Mutation Research/DNA Repair Reports最新文献

The DNA-damaging capacity and the mutagenicity of 6 disinfectants were studied by liquid rec-assay and Ames test. 5 disinfectants were found to be positive in DNA-damaging capacity while only one of them showed clear mutagenicity in the Ames test. Liquid rec-assay by direct incubation with S9 mix was the most sensitive method and gave the best correlation between the growth ratio (R 50) and the time lag, both of which compared Rec⁺ and Rec⁻. liquid rec-assay may be useful for detecting the DNA-damaging capacity of chemicals with a strong killing effect.

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously.

The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt⁺ colonies were selected, and cell lines esttablished. Several times were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95 000-dalton UvrA-related protein. The expression of uvrA was found to be 50–100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the “mammalian” UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA⁺ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA⁺ cell lines were examined for their UV_resistant phenotye and not unexpectedly were found not to be reverted to a state of repair proficiency.

Chinese hamster ovary cells and two UV-hypersensitive derivatives were used to determine the importance of DNA excision repair for split-dose recovery. In the wild-type cells 75% of the maximum theoretical recovery was observed when the fractions were delivered at 2-h intervals. Very little recovery was evident in the two hypersensitive cell lines. Using radioimmunoassays specific for (6-4)photoproducts and cyclobutane dimers, the ability of UV-irradiated repair-deficient cells representing 5 complementation groups to repair these 2 photoproducts was determined. Removal of antibody-binding sites specific for (6-4)photoproducts was 80% complete in 6 h and was defectiev in the UV-sensitive cells. In contrast, only 20-–60% of antibody-binding sites specific for cylcobutane dimers were removed 18 h post-irradiation, and the extent of removal was the same in normal and defective cell lines. We conclude that repair pf (6-4)photoproducts accounts for split-dose recovery. In addition, we conclude thtat a consequences of DNA repair in CHO cells is modification rather that reemoval of cylobutane dimers.

Five UV-sensitive mutants obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment of the Staphyloccocus epidermidis W5 strain were characterized phenotypically by assaying their UV- and MNNG-sensitivities, lysogenic inducibility, host-cell reactivation and Weigle reactivation capacities. The results were compared with those of well-characterized Escherichia coli strains, permitting the identification of: 2 mutants that behave as Uvr⁻ Umu⁻; 1 mutant that appears analogous to Uvr⁻; 1 mutant that resembles LexA⁻ and 1 mutant that exhibits a RecA⁻ phenotype. The study of these mutants can contribute to the understanding of the repair mechanisms in S. epidermidis.

Do damage-inducible responses in mammalian cells alter the interaction of lesions with replication forks? We have previously demonstrated that preirradiation of the host cell mitigates UV inhibition of SV40 DNA replication; this mitigation can be detected within the first 30 min after the test irradiation. Here we test the hypothesis that this mitigation involves either (1) rapid dimer removal, (2) rapid synthesis of daughter strands past lesions (trans-dimer synthesis), or (3) continued progression of the replication fork beyond a dimer.

Cells preirradiated with UV were infected with undamaged SV40, and the effects of UV upon viral DNA synthesis were measured within the first hour after a subsequent test iirradiation. In preirradiated cells, as well as in non-preirradiated cells, pyrimidine dimers block elongation of daughter strands; daughter strands grow only to a size equal to the interdimer distance along the parental strands. There is, within this first hour after UV, no evidence for trans-dimer synthesis, nor for more rapid dimer removal either in the bulk of the parental DNA or in molecules in the replication pool. Progression of the replication forks was analyzed by electron microscopy or replicating SV40 molecules. Dimers block replication-fork progression in preirradiated cells to the same extent as in non-preirradiated cells. These experiments argue strongly against the hypothesis that preirradiation of host cells results in either the rapid removal of dimers, trans-dimer synthesis, or continued replication-fork progression beyond dimers.

A DNA-repair mutant was characterized that has the extraordinary and interesting properties of extreme sensitivity to UV killing combined with a high level of nucleotide excision repair. The mutant V-H1 isolated from the V79 Chinese hamster cell line appeared very stable, with a reversion frequency of about 3.5 × 10⁻⁷. Genetic complementation analysis indicates that V-H1 belongs to the first complementation group of UV-sensitive Chinese hamster ovary (CHO) mutants described by Thompson et al. (1981). This correponds with data on cross-sensitivity and mutation induction after UV irradiation published by this group. Surprisingly, the mutant V-H1 shows only slightly reduced (to ∼ 70%) unscheduled DNA synthesis (UDS) after UV exposure, while the other two mutants of this complementation group are deficient in UDS after UV. In agreement with the high residual UDS, in V-H1 also the amount of repair replication in response to UV treatment is relatively high (∼ 50%). It has also been shown that the incision step of the nucleotide excision pathway takes place in V-H1 (with a lower rate than observed in wild-type cells), whereas another mutant (UV5) of the same complementation group is deficient in incision.

This heterogeneity within the first complementation group indicates that the repair gene of this complementation group may have more than one functionally domain or that the gene is not involved in the incision per se but is involved in e.g. preferential repair of active genes.

We partially depleted the O⁶-methylguanine-DNA methyltransferase activity in four O⁶-methylguanine (O⁶-mGua) repair-proficient (Mer +) human cell strains with exogenous O⁶-mGua (2 mM for 3 h, a non-toxic regimen) and then challenged them with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MT-partially depleted HT29 cells removed O⁶-mGua from DNA at about half the rate of control cells, while removal of 3-methyladeninwas unaffected. In spite of partial depletion, of MT, however, cell killing by MNNG in a colony-forming assay with HT29, A549, A498 or KD cells was not greatly affected. (This is in contrast to the dramatic potentiation of CNU cytotoxicity observed previously.) In an attempt to sensitize Mer + strains to killing by MNNG, we treated cells with O⁶-mGua following MNNG exposure (0.4 mM for 4 days), in addition to the pre-MNNG treatment of 2 mM O⁶-mGua for 3 h. This sensitized KD and HT29 cells 2-fold to killing by MNNG, based on the dose at 10% survival, but did not sensitive Mer - A1336. However, post-treatment alone was as effective as combined pre- and post-treatment in sensitizing KD cells to killing. Thus, when the O⁶-mGua post-tretment was begun, > 50% of O⁶-mGua was already removed from cell DNA. Our finding may be accounted for by at least two schemes, one in which nonlethal O⁶-mGua are removed from DNA rapidly, while potentially lethal O⁶-mGua are repaired later. The other scheme proposes that exogenous O⁶-mGua increases the lethality of a non-O⁶-mGua lesion by reducing its repair both in Mer + and Mer − cells. Both schemes are consistent with the hypothesis that O⁶-mGua may be a lethal DNA lesion in human cells.

Chinese hamster ovary cells and human P3 teratocarcinoma cells were exposed to superoxide anion (O₂⁻) generated by the addition of potassium superoxide (KO₂). DNA from the cells was examined by alkaline elution techniques for the production of single-strand breaks, as well as for the production of double-strand breaks and DNA-protein cross-links. It was demonstrated that KO₂ produced only single-strand breaks in DNA in both cell lines, in a dose-dependent manner. The number of breaks was reduced by the prior addition of a metal chelator, indicating that some of the breaks may have been caused by the metal-catalyzed (Fenton reaction) reduction products, hydrogen peroxide or hydroxyl radical. Catalase almost completely inhibited break induction by O₂⁻, evidence for a role of hydrogen peroxide. The results of this study indicate that O₂⁻ and its reduction products can damage intracellular mammalian DNA.

A comparative study of the effects of ultraviolet radiation on three Bacillus subtilis phages is presented. Phages φ29, SPP1 and SPO2c12 or their DNAs were irradiated by UVC (254 nm) and quantum yields for inactivation were calculated. For each phage, the purified DNA was found to be more sensitive than the intact virus when assayed in a uvr⁺ host. The data imply that this is because transfecting DNA is repaired less efficiently than DNA of the intact phage; rather than because of differences in sensitivity to lesion production. Even though φ29 has the smallest target size of the three phages, φ29 and its DNA are the most sensitive. Phages SPO2 and SPP1 code for gene products which complement the repair system of the host. The transfecting DNA of phage SPP1 is extremely sensitive to UV damage when assayed in a uvr⁻ host. This is attributed to the fact that in transfection SPP1 DNA must undergo recombination for productive infection to occur. The recombination process strongly interferes with the repair damaged DNA.