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-alk ylpurines 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 regula tory 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 relate d to proficient repair of nitrogen mustard-induced alkylation products in the actively transcribed dihydrofolate reductase gene in the Chine se hamster ovary B11 cells. Nuclear run-off transcription analysis ver ified that alpha-amanitin completely and selectively inhibited transcr iption by RNA polymerase II. At the drug exposure examined, nitrogen m ustard induced DNA damage capable of a complete transcription terminat ion in the RNA polymerase II-transcribed dihydrofolate reductase gene and reduced 28S rDNA transcription by a factor of 7.9. The transcripti on activity did partially recover following reincubation in drug-free medium; this recovery was about 34 and 76% of ribosomal 28S gene trans cripts and dihydrofolate reductase gene transcripts, respectively, aft er 6 h of repair incubation. Alpha-amanitin significantly inhibited th e removal of nitrogen mustard-induced N-alkylpurines in the 5'-half of the essential, constitutively active dihydrofolate reductase gene, wh ile no effect of alpha-amanitin was observed on the lesion removal fro m a noncoding region 3'-flanking to the gene and from the genome overa ll. In the actively transcribed gene region, about 77% of N-alkylpurin es 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 alph a-amanitin treatment. From these results we suggest that gene-specific repair of nitrogen mustard-induced N-alkylpurines is dependent on ong oing activity of the transcribing RNA polymerase II. The findings are discussed in terms of the current ideas about the mechanism of prefere ntial DNA repair.