Open AccessIdentification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase.
Author(s) -
Marc R. Gartenberg,
James C. Wang
Publication year - 1993
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.90.22.10514
Subject(s) - dna supercoil , topoisomerase , saccharomyces cerevisiae , dna , circular bacterial chromosome , dna clamp , biology , plasmid , protein–dna interaction , in vitro recombination , recombinase , microbiology and biotechnology , dna replication , biophysics , biochemistry , yeast , molecular cloning , transcription factor , gene , dna binding protein , peptide sequence , recombination , reverse transcriptase , rna
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 delta 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-microns 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.