2026

Transketolase promotes RNF20-dependent H2BK120 ubiquitination and DNA repair through a non-enzymatic adaptor function.

Efficient repair of DNA double-strand breaks (DSBs) is essential for maintaining genome stability and conferring tumor radioresistance. Histone H2B monoubiquitination at lysine 120 (H2BK120ub), catalyzed by the RNF20/RNF40 E3 ligase complex, promotes DSB repair by coordinating chromatin remodeling and repair factor recruitment. Here we identify transketolase (TKT) as a non-enzymatic regulator of DNA damage repair in colorectal cancer cells. TKT enhances DNA repair efficiency and radioresistance independently of its catalytic activity by facilitating RNF20-dependent H2BK120ub. Mechanistically, TKT interacts with both the RNF20/RNF40 complex and the FACT chromatin remodeling complex, functioning as a non-enzymatic adaptor that facilitates FACT-RNF20 association and RNF20 foci formation following DNA damage. Disruption of this TKT-FACT-RNF20 axis impairs RNF20 foci formation and H2BK120ub induction, increases DNA damage, and enhances radiosensitivity. These findings reveal a previously unrecognized non-enzymatic adaptor function of TKT in DNA damage repair and highlight a potential vulnerability in radioresistant tumors.

DNA repair↗
PubMed1976

The effect of hyperthermia on DNA repair.

In the past there were many individual observations on the value of hyperthermia in the treatment of human neoplasia but most of the information about the value of hyperthermia as a single agent or in the combined modality approach has come from laboratory investigations. Dose response curves for cell survival after exposure to heat are similar in shape to cell survival curves obtained after irradiation or treatment with some cytostatic agents. The shoulder in such curves suggests that repair of sublethal or potentially lethal damage takes place after hyperthermic treatment. On the level of molecular biology the process of cellular repair should correspond to repair of damage inflicted on deoxyribonucleic acid (DNA). We have shown by means of the BUdR assay that such DNA-repair synthesis does take place upon exposure to heat. Many investigations have provided evidence of a synergism between hyperthermia and ionizing irradiation or some cytostatic agents. It was suggested that such synergism might be caused by the inhibition of repair of sublethal damage by heat. After inflicting DNA damage by a strong alkylating agent (NA-AAF) we could demonstrate DNA-repair synthesis by means of the BUdR-assay during exposure to heat. At the present time results obtained by assaying DNA repair on the basis of cell survival and by means of the BUdR-assay are difficult to reconcile.

Acetoxyacetylaminofluorene↗
PubMed1979

Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea.

The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair:the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65--70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.

Bromodeoxyuridine↗
PubMed1977

Role of DNA repair in natural resistance of rat ascites hepatomas to nitrogen mustard.

Mechanism of natural resistance of rat ascites hepatomas to nitrogen mustard was examined with tumors, especially AH-13 and AH-44, sensitive and resistant to the chemical, respectively. There was found little difference in the uptake and binding of the chemical among the two lines. In contrast, DNA repair activity of the resistant line was higher than that of the sensitive line. This result suggested that natural resistance of AH-44 to the drug may be, at least partially, due to its higher activity of repairing DNA damage.

Animals↗
PubMed1977

Nonuniform distribution of DNA repair in chromatin after treatment with methyl methanesulfonate.

The distribution of methyl methanesulfonate induced DNA repair was measured in mouse mammary cell chromatin by digestion of "repair labeled" nuclei with micrococcal nuclease. The results indicate that there is a nonuniform distribution of DNA repair in chromatin. The chromatin fraction digested during the first 5 minutes of incubation with micrococcal nuclease appears to be a primary site of DNA repair after methyl methanesulfoante treatment. The observed nonuniform distribution of DNA repair in chromatin may be due to 1)a nonrandom alkylation of DNA in chromatin by methyl methanesulfonate or 2)areas in chromatin of increased accessibility for the repair enzymes to the DNA lesions.

Animals↗
PubMed1978

The nucleotide-permeable Escherichia coli cell, a sensitive DNA repair indicator for carcinogens, mutagens, and antitumor agents binding covalently to DNA.

Ether-permeabilized (nucleotide-permeable) Escherichia coli cells respond to alkylating and arylalkylating carcinogens with DNA excision repair, as assessed by their stimulation of DNA repair synthesis. In the present work, we have investigated whether DNA repair synthesis in ether-treated E. coli cells can serve as a general indicator to monitor the DNA-binding of carcinogens, mutagens and antitumor agents. Therefore, a standard assay was developed and comparative analyses were performed on 11 ultimate carcinogens, 10 proximate carcinogens, 2 tumor promoters, 6 mutagens, and 12 antitumor agents. All ultimate carcinogens (alkylating, acylating, arylalkylating agents) and mutagens (e.g., hydrogeen peroxide, acridine derivatives) caused DNA excision repair in wild type cells as measured by [3H] dTMP incorporation and simultaneously inhibited replicative DNA synthesis to various extents. Control experiments with the mutant cells uvrA and uvrB were performed to determine whether the pyrimidine-dimer-specific UV-endonuclease was involved in the removal of DNA damage. This was found to be true for the ultimate carcinogens (Ac)2 ONFln, mitomycin C, and for very reactive alkylating carcinogens. None of the ultimate carcinogens induced repair polymerization in mutant cells lacking the 5'-3' exonucleolytic activity of DNA polymerase I. Proximate carcinogens, such as Me2NNO, 4-nitroquinoline-1-oxide and aflatoxins, did not induce excision repair in the standard assay, probably because of the inability of E. coli to perform the activation steps necessary for covalent DNA-binding. However, Me2NNO, when pretreated with Udenfriend's hydroxylating mixture, gave rise to a low level of repair polymerization in ether-treated cells. Intercalating mutagens, such as quinacrine and ethidum bromide, inhibited replicative DNA synthesis. However, they were not found to be repair-inducers. THE TUMOR PROMOters TPA and phorbol-12,13-didecanoate did not cause excision repair, even when applied at high concentrations, nor did they inhibit repair synthesis stimulated by MeNOUr or (Ac)2 ONFln. The antitumor agents may be classified into two groups on the basis of the influence they exert on DNA synthesis: members of the first group (involving BCNU and bleomycin) stimulate repair polymerization and, in addition, inhibit DNA replication. These compounds are known to bind covalently to DNA. The second group of drugs (including adriamycin and cis-Pt(II)diammine complexes) inhibits DNA replication without stimulating repair synthesis. The predominant DNA-interaction of these compounds is known to be a non-covalent (i.e., intercalative, electrostatic) binding. Our experiments show that the ether-permeabilized E. coli cell can be successfully used to test ultimate carcinogens, mutagens and antitumor agents for repair-inducing and replication-inhibiting activity. The standard test might be extended to pre- and proximate carcinogens, provided these can be suitably activated.

Antineoplastic Agents↗
PubMed1976

Studies on DNA repair in early spermatid stages of male mice after in vivo treatment with methyl-, ethyl-, propyl-, and isopropyl methanesulfonate.

In vivo DNA repair occurring in early spermatid stages of the mouse has been studied with four mutagens that are chemical homologs: MMS, EMS, PMS and IMS. Using the well-studied sequence of events that occurs during spermatogenesis and spermiogenesis in the mouse, aatids was measured by the unscheduled incorporation of [3H]dT into these germ cells which were recovered from the caudal epididymides 16 days after chemical treatment. Purification of the caudal sperm DNA at this time verified that the [3H]dT was incorporated into the DNA. For each chemical mutagen a study was made on the level of DNA repair occurring in early spermatids as a function of the administered, in vivo dose. Within experimental errors, all four chemicals produced a linear increase in DNA repair in early spermatids with increasing dose. Only the highest dose of MMS (100 mg/kg) produced a greater repair response than expected for a linear curve. At equimolar doses the most effective chemical in inducing DNA repair was MMS, followed by EMS, IMS and PMS. When testicular injections of [3H]dT were given at the same time as the intraperitoneal injections of the mutagens, the amount of unscheduled incorporation of [3H]dT into the DNA of early spermatids was maximized. Since [3H]dT has been shown to be available for incorporation into germ-cell DNA for only approximately 1 h after injection, all four mutagens must reach the DNA of early spermatids and begin producing "repairable" lesions within 1 h after treatment. The amount of DNA repair occurring at later times after chemical treatment of early spermatids was studied by testicular injections of [3H]dT 1/2, 1, 2 and 3 days after chemical treatment. Repair was still occurring in the early spermatids at 3 days post-treatment; this repair is most likely a manifestation of the finite rate of the repair process rather than resulting from newly alkylated DNA. For MMS and EMS there was a rapid decrease in the level of DNA repair in the first 1/2 day following treatment. This was followed by a much slower, exponential decrease in the level of repair out to 3 days post-treatment. The curves suggest that the amount of repair is proportional to the number of repairable lesions still present in the DNA. For PMS and IMS the level of repair decreases rapidly in the first 1/2 day after treatment and thereafter remains relatively constant through 3 days post-treatment. With all four mutagens, DNA repair in early spermatids was detectable at doses 5 to 10 times lower than those required to observe other genetic end points such as dominant lethals, translocations and specific-locus mutations in any germ-cell stage. The sensitivity of detection of in vivo DNA repair in the germ cells of male mice makes such a system a useful adjunct to other genetic tests for studying chemical mutagenesis in mammals.

1-Propanol↗
PubMed2022

Preparation of Nucleosome Core Particles Complexed with DNA Repair Factors for Cryo-Electron Microscopy Structural Determination.

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Chromatin↗
PubMed1979

Effects of opiates and demographic factors on DNA repair synthesis in human leukocytes.

DNA repair synthesis in leukocytes stressed by far UV irradiation was studied in 90 normal individuals, 38 street-heroin addicts, and 18 methadone maintenance patients. Age, sex, coffee use, and alcohol use had no significant effect on the maximal repair synthesis response of the control subjects, but smoking tobacco significantly decreased the mean response and variance when compared with nonsmoking controls. Heroin addiction had an even more pronounced negative effect, and this may be related to the high rate of chromosome aberrations found in this population. Half of the addicts tested were incapable of repairing UV fluences one-quarter as large as those repaired by the control subjects (5 J/m2 and 20 J/m2, respectively) in the 2-hr assay period. Long-term methadone treatment ameliorated the effects of the street heroin, just as it resulted in a decrease of the chromosome aberration frequency.

Adolescent↗
PubMed1977

DNA repair characteristics and skin cancers of xeroderma pigmentosum patients in Japan.

Fifty xeroderma pigmentosum patients in Japan were examined for clinical characteristics and DNA repair of their cells, Skin cancers developed in 22 patients. Most of the patients without skin cancers were children, except for 5 older patients who had intermediate or nearly normal levels of DNA repair in their cells. All patients younger than 10 years old had no or very low activity of unscheduled DNA synthesis after ultraviolet light irradiation. Three genetic complementation groups, A, D, and E, and variants were found. Many Group A patients and no Group C patients characterized Japanese patients, compared with those in Europe and the United States, where Group C patients were most frequent. The high frequency of patients with low DNA repair capacities in their cells may account for the apparent high frequency of xeroderma pigmentosum patients in Japan. Age distribution of the cancer-bearing patients and their DNA repair characteristics suggest that almost all xeroderma pigmentosum patients will develop skin cancers unless their cells have nearly normal levels of DNA repair.

Adolescent↗
PubMed1975

Genetic heterogeneity in xeroderma pigmentosum: complementation groups and their relationship to DNA repair rates.

Fibroblast strains from 12 patients with xeroderma pigmentosum had lower than normal rates of DNA repair, as determined by autoradiographic studies of ultraviolet-induced unscheduled nuclear DNA synthesis. The nuclei in binuclear cells, obtained by fusing fibroblasts from certain pairs of these strains, had a greater rate of DNA repair than the nuclei of either strain's unfused mononuclear cells. These results indicate that complementary corrections of the strains' repair defects had occurred in the fused cells. Four complementation groups were found, indicating that at least four mutations caused decreased DNA repair among these 12 strains. The unfused mononuclear cells of each group had a characteristic rate of repair that differed from the rates of the other groups.

Autoradiography↗
PubMed1977

Loss of DNA repair capacity during successive subcultures of primary rat fibroblasts.

Cultures of fibroblasts from newborn rats and successive subcultures of these cells were treated with 4-nitroquinoline-1-oxide to induce DNA repair. DNA from the cultures was examined by velocity sedimentation in alkaline sucrose gradients immediately after drug treatment and after a post-treatment incubation period of 3 h. Early passage cells were able to repair the damage that appeared as single strand breaks, however, by the seventh subculture this activity was not apparent. Measurements of repair synthesis showed a partial loss of this capacity with successive subculture. The results fit a model in which 4NQO causes two kinds of DNA modification, one of which is alkali labile and appears as a single-strand break. Both modifications are subject to excision repair, but each is recognized initially by a specific endonuclease. In the late passage cells, the endonuclease specific for the alkali labile modification is absent.

4-Nitroquinoline-1-oxide↗
PubMed1979

DNA repair in lens cells during chick embryo development.

When chick lens epithelium is cultured in vitro, differentiation into lens fiber cells is accompanied by DNA degradation. This phenomenom of terminal differentiation was studied in the epithelium from embryos at the 6th and 11th days of development. DNA size and the ability of the cells to repair DNA damage induced by X-rays were analysed in alkaline sucrose gradients. In the 6-day epithelium a rapid degradation and complete lack of DNA repair were recorded. Similar observations have been made in previous studies on the 11-day sample, but here degradation is progressive and occurs after a lag of several days. In the younger epithelium, internal irradiation by [3H]thymidine also had a drastic effect resembling that caused by X-rays. In order to assess the process of differentiation in our experimental system the synthesis of delta- and alpha-crystallins was monitored. Stage-related modifications in the rates of synthesis were recorded. The results confirm that the DNA repair system is impaired during terminal differentiation. The differences observed between the two stages may reflect either a developmental modification in DNA repair mechanisms or a change in the relative proportions of differentiating cells. An hypothesis is proposed in support of the latter case.

Age Factors↗
PubMed1978

In vivo DNA repair after N-methyl-N-nitrosourea administration to rats of different ages.

DNA repair time-course was studied after injury by N-methyl-N-nitrosourea (MNU) in rat liver cells of animals of different ages and in fetuses using hydroxyurea (HU) as inhibitor of scheduled DNA synthesis. DNA repair was a rapid phenomenon, more so in young adults than in newborns, and was not detectable in fetuses. A correlation seems to exist among organ sensitivity to carcinogen, age of animal and DNA repair.

Age Factors↗