Inhibition of transcription and strand-specific DNA repair by alpha-amanitin in Chinese hamster ovary cells.
Recent studies have shown preferential repair of UV-induced cyclobutane pyrimidine dimers (CPD) in the transcribed strand of the expressed dihydrofolate reductase (DHFR) gene in human and rodent cells. We have tested the hypothesis that the strand-specific repair of such transcription-blocking lesions is dependent upon concurrent transcription. Chinese hamster ovary (CHO) B11 cells with an amplified DHFR gene were treated with alpha-amanitin before irradiation with UV (254 nm) and during post-irradiation incubation. Nuclear run-off analysis verified inhibition of transcription in the DHFR gene. CsCl density gradient analysis showed that alpha-amanitin at the levels used does not significantly interfere with overall genomic repair replication or semiconservative replication. However, we did observe a dramatic reduction in the removal of CPD from the transcribed strand in the 14 kb KpnI fragment within the DHFR gene in treated cells. We conclude that strand-specific repair of an active gene in CHO cells is dependent upon the activity of the transcribing RNA polymerase. Our results support the model that transcription complexes stalled at CPD signal the repair machinery to achieve efficient repair of the transcribed strand in active genes.
Christians
F C
FC
Department of Biological Sciences, Stanford University, CA 94305-5020.
Hanawalt
P C
PC
D000546:Amanitins / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004260:DNA Repair / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects; D004275:DNA, Ribosomal / Q000235:genetics; D005796:Genes; D006801:Humans; D066298:In Vitro Techniques; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects* / Q000528:radiation effects; D014466:Ultraviolet Rays
D000546:Amanitins; D004275:DNA, Ribosomal; D013762:Tetrahydrofolate Dehydrogenase