Mutation Research/DNA Repair Reports最新文献

Recently, two human DNA-repair genes have been cloned which complement the defects in complementation groups 1 and 2 of the CHO mutants which are sensitive to ultraviolet light and deficient in the incision step of excision repair. Here we report human gene transfer-mediated complementation of a group 4 CHO mutant sensitive to ultraviolet light and mitomycin C (MMC). The transfectants generated by transfecting human DNA into the repair-deficient cell line demonstrate the repair-proficient phenotype, as they have wild-type levels of resistance to UV light and MMC and are competent in performing the incision step of excision erpair in response to UV irradiation. 3 of the 8 transfectants isolated display no detectable human repetitive sequences, while the other 5 contain varying amounts of human repetitive DNA. As the evidence suggests that all of the transfectants are repair-proficient as a result of the uptake of humand DNA, we conclude that the human gene that complements the repair defect in group 4 CHO mutants contains no highly abundant human repetitive sequences. This imposes the necessity of developing cloning strategies involving the identification of sequences that flank the gene.

Excision repair kinetics of UV-induced pyrimidine dimers in DNA of phytohemagglutinin (PHA)-stimulated human peripheral blood lymphocytes were compared to unstimulated lymphocytes using a dimer-specific endonuclease from Micrococcus luteus in conjunction with agarose gel electrophoresis. Removal of pyrimidine dimers could be detected within 6 h after irradiation only PHA-stimulated lypmhocytes. However, incorporation of [³H]thymidine as UV-induced unscheduled DNA synthesis was detected in the unstimulated lymphocytes in the 6-h period. The number of pyrimidine dimers remaining in unstimulated lymphocytes was approximately 85% after 24 h as compared to less than 25% in stimulated cells.

In order to discover whether the nuclear recombinational repair pathway also acts on lesions induced in mitochondrial DNA (mtDNA), the possible role of the RAD50, −51, −52, −55 and −56 genes on the induction of rho⁻ mutants by radiations was studied. Such induction appeared to be independent of this pathway. Nevertheless, an efficient induction of respiration-deficient mutants was observed in γ-irradiated rad52 diploids. We demonstrate that these mutants do not result from a lack of mtDNA repair, but from chromosome losses induced by γ-rays. Such an impairment of the respiratory ability of diploids by chromosome losses was effectively observed in the aneuploid progeny of unirradiated RAD⁺ cdc6 diploids incubated at the restrictive temperature.

A mutant of Salmonella typhimurium with a reduced response to mutation induction by 9-aminoacridine (9AA) has been isolated. The mutation (dam-2) is located in the DNA adenine methylase gene. The dam-2 mutant strain exhibits a level of sensitivity to 2-aminopourine(2AP) intermediate between that of the dam⁺ and the DNA adenine methylation-decifit dam-1 strain, and 2AP sensitivity was reversed by introduction of a muH mutation or the plasmid pMQ148 (which carries a functional Escherichia coli dam⁺ gene). However, the dam-2 strain is not grossly defective in DNA adenine methylase activity. Whole cell DNA appears full methylated at -GATC- sites.

The levels of 9AA required to induced equivalent levels of frameshift mutagnesis in the dam-2 strain were approximately 2-fold higher than for the dam⁺ strain. Introduction of pMQ148 dam⁺ reduced the level of 9AA required for induction of frameshift mutations 4-fold in the dam-2 strain and 2-fold in the dam⁺ strain. The dam-2 mutation had no effect on the levels of ICR191 required for induction of frameshift mutation, but introduction of pMQ148 reduced the ICR191-induced mutagenesis 2-fold. The dam⁺ / pMQ148, dam-2/ pMQ148 and dam-1 / pMQ148 strains showed identical dose-receptor identical dose-response curves for both 9AA and ICR191. These results are consistent with a slightly reduced )(dam-2) or increased (PmQ148) rate of methylation at the replication fork.

The 2AP sensitivity of the dam-2 strain cannot be simply explained. Futhermore, addition of methione to the assay medium reverses the 2AP sensitivity of the dam-2 strain, but has no effect on 9AA mutaganesis.

The repair of DNA alkyalation damage in human cells is poorly understood. We have adapted the alkaline elution technique for use with human peripheral blood lymphocytes in culture. We have also established conditions necessary for short-term culture of human lymphocytes. Lymphocyte growth which can be maintained for up to 30 days is dependent upon irradiated TK6 feeder cells and T-cell growth factor (crude TCGF). The amount of damage induced by a given concentration of methyl methanesulfonate (MMS) is dependent upon cell number per cent ml of growth medium. The DNA damage measured, in lymphocytes, by alkaline elution is a composite of single breaks and alkali-labile lesions. Repair of this damage after appropriate recovery periods is also detectable. The irradiated feeder TK6 cells do not contribute to the number of strand breaks detected or the amount of recovery after treatment. This method offers a quick and reproducible means of detecting DNA damage and repair in human T-lymphocytes.

The UV-sensitive mutant V-B11, isolated from the V79 Chinese hamster cell line (Zdzienicka and Simons, 1987) was further characterized. V-B11 has a slightly increased cross-sensitivity to 3me4NQO, whereas no increased sensitivity towards 4NQO was observed. A slightly increased sensitivity towards EMS and MMS was also found. The mutant shows a defect in the ability to perform the incision step of nucleotide-excision repair after UV irradiation: 2 h after UV exposure, the accumulation of incision breaks in V-B11, in the presence of HU and araC, was about 30% of that found in wild-type V79 cells. V-B11 was crossed to a panel of 6 UV-sensitive Chinese hamster ovary (CHO) cells, which represents all the previously identified 6 complementation groups of UV-sensitive Chinese hamster mutants. Since in all crosses complementation has been observed, V-B11 appears to be the first mutant of a new, 7th, complementation group.

The DNA unwinding technique has been used to measure the induction and repair of DNA strand breaks by X-rays in the X-ray sensitive (xrs 5) mutant and its parent CHO K1 line of Chinese hamster cells. Results show that frequency of induction of DNA strand breaks was the same for both cell lines. The repair of single-strand breaks was found to be slightly slower in xrs 5 over the first 20 min after X-ray exposure, but the level of repair of ssb reached after an incubation of 1h following X-ray exposure in xrs 5 was the same as in CHO K1. Our results also show that the rate of repair of DNA double-strand breaks in xrs 5 cells was clearly slower than that in CHO K1, supporting the conclusion of Kemp et al. (1984) who used the neutral elution technique, that xrs 5 is defective in the repair pathway of DNA double-strand breaks.

A combination of specific rifampicin-resistant (rpo B87) and nalidixic acid-resistant (gyrA87) mutations results in a marked increase in the survival of Escherichia coli against mitomycin C-induced lethality in mutants defective for SOS induction and excision repair. Although the response does not seem to be obligatorily dependent upon the RecA protein, the efficiency is markedly increased in its presence, even in a conventionally inactive form. This response is not elicited against lethality due to ultraviolet radiation or N-methyl-N′-nitro-N-nitrosoguanidine exposure. The combination of rpo B87 and gyrA87 mutations also greatly alleviates post-mitomycin C degradation of DNA under SOS non-inducible conditions. It is proposed that the rpo B subunit of RNA polymerase and gyrA subunit of DNA gyrase could participate in the repair of certain types of DNA damage, such as cross-links, in a mode independent of SOS-regulated excision repair and post-replication repair.

The ultraviolet sensitivity of Potorous tridactylus male kidney (PtK-2) cells is markedly increased by post irradiation treatment for 24 h with 5 μM emetime (which inhibits protein synthesis by acting on the 40S ribosomal subunit), or with 5 μM cycloheximide (which inhibits by interaction with the 60S subunit), or with the RNA polymerase II inhibitor 5,6,-dichloro-1-ß-ribofuranosylbenzimidazole at 50 μM. All 3 treatments give the same sensitivity, while unirradiated cells are little affected. Shortening the time of treatment, or delaying application of the drugs decreases their effect on the same time schedule. Preirradiation of cells, with no drug treatment in the following 8 h, diminishes the sensitivity to a subsequent irradiation with protein synthesis blocked afterwards. Photoreactivation immediately following such preirradiation eliminates its desensitizing effect. Inhibiting protein synthesis after irradiation also markedly reduces the frequency of UV-induced mutants in the surviving population. These facts suggest that gene expression in the period following irradiation facilitates recovery from radiation damage, with an increased probability of mutation, reminiscent of the “SOS response” in Escherichia coli.

Mitomycin C-sensitive mutants MCN 151 (complementation group I) and MCE 50 (complementation group II) derived from mouse lymphoma L5178Y cells were found to be also highly sensitive to the lethal effects of other DNA cross-linking agents, such as photoaddition of 8-methoxypsoralen (8-MOP) and cis-diamminedichloroplatinum II (cis-DDP). They were less sensitive to the monofunctional derivative 3-carbethoxypsoralen (3-CPs) and to trans-DDP than their bifunctional counterparts.

Incorporation levels of labeled 8-MOP or 3-CPs in wild-type cells and 2 mutants were almost the same, indicating that the sensitivity is not caused by differential incorporation of the agents.

The rates of photoinduced mutations to 6-thioguanine resistance in the mutants, per unit dose of 8-MOP, were about 4 times higher for MNC 151 and 3 times higher for MCE 506 than that in L5178Y cells. However, the rates of induced mutations per viable cells in the mutants were nearly equal to those in wild-type cells.

Cross-link repair was compared between mutants and wild-type cells by using the alkaline sucrose-gradient gradient sedimentation technique. The results show that normal cells and both mutants are able to incise the cross-linked DNA, which is the fisrst step of cross-link repair.