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[en] 7 strains of human primary fibroblasts were chosen from the complementation groups A through G of xeroderma pigmentosum; these strains are UV-sensitive and deficient in excision repair of UV damage on the criterion of unscheduled DNA synthesis (UDS). They were compared with normal human fibroblasts and one xeroderma pigmentosum variant with regard to their capacity to remove pyrimidine dimers, induced in their DNA by UV at 253.7 nm. The XP variant showed a normal level of dimer removal, whereas 6 of the other XP strains had a greatly reduced capacity to remove this DNA damage, in agreement with their individual levels of UDS. Strain XP23OS (complementation group F), however, only showed a 20% reduction in the removal of dimers, which is much less than expected from the low level of UDS in this strain. (Auth.)

[en] Cultured human and embryonic chick fibroblasts possess different enzyme-mediated processes to repair cyclobutyl pyrimidine dimers induced in their deoxyribonucleic acid (DNA) by ultraviolet (uv) radiation. While dimers are corrected in human cells by excision repair, a photoenzymatic repair process exists in embryonic chick cells for the removal of these potentially deleterious uv photoproducts. We have utilized a sensitive enzymatic assay to monitor the disappearance, i.e., repair, of dimer-containing sites in fused populations of human and chick cells primarily consisting of multinucleate human/chick heterokaryons. Fused cultures were constructed such that uv photoproducts were present only in chick DNA when evaluating excision repair and only in human DNA when evaluating photoenzymatic repair. Based on the kinetics of site removal observed in these cultures we are led to conclude the following: Within heterokaryons per se the photoreactivating enzyme derived from chick nuclei and at least one excision-repair enzyme (presumably a uv endonuclease) derived from human nuclei act on uv-damaged DNA in foreign nuclei with an efficiency equal to that displayed toward their own nuclear DNA. Hence, after cell fusion these chick and human repair enzymes are apparently able to diffuse into foreign nuclei and once therein competently attack uv-irradiated DNA independently of its origin. In harmony with the situation in nonfused parental cultures, in heterokaryons the chick photoenzymatic repair process rapidly removed all dimer-containing sites from human DNA including the residual fraction normally acted upon slowly by the human excision-repair process. (U.S.)

[en] A sensitive enzymatic assay has been utilized to monitor repair of uv-induced damage to DNA in primary human and embryonic chick cells in multinucleate heterokaryons artificially derived from both. The assay exploits the unique ability of a purified repair endonuclease to attack uv-irradiated DNA at sites containing pyrimidine dimers. These nuclease-susceptible sites are subsequently observed as single-strand scissions by velocity sedimentation in alkaline sucrose gradients. Incubation of uv-damaged cultures followed by extraction and enzymatic analysis of the radioactively labeled DNA enables one to trace the disappearance of such sites in vivo and hence to monitor endogenous repair activity. When uv-irradiated human cells are incubated in the dark, the curve for site removal exhibits a two-phase exponential decline; i.e., there exists a fast component responsible for elimination of 60 percent of the initial damage and a second one approximately 7 times slower in rate. The removal of sites is not further enhanced by exposing cells to blacklight during post-uv incubation. Conversely, uv-damaged chick cells rid their DNA of all nuclease-susceptible sites rapidly (i.e., at an exponential rate approximately 13 times faster than the fast component of site removal in human cells) when incubated under blacklight but not when kept in the dark. These data indicate the presence in human and embryonic chick cells of distinct enzymatic mechanisms for the elimination of dimer-containing sites. Whereas human fibroblasts rely heavily on a light-independent process, excision-repair, chick fibroblasts possess a light-dependent mechanism, presumably photoenzymatic repair

[en] The induction and removal of UV-induced photolesions was investigated in confluent human fibroblasts employing two different approaches. Photolyase and highly purified E. coli Uvr A,B and C proteins were used to measure the frequency of 6-4 photoproducts (6-4PP) in transcriptionally active and inactive genes. At a UV-dose range of 20-60 J/m² 6-4PP were induced at about 4-fold lower frequency then cyclobutane pyrimidine dimers (CPD). In normal cells exposed to 30J/m² 6-4PP were induced at about 4-fold lower frequency then cyclobutane pyrimidine dimers (CPD). In normal cells exposed to 30J/m², the repair of 6-4PP was very rapid in both active and inactive genes when compared to CPD removal. No strand specific repair of 6-4PP in active genes was observed, although repair of 6-4PP occurred preferentially in the active genes when compared to inactive X-chromosomal genes. In xeroderma pigmentosum group C cells 6-4PP were selectively removed from the transcribed strand of active genes. In these cells the kinetics of repair of CPD and 6-4PP from the transcribed strand of active genes was very similar. Our results indicate that 6-4PP can be removed by a transcription coupled repair pathway, but that repair of 6-4PP by the global repair system is much more efficient. The sam conclusion can be drawn from studies aimed to determine BUdR labelled repair sites in genomic fragments. The results of these studies indicate a lack of strandspecific repair of 6-4 PP in active genes in normal cells at a relative low UV-dose of 10J/m²

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[en] Treatment of mammalian cells with buthionine sulphoximine (BSO) or diethyl maleate (DEM) results in a decrease in the intracellular GSH (glutathione) and NPSH (non-protein-bound SH) levels. The effect of depletion of GSH and NPSH on radiosensitivity was studied in relation to the concentration of oxygen during irradiation. Single- and double-strand DNA breaks (ssb and dsb) and cell killing were used as criteria for radiation damage. Under aerobic conditions, BSO and DEM treatment gave a small sensitization of 10-20% for the 3 types of radiation damage. Also under severely hypoxic conditions (0.01 μM oxygen in the medium) the sensitizing effect of both compounds on the induction of ssb and dsb and on cell killing was small (0-30%). At somewhat higher concentrations of oxygen (0.5-10 μM) however, the sensitization amounted to about 90% for the induction of ssb and dsb and about 50% for cell killing. These results strengthen the widely accepted idea that intracellular SH-compounds compete with oxygen and other electron-affinic radiosensitizers with respect to reaction with radiation-induced damage, thus preventing the fixation of DNA damages by oxygen. These results imply that the extent to which SH-compounds affect the radiosensitivity of cells in vivo depends strongly on the local concentration of oxygen. (Auth.)

No abstract available