THE SENSITIVITY OF HUMAN FIBROBLASTS TO N-ACETOXY-2-ACETYLAMINOFLUORENE IS DETERMINED BY THE EXTENT OF TRANSCRIPTION-COUPLED REPAIR, AND ORTHEIR CAPABILITY TO COUNTERACT RNA-SYNTHESIS INHIBITION/

Nucleotide excision repair (NER) mechanism is the major pathway respon sible for the removal of a large variety of bulky lesions from the gen ome, Two different NER subpathways have been identified, i.e. the tran scription-coupled and the global genome repair pathways, For DNA-damag e induced by ultraviolet light both transcription-coupled repair and g lobal genome repair are essential to confer resistance to cytotoxic ef fects, To gain further insight into the contribution of NER subpathway s 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 inac tive genes in normal human cells, XP-C cells (only transcription-coupl ed 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 ce lls and, like normal cells, they are able to recover RNA synthesis rep ressed by the treatment, In contrast, XP-A cells are sensitive to NA-A AF and unable to recover from RNA synthesis inhibition. Repair of dG-C 8-AF in the active ADA gene proceeds in a biphasic way and without str and specificity, with a subclass of lesions quickly repaired during th e first 8 h after treatment, Repair in the inactive 754 gene occurs mo re slowly than in the ADA gene, In XP-C cells, repair of dG-C8-AF in t he 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 7 54 gene in XP-C cells is virtually absent, Consistent with these resul ts we found that repair replication in XP-C is drastically reduced whe n compared with normal cells and abolished by alpha-amanitin indicatin g that the repair in XP-C cells is mediated by transcription-coupled r epair only, Our data suggest that dG-C8-AF is a target for transcripti on-coupled repair and that this repair pathway is the main pathway or recovery of RNA synthesis inhibition conferring resistance to cytotoxi c effects of NA-AAF. In spite of this, repair of dG-C8-AF in active ge nes in normal cells by transcription-coupled repair and global genome repair is not additive, but dominated by global genome repair, This in dicates that the subset of lesions which are capable of stalling RNA p olymerase II, and are, therefore, a substrate for TCR, are also the le sions which are very efficiently recognized by the global genome repai r system.