Rapid induction of c-fos transcription reveals quantitative linkage of RNA polymerase II and DNA topoisomerase I enzyme activities.
The functional association between DNA topoisomerase I and gene activity has been analyzed using the tightly regulated c-fos proto-oncogene, which undergoes rapid transitions between active and inactive states of transcription. We show that the topoisomerase I inhibitor camptothecin can be used to measure topoisomerase I activity throughout the transcription cycle of the c-fos gene. Upon induction of c-fos transcription in the presence of camptothecin, topoisomerase I cleavages spread through the gene in the 5' to 3' direction and concomitantly transcriptional elongation is retarded. Parallel kinetic measurements of RNA polymerase II activity and topoisomerase I activity demonstrate a quantitative and temporal link between the two enzymes. Our results argue that topoisomerase I quantitatively relieves the torsional consequences of transcriptional elongation in intact cells.
Stewart
A F
AF
Institut für Zell-und Tumorbiologie, Deutsches Krebsforschungszentrum im Neuenheimer Feld 280, Heidelberg, Federal Republic of Germany.
Herrera
R E
RE
Nordheim
A
A
D000818:Animals; D015152:Blotting, Northern; D000001:Calcimycin / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos; D011519:Proto-Oncogenes / Q000187:drug effects; D012319:RNA Polymerase II / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D013950:Thymus Gland / Q000201:enzymology; D014158:Transcription, Genetic
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D000001:Calcimycin; D011505:Protein-Tyrosine Kinases; D012319:RNA Polymerase II; D004264:DNA Topoisomerases, Type I; D002166:Camptothecin; D004121:Dimethyl Sulfoxide