Identification of processes that influence negative supercoiling in the human c-myc gene.
DNA elements with sequences suitable for Z-DNA formation are found frequently at various positions in chromatin. Z-DNA formation in these sequences depends largely on the level of local negative supercoiling. We can use binding of a Z-DNA specific antibody at low concentrations in metabolically active permeabilized nuclei to detect naturally occurring Z-DNA formation. Previously we identified three sequence elements in the human c-myc gene that adopt the Z-DNA conformation in the transcribed gene. The three elements are found far upstream (Z1), close to the main transcription start site (Z2) and in the first intron (Z3). Here we measure the persistence of Z-DNA at these three sites under the influence of various metabolic inhibitors. This provides some insight into the varying levels of negative supercoiling. alpha-Amanitin, an inhibitor of transcription, reduced the persistence of Z-DNA in all three elements. Aphidicolin, an inhibitor of replication, increased the persistence of Z-DNA in one element without significantly influencing the other two elements. When camptothecin an inhibitor of topoisomerase I was added in the presence of alpha-amanitin, the persistence of Z-DNA was extended in all three elements. However, in the presence of aphidicolin no effect of camptothecin on Z-DNA formation was observed.
Wölfl
S
S
Massachusetts Institute of Technology, Department of Biology, Cambridge, USA.
Wittig
B
B
Dorbic
T
T
Rich
A
A
D000546:Amanitins / Q000494:pharmacology; D016590:Aphidicolin / Q000494:pharmacology; D002166:Camptothecin / Q000494:pharmacology; D002460:Cell Line; D004256:DNA Polymerase I / Q000037:antagonists & inhibitors; D004278:DNA, Superhelical / Q000096:biosynthesis* / Q000737:chemistry; D016259:Genes, myc; D006801:Humans; D008968:Molecular Conformation; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping
D000546:Amanitins; D004278:DNA, Superhelical; D016590:Aphidicolin; D004256:DNA Polymerase I; D002166:Camptothecin