Mutation Research/DNA Repair Reports最新文献

The damage-directed strand incision step in the nucleotidyl DNA excision-repair pathway (NDERP) was characterized in quiescent monolayer cultures of human fibroblasts in which the plasma membrane was selectively permeabilized with saponin. When permeable normal human fibroblasts (NHF) were incubated in a DNA-repair assay mixture lacking the deoxyribonucleoside triphosphate precursors, the numbers of UV-dependent DNA-strand breaks were increased by about 9-fold consistent with the uncoupling of incision from gap-filling DNA synthesis and ligation. In uncoupled NHF omission of ATP reduced the numbers of UV-dependent strand breaks by 84% confirming the requirement for ATP for reparative strand incision. Time-course experiments indicated that the maximum rate of strand incision occurred in the first 10 min of incubation of permeable cells and diminished to 16–28% of this rate between 30 and 60 min of incubation. The initial rate of incision in permeable NHF was estimated to be 20% of that seen in intact fibroblasts. Dose-response studies indicated an initial saturation of strand incision activity at fluences between 10 and 25 J/m². In permeable group A xeroderma pigmentosum fibroblasts (XPA) few UV-dependent incisions were produced after 10–25 J/m². In the xeroderma pigmentosum variant (XPV) strain that we studied, strand incisions saturated at a plateau level that was about twice that seen in the NHF strain suggesting the preservation of a higher level of incision activity after permeabilization. After fluences above 50 J/m² additional strand incision was observed in all cell strains reflecting the activity of a damage-dependent endodeoxyribonuclease that is independent of the NDERP. Saponin-treated fibroblasts were also permeable to pancreatic deoxyribonuclease I and the UV-DNA endonuclease from M. luteus indicating that these preparations may be used for in vitro complementation.

Thus far, our studies in Drosophila have concentrated primarily on the various enzymes involved in the in vitro repair of modified or nonconventional DNA substrates. In some cases, our findings have led us to investigate events that may not have a bearing on DNA repair, but rather may be associated with developmental signals important to the maturation of the organism. As appealing as some of these models seem, however, they must await confirmation through detailed genetic studies before any substantial conclusions can be drawn. This combination of genetic and biochemical knowledge makes Drosophila an exciting organism for an eventual detailed understanding of the developmental expression and cellular location of DNA-repair systems.

In recB recC sbcB mutants genetic recombination is dependent upon the recF gene. recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background. Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background. In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages. In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101. These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4′-hydroxy-methyl-4,5′,8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S₁ nuclease digestion of ³H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.

Fibroblasts obtained from biopsied material and lymphocytes from patients wwith homocystinuria were studied for repair activity using the criterion of repair of DNA breaks induced by 4-nitroquinoline 1-oxide and γ-irradiation and criteria of reactivation and induced mutagenesis of vaccinia virus. Lymphocytes showed defective DNA repair for all these criteria. In fibroblast cultures, the inhibition of cell-repair activity for the γ-type was retained. The number of spontaneous and γ-induced virus mutations increased as passaging of fibroblasts proceeded.

The occurence of a RecA-like activity similar to the one detected in the fibroblast cell line GM1492 derived from a patient suffering from the autosomal recessive disease Bloom's syndrome has been investigated in cell extracts of different origin. The formation of D-loop containing joint molecules from ΦX174 RFI DNA and fragments of ΦX174 single-stranded DNA by partially purified extracts was measured by a filter binding assay.

The RecA-like activity, dependent on ATP and Mg²⁺, was detected at an elevated level only in the human and rodent cell lines, GM1492 and CHO respectively. The level of activity in DNA-cellulose-purified cell extracts from these cell lines was 4–7-fold higher compared to normal human fibroblasts. Low levels of activity were also detected in extracts from two additional Bloom's syndrome fibroblast cell lines, Fanconi's anemia fibroblasts, virus-(Epstein-Barr virus, Simian virus 40) transformed human cells and human placenta. Cell extracts from rat testis, spleen and calf thymus were also of low activity.

The fate of 8-methoxypsoralen (8-MOP)-photoinduced DNA interstrand crosslinks was followed by alkaline elution in Fanconi's anemia (FA) fibroblasts belonging to complementation groups A (FA 150 and FA 402) and B (FA 145) in comparison to a normal (1 BR/3) and a heterozygote (F 311) cell line. Clonogenic cell survival to 8-MOP photoaddition was established in parallel for all cell lines. In comparison to normal cells, group A FA cells demonstrated a higher photosensitivity than group B cells (sensitivity index 2.3 and 1.5, respectively), the heterozygote cell line being only slightly more sensitive.

FA cells from both groups A and B demonstrated an incision capacity of crosslinks, the kinetics and extent of which being, however, different from that of normal or heterozygote cells. The incision is slower in FA cells and, at 24 h of post-treatment incubation, the amount of crosslinks incised is clearly lower than that observed in normal cells for group A cells, whereas in group B cells incision approaches the level of normal cells. These results correlate with survival as well as with rates of DNA semi-conservative synthesis after 8-MOP photoaddition.

Sensitivities of several murine cell strains to killing by the DNA cross-linking agents 1-(2-chloroethyl)-1-nitrosourea (CNU), cis-diamminedichloroplatinum (II) (Cis-Pt) and mitomycin C (MMC) were measured by post-treatment colony-formation. Virally-transformed murine cells were usually more sensitive to cell killing by these agents than were the parental 3T3 cell strains. The hypersensitivity to CNU of some virally-transformed murine cell strains correlated well with the reduced ability to repair O⁶-methylguanine (O6mGua), a phenomenon similar to that in human cells. The loss of ability to repair O6mGua, as well as the increased sensitivity of transformed strains to cell killing, may not be due to a mutation but rather due to a change of gene expression associated with transformation by viruses or activation of oncogenes.

Pyrimidine dimer-DNA glycosylase activity prepared from Micrococcus luteus has been used to develop an enzyme-sensitive site assay for the detection and quantification of closely opposed pyrimidine dimers in the nuclear DNA of UV-irradiated yeast. With this assay, closely opposed dimers were found to be induced as a linear function of dose from 0 to 200 J/m² (254 nm). Closely opposed dimer frequencies decreased during the incubation of UV-irradiated, excision repair-proficient cells under liquid-holding conditions in the dark and during post-irradiation exposure of excision-deficient cells to photoreactivating light. Incubation of excision-deficient cells in the dark had no effect on the frequency of closely opposed dimers for up to 16 h. These results indicate that closely opposed dimers in UV-irradiated yeast are subject to repair by enzymatic photoreactivation and/or by dark-repair processes dependent, at least in part, upon functions necessary for normal excision repair. The genetic and biochemical implications of these results are discussed.

Fibroblast cells from two Japanese patients with Bloom's syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.