Ongoing activity of RNA polymerase II confers preferential repair of nitrogen mustard-induced N-alkylpurines in the hamster dihydrofolate reductase gene.
Recently, it has been demonstrated that nitrogen mustard-induced N-alkylpurines are excised rapidly from actively transcribing genes, while they persist longer in noncoding regions and in the genome overall. It was suggested that transcriptional activity is implicated as a regulatory element in the efficient removal of lesions. By treating cells or not with the transcription inhibitor alpha-amanitin, we have explored whether ongoing activity of RNA polymerase II was coordinately related to proficient repair of nitrogen mustard-induced alkylation products in the actively transcribed dihydrofolate reductase gene in the Chinese hamster ovary B11 cells. Nuclear run-off transcription analysis verified that alpha-amanitin completely and selectively inhibited transcription by RNA polymerase II. At the drug exposure examined, nitrogen mustard induced DNA damage capable of a complete transcription termination in the RNA polymerase II-transcribed dihydrofolate reductase gene and reduced 28S rDNA transcription by a factor of 7.9. The transcription activity did partially recover following reincubation in drug-free medium; this recovery was about 34 and 76% of ribosomal 28S gene transcripts and dihydrofolate reductase gene transcripts, respectively, after 6 h of repair incubation. alpha-Amanitin significantly inhibited the removal of nitrogen mustard-induced N-alkylpurines in the 5'-half of the essential, constitutively active dihydrofolate reductase gene, while no effect of alpha-amanitin was observed on the lesion removal from a noncoding region 3'-flanking to the gene and from the genome overall. In the actively transcribed gene region, about 77% of N-alkylpurines were removed 21 h following drug exposure of cells not treated with alpha-amanitin and about 47% in 21 h in alpha-amanitin treated cells. The global semiconservative replication seemed unaffected by the alpha-amanitin treatment. From these results we suggest that gene-specific repair of nitrogen mustard-induced N-alkylpurines is dependent on ongoing activity of the transcribing RNA polymerase II. The findings are discussed in terms of the current ideas about the mechanism of preferential DNA repair.
Wassermann
K
K
Department of Toxicology and Biology, National Institute of Occupational Health, Copenhagen, Denmark.
Damgaard
J
J
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair / Q000187:drug effects*; D008466:Mechlorethamine / Q000494:pharmacology*; D012313:RNA / Q000096:biosynthesis*; D012319:RNA Polymerase II / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects*
D000546:Amanitins; D008466:Mechlorethamine; D012313:RNA; D013762:Tetrahydrofolate Dehydrogenase; D012319:RNA Polymerase II