Mutation Research/DNA Repair Reports最新文献

The formation of DNA strand breaks was characterized in human fibroblasts prepared by several methods. In quiescent monolayer cultures of normal human fibroblasts (NHF), exposure to 254 nm radiation (UV) caused the rapid appearance of DNA strand breaks as monitored by alkaline elution analysis. Maximal levels of DNA breaks were seen 30 min after 10 J/m²; thereafter, strand breaks disappeared. Breakage soon after irradiation appeared to saturate at fluences above 10 J/m². Xeroderma pigmentosum fibroblasts belonging to complementation group A (XPA) did not display this response which reflects operations of the nucleotidyl DNA excision repair pathway. When fibroblasts strains were released from culture dishes by enzymatic digestion with trypsin or by scraping with a rubber policeman, UV-dependent DNA breakage displayed altered dose and time responses. Few breaks were detected in detached preparations of NHF after 10 J/m² indicating inactivation of nucleotidyl DNA excision repair. The fluence response in detached fibroblasts was linear up to an incident fluence of 100 J/m². Moreover, after 25 or 50 J/m², strand breaks accumulated as a linear function of time for up to 2 h after irradiation. This UV-dependent and time-dependent incision activity was also observed in XPA monolayers and released-cell preparations. In permeable fibroblast preparations, DNA breaks accumulated in unirradiated cells that had been released with trypsin or by scrapping. Permeabilization in situ using saponin to open the plasma membrane produced a cell preparation that accumulated fewer UV-independent breaks. In saponin-permeabilized NHF that were irradiated with 10 J/m², UV-dependent strand incision activity occurred at about 30% of the rate of incision seen in intact monolayer NHF. These results reveal at least 3 DNA strand incision activities in human fibroblast preparations of which only one reflects operation of the nucleotidyl DNA excision repair pathway.

We have investigated the effects of fluctuations in deoxynucleoside triphosphate (dNTP) pool size on DNA repair and, conversely, the effect of DNA repair on dNTP pool size. In confluent normal human skin fibroblasts, dNTP pool size was quantitated by the formation of [³H]TTP from [³H]thymidine; DNA repair was examined by repair replication in cultures irradiated with UV light. As defined by HPLC analysis, the [³H]TTP pool was formed within 30 min of the addition of [³H]thymidine and remained relatively constant for the next 6 h. Addition of 2–10 mM hydroxyurea (HU) caused a gradual 2–4-fold increase in the [³H]TTP pool as HU inhibited DNA synthesis but not TTP production. No difference was seen between the [³H]TTP pool size in cells exposed to 20 M/m² and unrradiated controls, although DNA-repair synthesis was readily quantitated in the former. This result was observed even though the repair replication protocol caused an 8–10-fold reduction in the size of the [³H]TTP pool relative to the initial studies. In the UV excision-repair studies the precense of hydroxyurea did not alter the specific activity of [³H] thymidine 5'-monophospahte incorporated into parental DNA due to repaier replication. These results suggest that fluctuations in the deoxynucleoside triphosphate pools do not limit the extent of excision-repair sythesis in human cells and demonstrate that DNA nucleotide excision-repair synthesis does not significantly diminish the size of the [³H]TTP pool.

In an E. coli strain carrying two mutations, one in the dnaC gene involved in initiation of DNA replications and another in the uvrB gene which affects the excision-repair system, it has been shown that the SOS response cannot be induced by UV. This is probably due to the absence of any inducing signal (Salles and Defais, 1984). The capacity to induce the SOS network was followed using RecA protein amplification as a probe. When breaks were produced in DNA, RecA protein induction was restored. We describe here a strain in which both RecA protein and β-galactosidase from a sfiA::lacZ fusion can be measured simultaneously in the same bacterial extract. In conditions in which no replications proceeds, this strain can be used to detect the ability of chemicals to produce free radical-mediated DNA breaks in vivo.

The biological activity of a series of nitopyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a st-a mutant of polyoma virus. Concentrations of 10–30 μg/ml of 1-nitropyrene (1-NP) induced viral replications, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 μ/ml) to the culture medium. The response was less than that obtained with 0.1 μg/ml of the activated metabolite of benzo[a]pyrene (BP), BP trans-7,8-dihydrodiol-9,10 epoxide (anti) (BPDE). A series of di-, tri-, and tetra-nitropyrenes were also found to induce polyoma DNA replicatin, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5–2.0 μg/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 μg/ml inidcated that this compound was approximately equipotent with BPDE for induction of polyoma DNA synthesis. Studies of drug metabolism, DNA binding and DNA adduct formation indicate that 1,6-DNP is metabolized in this cell line, binds to DNA, and forms stable adducts. The level of DNA modifications seen with, 1,6-DNP is higher than that observed under comparable conditions with an equivalent dose of BPDE. These findings provide additional evidence that the nitropyrene class of compounds can exert biological effects in mammalian cells, and that the dinitropyrenes are more potent than 1-NP.

Male NMRI mice were fed a diet containing a complete mixture of amino acids or a mixture deficient in methionine-cysteine or lysine (30% of the control level) for a period of 6 days. During the feeding period all mice received dimethylnitrosamine in the drinking water ad libitum. The exposure avaraged 1 mg dimethylnitrosamine/kg body weight and day. The concentration of O⁶-methylguanine-DNA methyltransferase was measured in liver extracts. It decreased significantly in the methionine-cysteine deficient mice. When DNA from the liver was analyzed for alkylated purine bases the mice received a single dose of ¹⁴C-labeled dimethylnitrosamine (0.5 or 1 mg/kg body weight)_at 120 min before sacrifice. The concentration of O⁶-methylguanine increased significantly over the control level upon feeding the deficient diets and was restored to the concentration of the controls by refeeding lysine for 2 days following 6 days of lysine deficiency. The increased ration of O⁶-methylguanine to N-7-methylguanine indicated that methylation of guanine in the N-7 position was not subject to variation by the intake of dimethylnitrosamine during the dietary deficiencies. The results demonstrate the requirement for a balanced composition of amino acids in the diet to maintain a sufficient concentration of O⁶-methylguanine-DNA methyltransferase in the cells and thus to permit efficient removal of the methyl group from the O-6 position of guanine in DNA after exposure to dimethylnitrosamine.

To determine the prevalance of the repaire enzyme HMU-DNA hlycosylase we assayed its activity in whole cell extracts of several bacterial species, the eukaryotic yeast Saccharomyces cerevesiae, mammalian cell line and murine tissue. Enzyme activity was constitutively present in murine, hasmter and human cell lines. It was not inducible by exposing cells to oxidative stress from ionizing radiation or by incubating cells with the 2′-deoxynucleoside of HMU, HMdU. In murine tissue, enzyme activity was highest in brain and thymus. HMU-DNA glycosylase activity was not detectable in bacteria or yeast nor could activity be detected after exposure of cells to H₂O₂. These results suggest that, in contrast to other DNA-repair enzymes, HMU-DNA glycosylase is a differentiated function limited to higher eukaryotic organisms.

We are examining the effects of preirradiation of host (monkey) cells upon the replication of UV-damaged SV40. Control cells and cells preirradiated with low fluences (5 or 10 J/m²) of UV were infected with undamaged SV40, and the immediate effects of a subsequent irradiation were determined. UV inhibited total SV 40 DNA synthesis (incorporation of thymidine into viral DNA) in both preirradiated and control cells, but the extent of inhibition was less in the preirradiated cells. A test fluence of 60 J/m² to SW40 replicating in preirradiated cells reduced synthesis only as much as a test fluence of 25 J/m² in control cells. The fraction of recently replicated SV40 molecules that re-entered the replication pool and subsequently completed one round of replication in the first 2 h after UV was also decreased less in the prerradiated cells. Thus preirradiation of the host cell mitigates the immediate inhibitory effects of a subsequent UV exposure upon SV40 replication.

Nonsense-defective auxotrophic strains of Escherichia coli B/r were used to study mutation frequency decline (MFD) after mutagenesis with ethyl methanesulfonate (EMS). The mutation frequencies for prototrophic revertants that were either converted or de novo glutamine tRNA suppressor mutations declined as treated auxotrophic parental cells were incubated with glucose but without required amino acids (a condition typically producing MFD). The decline for converted suppressor mutations was more rapid than the decline for de novo suppressor mutations after low or moderate EMS treatment, but both suppressor mutation types showed the same slow decline after extensive treatment. The declines for both types of suppressor mutation were eliminated in uvrA-defective cells, and the rapid decline seen for converted suppressor mutations appeared as a slow decline in mfd-defective cells. The results are interpreted that true MFD (the rapid process) affects only the EMS-induced converted glutamine tRNA suppressor mutations. This would account for the rapid decline that is blocked in cells with an mfd defect and in cells with deficient excision repair activity (uvrA or excessive DNA damage). In addition, a second non-specific antimutation mechanism is proposed that is dependent on excision repair only and accounts for the slow decline seen with converted suppressor mutations in some instances and with de novo suppressor mutations at all times. The true MFD mechanism may consist of a physiologically dependent facilitated excision repair specifically for premutational residues located in the transcribed strand of the target DNa sequence (for O⁶-ethylguanine in cells treated with ethyl methanesulfonate or pyrimidine- pyrimidine photoproducts after UV irradiation.