Mutation Research/DNA Repair Reports最新文献

Sodium arsenite at a non-toxic concentration was found to inhibit strongly mutagenesis induced by ultraviolet light (UV), 4-nitroquinoline-1-oxide (4NQO), furylfuramide (AF-2) and methyl methanesulfonate (MMS) as well as spontaneous mutation in the reversion assay of E. coli WP2uvrA/pKM101. The effect was not, however, seen in the case of the mutagenesis induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG).

In order to elucidate the mechanism of the mutation-inhibitory effect of sodium arsenite, its action on umuC gene expression and DNA-repair systems was investigated. It was found that sodium arsenite depressed β-galactosidase induction, corresponding to the umuC gene expression. For UV-irradiated E. coli strains possessing different DNA-repair capacities, sodium arsenite decreased the UV survival rates of WP2, WP2uvrA[uvrA] and WP67[uvrA polA], increased those of SOS-uninducible strains having either the recA⁺ or uvrA⁺ such as CM571 [recA], CM561 [lexA(Ind⁻)] and CM611[uvrA lexA (Ind⁻], and did not affect that of the uvrA recA double mutant, WP100.

From these results, we assume that sodium arsenite may have at least two roles in its antimutagenesis: as an inhibitor of umuC gene expression, and as an enhancer of the error-free repairs depending on the uvrA and recA genes.

Normal human fibroblasts treated with r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) yielded DNA polymerase alpha with elevated levels of activity, incorporated [³H]thymidine as a function of unscheduled DNA synthesis, and exhibited restoration of normal DNA-strand length as a function of unscheduled DNA synthesis. Lipoprotein-deficient fibroblasts treated with BPDE did not show elevated levels of DNA polymerase alpha activity, exhibited minimal [³H]thymidine incorporation, and had fragmented DNA after 24 h of repair in the absence of lipoprotein or phosphatidylinositol supplementation. When DNA polymerase beta activity was inhibited, cells with normal lipoprotein uptake exhibited [³H]thymidine incorporation into BPDE-damaged DNA but did not show an increase in DNA-strand length. DNA polymerase alpha activity and [³H]thymidine incorporation in lipoprotein-deficient fibroblasts increased to normal levels when the cells were permeabilized and low-density lipoproteins or phosphatidylinositol were introduced into the cells. DNA polymerase alpha isolated from normal human fibroblasts, but not from lipoprotein-deficient fibroblasts, showed increased specific activity after the cells were treated with BPDE. When BPDE-treated lipoprotein-deficient fibroblasts were permeabilized and ³²P-ATP was introduced into the cells along with lipoproteins, ³²P-labeled DNA polymerase alpha with significantly increased specific activity was isolated from the cells. These data suggest that treatment of human fibroblasts with BPDE initiates unscheduled DNA synthesis, as a function of DNA excision repair, which is correlated with increased activity of DNA polymerase alpha, and that increased DNA polymerase alpha activity may be correlated with phosphorylation of the enzyme in a reaction that is stimulated by low-density lipoprotein or by the lipoprotein component, phosphatidylinositol.

Mechanisms for induction of single-strand scissions in DNA by S9-activated benzo[a]pyrene (B[a]P) and their repair in cultured Chinese hamster V79 cells were investigated with inhibitors of DNA-repair synthesis using alkaline sucrose gradient sedimentation analysis.

The marked induction of single-strand scissions in DNA was observed following 3 h treatment of V79 cells with 5 μg/ml of B[a]P. These DNA lesions were repaired to the control level within 4 h after removal of B[a]P. The simultaneous addition of inhibitors of DNA-repair synthesis, 1-β-D-arabinofuranosylcytosine (araC) plus hydroxyurea with B[a]P did not increase the formation of DNA single-strand scissions. When these inhibitors were added after removal of B[a]P, however, they significantly blocked the rejoining of DNA-strand scissions. On the other hand, when aphidicolin, a specific inhibitor of DNA polymerase α, was used instead of araC, a partial inhibition of the rejoining was observed, and further addition of 2′,3′-dideoxythymidine, an inhibitor of DNA polymerase β, augmented the inhibitory effect. These results indicate that B[a]P-induced single-strand scissions of DNA in V79 cells could be repaired mostly by excision repair which involved DNA polymerase α and a non-α polymerase, presumably polymerase β.

The colony-forming ability of 10 normal human fibroblast cell strains and of 10 strains representing 3 degenerative diseases of either nerve or muscle cells was determined after exposure of the cells to X-rays or β-particles from tritiated water. Both methods of irradiation yielded similar comparative results. The fibroblast strains from the 5 Usher's syndrome patients and from 1 of the 2 Huntington's disease patients were hypersensitive to radiation, while those from the 3 Duchenne muscular dystrophy patients and the second Huntington's disease patient had normal sensitivity to radiation. These results indicate both disease-specific and strain-specific differences in the survival of fibroblasts after exposure to ionizing radiation.

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of β-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.

By making use of the temperature-sensitive mutant recB270, we showed that the RecBCD enzyme is needed for repair between 1 and 4 h after UV exposure. recB-dependent prophage inactivation (Petranović et al. (1984), Mol. Gen. Genet., 196, 167–169) takes place in all dying cells during the same period of time. The kinetics of decrease in the yield of recombinants in phage-propage crosses resemble those of prophage inactivation in UV-irradiated bacteria. This indicates that recombination processes (including site-specific recombination required for prophage excision) are blocked in cells destined to die. On the basis of our results, we suggest that a large fraction of damaged cells is rescued by the RecA−RecBCD recombination pathway. If repair is unsuccessful, RecA−RecBCD recombination intermediated persist in the irradiated cells leading to prophage inactivation.

The rep-38 and mmrA1 mutations are located very close to each other (∼85 min), and have been suggested to be allelic. To address this question we have compared the phenotypes of the mmrA1 and rep-38 mutants. Both the mmrA1 and rep-38 mutations blocked the enhanced killing and inhibition of postreplication repair by rich growth medium that occurs in UV-irradiated Escherichia coli K-12 uvrA cells, i.e., the mmrA1 and rep-38 strains did not show minimal medium recovery (MMR). However, ΦX174 bacteriophage propagated well in mmrA1 strains, but not in rep-38 strains; a rep mutation sensitized a uvrA strain to UV irradiation, but a mmrA mutation did not. During chloramphenicol treatment, the rep-38 strain showed a larger amount of residual DNA synthesis than observed in the mmrA1 strain. The mmrA1 mutation appears to be a dominant mutation. This was determined by the failure of either plasmid pLC44-7 or episome F′KLF11, both of which carry the mmrA⁺ gene, to complement the Mmr⁻ phenotype of a uvrA mmrA strain. Plasmid pLC44-7 is known to complement the rep-38 mutation, suggesting that rep-38 is a recessive mutation. Although certain of the phenotypes of the rep and mmrA mutants are similar, a number are quite different. These differences suggest that these two mutations are not allelic.

The mutagenic potential of 9-[(3-dimethylaminopropyl)amino]-acridine and its 1-, 2-, 3- and 4-nitro derivatives was studied in several strains of Salmonella typhimurium carrying the frameshift marker hisC3076. The strains all carried deep rough (rfa) mutations, and were either wild-type with respect to DNA repair capacity or carried recA, uvrB, polA1 or polA3 (amber) mutations. Derivatives with and without plasmid pKM101 were also studied.

The des-nitro compound resembled 9 aminocridine and other simple intercalating compounds. Both toxicity and mutagenesis were apparently unaffected by the uvrB and recA mutations or by the presence of plasmid pKM101. However, mutagenicity was reduced by the polA1 mutation, and virtually eliminated by the polA3 mutation. The drug was substantially more toxic in the latter, slightly more toxic in the former, of these polA⁻ strains. Plasmid pKM101 enhanced mutagenesis and protected from toxicity in both polA1⁻ and polA3⁻ strains, although it did not restore either of these parameters to the level in the wild-type strain. The 2-nitro compound was generally similar to the des-nitro compound, except that it was considerably more toxic and apparently non-mutagenic in the recA-bearing strain. By contrast, mutagenicity of the 3- and 4-nitro compounds was enhanced by the uvrB mutation and by the presence of the plasmid. These compounds were highly toxic but non-mutagenic in the recA⁻ strain, and showed some increased toxicity in polA1⁻ and polA3⁻ strains.

The 1-nitro compound has been previously found to cross-link DNA. Unlike well-characterised cross-linkers such as mitomycin C it was highly mutagenic in the uvrB⁻ strain, and this mutagenesis was enhanced by plasmid pKM101, but eliminated by the recA mutation. At high doses, where the drug was completely toxic towards uvrB⁻ or recA-carrying strains, it became mutagenic in the DNA-repair-proficient strains. This ‘high-dose’ mutagenesis was enhanced by plasmid pKM101, but reduced by the polA1 mutation and almost eliminated by the polA3 mutation. Although there are several possible interpretations of these data, they are compatible with the suggestion that the lesion induced by high doses (but not by low doses) of nitracine is a cross-link, but that this is not the major mutagenic lesion.

Exposure to UV light is known to produce lesions that block DNA polymerases at least on the leading strand. If several lesions are present in adjacent replicons, it is likely that sections of DNA would remain unreplicated because of the presence for blocking lesions. For cells to multiply and survive these areas must eventually be replicated. One mechanism that has been postulated to be involved in the replication of DNA between two blocking lesions is the activation of alternative sites of replicon initiation.

To detect the existence of alternative sites of replicon initiation we employed the high specific/low specific activity labelling protocol first used by Huberman and Riggs (1968) for DNA fiber autoradiography. After development of the autoradiographs, the distances between adjacent sites of replicon initiation (inter-origin distances) were measured. In both wild-type Chinese hamster ovary (CHO) cells and UV-5 CHO cells, which exhibit no excision repair abilities, the inter-origin distances were, on average, shorter in cells exposed to UV, indicating that exposure to UV results in the activation of alternative sites of initiation. This activation appears to occur immediately after UV in both cell lines, but persist for a longer time in the excision-deficient line.