amanitins and Xeroderma-Pigmentosum

The ubiquitous process of nucleotide excision repair includes an obligatory step of DNA repair synthesis (DRS) to fill the gapped heteroduplex following excision of a short (approximately 30-nucleotide) damaged single-strand fragment. Using 5-iododeoxyuridine to label repair patches during the first 10-60 min after UV irradiation of quiescent normal human fibroblasts we have visualized a limited number of discrete foci of DRS. These must reflect clusters of elementary DRS patches, since single patches would not be detected. The DRS foci are attenuated in normal cells treated with alpha-amanitin or in Cockayne syndrome (CS) cells, which are specifically deficient in the pathway of transcription-coupled repair (TCR). It is therefore likely that the clusters of DRS arise in chromatin domains within which RNA polymerase II transcription is compartmentalized. However, we also found significant suppression of DRS foci in xeroderma pigmentosum, complementation group C cells in which global genome repair (GGR) is defective, but TCR is normal. This suggests that the TCR is responsible for the DRS cluster formation in the absence of GGR. The residual foci detected in CS cells indicate that, even at early times following UV irradiation, GGR may open some chromatin domains for processive scanning and consequent DRS independent of transcription.

Topics: Amanitins; Cell Nucleus; Cells, Cultured; Chromatin; Chromosomes; Cockayne Syndrome; DNA; DNA Damage; DNA Repair; Fibroblasts; Humans; Idoxuridine; Interphase; Metaphase; Mutation; Nucleic Acid Synthesis Inhibitors; RNA Polymerase II; Transcription, Genetic; Ultraviolet Rays; Xeroderma Pigmentosum

Nucleotide excision repair (NER) mechanism is the major pathway responsible for the removal of a large variety of bulky lesions from the genome. Two different NER subpathways have been identified, i.e. the transcription-coupled and the global genome repair pathways. For DNA-damage induced by ultraviolet light both transcription-coupled repair and global genome repair are essential to confer resistance to cytotoxic effects. To gain further insight into the contribution of NER subpathways in the repair of bulky lesions and in their prevention of biological effects we measured the rate of repair of dG-C8-AF in active and inactive genes in normal human cells, XP-C cells (only transcription-coupled repair) and XP-A cells (completely NER-deficient) exposed to NA-AAF. XP-C cells are only slightly more sensitive to NA-AAF than normal cells and, like normal cells, they are able to recover RNA synthesis repressed by the treatment. In contrast, XP-A cells are sensitive to NA-AAF and unable to recover from RNA synthesis inhibition. Repair of dG-C8-AF in the active ADA gene proceeds in a biphasic way and without strand specificity, with a subclass of lesions quickly repaired during the first 8 h after treatment. Repair in the inactive 754 gene occurs more slowly than in the ADA gene. In XP-C cells, repair of dG-C8-AF in the ADA gene is confined to the transcribed strand and occurs at about half the rate of repair seen in normal cells. Repair in the inactive 754 gene in XP-C cells is virtually absent. Consistent with these results we found that repair replication in XP-C is drastically reduced when compared with normal cells and abolished by alpha-amanitin indicating that the repair in XP-C cells is mediated by transcription-coupled repair only. Our data suggest that dG-C8-AF is a target for transcription-coupled repair and that this repair pathway is the main pathway or recovery of RNA synthesis inhibition conferring resistance to cytotoxic effects of NA-AAF. In spite of this, repair of dG-C8-AF in active genes in normal cells by transcription-coupled repair and global genome repair is not additive, but dominated by global genome repair. This indicates that the subset of lesions which are capable of stalling RNA polymerase II, and are, therefore, a substrate for TCR, are also the lesions which are very efficiently recognized by the global genome repair system.

Topics: Acetoxyacetylaminofluorene; Amanitins; Carcinogens; Cell Line; DNA Adducts; DNA Damage; DNA Repair; Fibroblasts; Humans; Kinetics; RNA; Transcription, Genetic; Xeroderma Pigmentosum

Induction of p53 in u.v.-irradiated primary human fibroblasts was monitored by immunostaining and Western blotting. Minimum u.v. doses required for induction of nuclear accumulation of p53 (minimum response dose: MRD) were estimated in various cells with different DNA repair capacities. The MRD in repair deficient xeroderma pigmentosum (XP) group A cells is eightfold lower than in normal cells, indicating that nuclear accumulation of p53 is related to DNA repair capacity. Cells from patients with another u.v.-sensitive disorder, Cockayne syndrome (CS), which have normal repair capacity for the overall genome but have a specific defect in preferential repair of lesions in active genes, have the same low MRD as of XP-A cells. Furthermore, the MRD in XP-C cells, which have normal preferential repair but have defects in overall genome repair, is as high as that of normal cells. DNA damage induced by X-ray is repaired at similar rates in normal, XP and CS cells. In contrast to u.v.-irradiation, the minimum dose of X-rays that induces nuclear accumulation of p53 is the same in these cells. Inhibition of transcription with alpha-amanitin evokes nuclear accumulation of p53 both in normal cells and in XP cells. These results strongly suggest that u.v.-induced nuclear accumulation of p53 is evoked through DNA damage of actively transcribed genes. Nuclear accumulation of p53 is observed in any phase of the cell cycle at both low and high u.v. doses.

Topics: Amanitins; Cell Cycle; Cell Nucleus; Cells, Cultured; Cockayne Syndrome; DNA; DNA Damage; DNA Repair; Fibroblasts; Humans; Transcription, Genetic; Tumor Suppressor Protein p53; Ultraviolet Rays; Xeroderma Pigmentosum

alpha-Amanitin, an inhibitor of RNA polymerase II, has little effect on either UV-induced incision or repair synthesis in cultured normal human fibroblasts but almost completely inhibits both processes in xeroderma pigmentosum group C fibroblasts. Cycloheximide, at a concentration which inhibits protein synthesis by 75-80%, has no effect on incision or repair synthesis in either cell type, which argues that the effects of alpha-amanitin on repair occur at the level of transcription. Cot analysis demonstrates that UV-induced repair synthesis occurs at similar levels in highly repetitive, middle repetitive and single copy sequence in both normal and xeroderma group C cells. We conclude that normal cells must have at least two excision repair pathways for repair of UV-induced damage, one dependent on transcription and the other independent.

Topics: Amanitins; Cells, Cultured; Cycloheximide; DNA; DNA Repair; Humans; Repetitive Sequences, Nucleic Acid; Transcription, Genetic; Ultraviolet Rays; Xeroderma Pigmentosum