IDENTIFICATION OF PROCESSES THAT INFLUENCE NEGATIVE SUPERCOILING IN THE HUMAN C-MYC GENE

DNA elements with sequences suitable for Z-DNA formation are found fre quently at various positions in chromatin. Z-DNA formation in these se quences depends lamely 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 occurri ng Z-DNA formation. Previously we identified three sequence elements i n the human c-myc gene that adopt the Z-DNA conformation in the transc ribed gene. The three elements are found far upstream (Z1), close to t he main transcription start site (Z2) and in the first intron (Z3). He re 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 i nhibitor of transcription, reduced the persistence of Z-DNA in all thr ee elements. Aphidicolin, an inhibitor of replication, increased the p ersistence of Z-DNA in one element without significantly influencing t he other two elements. When camptothecin an inhibitor of topoisomerase I was added in the presence of alpha-amanitin, the persistence of Z-D NA was extended in all three elements. However, in the presence of aph idicolin no effect of camptothecin on Z-DNA formation was observed. (C ) 1997 Elsevier Science B.V.