Mutation Research/DNA Repair Reports最新文献

The ionising radiation-sensitive mutants irs 1, irs 2, irs 3, xrs-1 (or xrs-7, EM7 and XR-1 were fused to wild-type cells or to each other in pairs to create hybrid cells. These hybrids were checked chromosomally and their X-ray sensitivity tested. Each mutant was found to be recessive to wild-type and to complement the X-ray sensitivity of the other mutants. Thus there appear to be at least 6 complementation groups for ionising radiation sensitivity in Chinese hamster cells.

A dodecadeoxynucleotide of defined sequence containing O⁴-methylthymine was labeled at the 5′ end with [³²P] by the reaction with (γ-³²P]ATP and polynucleotide kinase. Extracts prepared from bacterial and mammalian sources such as the human cell lines, HeLa and HT29, and rat liver were incubated with the labeled, methylated dodecamer to determine the extent of repair of the lesion. The labeled, demethylated dodecamer was separated from the labeled methylated dodecamer on a reverse-phase column using a shallow methanol gradient. There was complete repair of O⁴-methylthymine by the E. coli alkyltransferase upon incubation for 4 h at 37°C. There was no detectable amount of demethylated product formed upon incubation with HeLa or HT29 cell extract for the same incubation period. There was also no repair of the O⁴-methylthymine lesion in the presence of crude rat-liver extract. However, the rat-liver extract alone degraded the methylated substrate completely, and the assay had to be conducted in the presence of NaF, AMP and unlabeled, nonmethylated dodecamer to prevent this. The results obtained from this assay, which is at least an order of magnitude more sensitive than previous methods, are in agreement with previous results that the mammalian alkyltransferase is specific for O⁶-alkylguanine repair.

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304–5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560–1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin.

Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313–319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.

The human DNA-excision repair gene ERCC-1 is cloned by its ability to correct the excision-repair defect of the ultraviolet light- and mitomycin-C-sensitive CHO mutant cell line 43-3B. This mutant is assigned to complementation group 2 of the excision-repair-deficient CHO mutants. In order to establish whether the correlation by ERCC-1 is confined to CHO mutants of one complementation group, the cloned repair gene, present on cosmid 34–43, was transfected to representative cell lines of the 6 complementation groups that have been identified to date. Following transfection, mycophenolic acid was used to select for transferants expressing the dominant marker gene Ecogpt, also present on cosmid 34–43. Cotransfer of the ERCC-1 gene was shown by Southern blot analysis of DNA from pooled (500–2000 independent colonies) transformants of each mutant. UV survival and UV-induced UDS showed that only mutants belonging to complementation group 2 and no mutants of other groups were corrected by the ERCC-1 gene. This demonstrates that ERCC-1 does not provide an aspecific bypass of excision-repair defects in CHO mutants and supports the assumption that the complementation analysis is based on mutations in different repair genes.

When pSV2-gpt or pSV2-neo plasmids are introduced into human cells by calcium phosphate coprecipitation, the yield of stable transformants (Gpt⁺ or Neo⁺) is increased by irradiating the respective plasmid DNA in vitro with UV (254) nm. To identify specific lesions that can increase the transforming activity of plasmids in human cells we examined pSV2 plasmids containing different types of damage. Of the lesions tested, cyclobutane pyrimidine dimers produced the greatest increase, and can nearly fully account for the effect of 254 nm UV on transformation. The enhancement of transformation produced by UV was not altered by the additional treatment of the plasmid DNA with T4 endonuclease V, an enzyme that nicks DNA specifically at pyrimidine dimers. Treatment of plasmid DNA with osmium tetroxide to produce thymine glycols, or with acid and heat to produce apurinic sites did not affect transformation frequency. The enhancement occurred in all the human cell lines tested, whether they contained or not sequences homologous to those in the plasmids, and was independent of the repair capacity of the recipient cells.

The capacity of a variety of human fibroblasts to incise DNA following exposure to far ultraviolet-light is determined from the rate of single-strand DNA break accumulation in the presence of DNA synthesis inhibitors. We have quantitated incision, one of the early steps in the UV excision repair pathway, in cells form normal, xeroderma pigmentosum groups C, D, G, H and variant individuals, and in the parents of one XPA patient. On the basis of the estimated initial rates of incision the different XP cells examined in this work can be marked as follows: XP variant $\text{⋙}\text{XPH}\text{>}\text{XPD}\text{>}\text{XPC}\text{>}\text{XPG}\text{>}\text{XPA}$. In each cell strain breaks accumulate immediately after irradiation over a range of 0.5–20 Jm⁻² with the exception of the XPC strain examined, where there is an initial delay of 15 min. The rate of incision in XPA heterozygote cells is roughly half that of normal fibroblasts. Analysis of the kinetics of break accumulation over short intervals after irradiation permits estimation of the apparent enzymatic parameters, K_m and V_max, for the incision step. The approximate values of K_m and V_max for normal and XP variant are similar while for the heterozygotes of an XPA individual K_m values are normal (around1 Jm⁻²), but there is only half the amount of normal enzyme activity. XPD and H cells express low levels of active enzyme, between 5 and 15% of that of the normal, but while the K_m of KPH is very similar to that of normal cells, that of two XPD strains examined is between 2- and 3-fold higher.

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m², maximal incorporation of [α³⁵S]dNTPs occurred at a lysolecithin concentration (∼ 80 μg/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the tottal cell population incorporated significant levels of ³⁵S into DNA. This result presumably reflected the fact that ∼ 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (< 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent K_m for all 4 dNTPs was 50–100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [³H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.

Ade⁻C is a Chinese hamster ovary cell line auxotrophic for purines because of a mutation in the de novo synthetic pathway. We now show that, in the absence of exogenous hypoxanthine, replicative DNA synthesis is rapidly shut down. Various aspects of DNA repair have been studied in purine-starved cells. Incision, the first step of excision repair of UV damage, appears normal, as do the later steps, repair synthesis (demonstrated following chemical damage as well as UV-irradiation) and ligation. However, removal of UV-induced pyrimidine dimers is not detected, and it seems that the repair that occurs is aberrant. This behaviour is associated with an increase in cell killing by UV light, and a several-fold increase in the frequency of mutations induced by UV.

DNA adduct formation and induction of mutations at gene loci, hypoxanthine-guanine-phosphoribosyltranferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O⁶-ethylguanine (O⁶-EtGua) and O⁶-ethylthymine (O⁴-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TG^r) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus.

When, however, the same analysis was applied to ouabain-resistant (our^r) mutants, it was found that, at similar levels of O⁶-EtGua and O⁴-EtThy, DES induced many more oua^r mutants than ENU. This result supports the notion that primary DNA lesions other than O⁶-EtGua and O⁴-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.

DNA endonuclease activities from the chromatin of normal human and xeroderma pigmentosum, complementing group A (XPA), lymphoblastoid cells were examined on DNA treated with 8-methoxy-psoralen (8-MOP) or 4,5′,8-trimethylpsoralen (TMP) plus long wavelength ultraviolet (UVA) light, which produce monoadducts and DNA interstrand cross-links, and angelicin plus UVA light, which produces mainly monoadducts. 9 chromatin-associated DNA endonuclease activities were isolated from normal and XPA cells and assayed for activity on PM2 bacteriophage DNA that had been treated with 8-MOP or TMP in the dark and then exposed to UVA light. Unbound psoralen was removed by dialysis and a second dose of UVA light was given. Cross-linking of DNA molecules was confirmed by alkaline gel electrophoresis. In both normal and XPA cells, two DNA endonuclease activities were found which were active on 8-MOP and TMP plus UVA light treated DNA. One of these endonuclease activities, pI 4.6, is also active on intercalated DNA and a second one, pI 7.6, is also active on UVC (254 nm) light irradiated DNA. The major activity against angelicin plus UVA light treated DNA in both normal and XPA cells was found in the fraction, pI 7.6. The levels of activity of both of these fractions on all 3 psoralen-damaged DNAs were similar between normal and XPA cells. These results indicate that in both normal and XPA cells there are at least two different DNA endonucleases which act on both 8-MOP and TMP plus UVA light treated DNA.