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Identification of Barriers to Rotation of DNA Segments in Yeast from the Topology of DNA Rings Excised by an Inducible Site-Specific Recombinase
Marc R. Gartenberg and James C. Wang
Proceedings of the National Academy of Sciences of the United States of America
Vol. 90, No. 22 (Nov. 15, 1993), pp. 10514-10518
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2363257
Page Count: 5
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Controlled excision of DNA segments to yield intracellular DNA rings of well-defined sequences was utilized to study the determinants of transcriptional supercoiling of closed circular DNA in the yeast Saccharomyces cerevisiae. In Δ top1 top2ts strains of S. cerevisiae expressing Escherichia coli DNA topoisomerase I, accumulation of positive supercoils in intracellular DNA normally occurs upon thermal inactivation of DNA topoisomerase II because of the simultaneous generation of positively and negatively supercoiled domains by transcription and the preferential relaxation of the latter by the bacterial enzyme. Positive supercoil accumulation in DNA rings is shown to depend on the presence of specific sequence elements; one likely cause of this dependence is that the persistence of oppositely supercoiled domains in an intracellular DNA ring requires the presence of barriers to rotation of the DNA segments connecting the domains. Analysis of the S. cerevisiae 2-μm plasmid partition system by this approach suggests that the plasmid-encoded REP1 and REP2 proteins are involved in forming such a barrier in DNA containing the REP3 sequence.
Proceedings of the National Academy of Sciences of the United States of America © 1993 National Academy of Sciences