Differential control of transcription-induced and overall DNA supercoiling by eukaryotic topoisomerases in vitro.
The global superhelical state of intracellular DNA is stringently controlled by topoisomerase action. Little is know, however, about topoisomerase-directed relaxation of localized DNA supercoiling generated by protein tracking processes such as transcription. Here we use transcription by a yeast Gal4 and phage T7 RNA polymerase fusion protein to induce localized supercoiling which, in turn, triggers site-specific DNA recombination by gamma delta resolvase. We demonstrate that only large amounts of eukaryotic topoisomerase I interfere, through supercoiling relaxation, with the topological coupling between transcription and recombination. The additional presence of a strong cleavage site for topoisomerase I has little influence on the relaxation of localized supercoiling. We also show that high levels of human topoisomerase II fail to compete with transcription-driven recombination. However, drastically reduced amounts of either enzyme completely suppress recombination of overall supercoiled DNA. Together, our results reveal a marked difference in topoisomerase requirement to relax transcription-induced and global DNA supercoiling. We discuss possible reasons for this difference and conclude that localized supercoiling frequently may escape relaxation by eukaryotic topoisomerases to mediate topological couplings between DNA transactions.
Wang
Z
Z
Department of Biology, University of Konstanz, Germany.
Dröge
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P
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004254:DNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics* / Q000378:metabolism*; D004268:DNA-Binding Proteins; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014158:Transcription, Genetic; D019895:Transposases; D014764:Viral Proteins
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D004254:DNA Nucleotidyltransferases; D019895:Transposases; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D004264:DNA Topoisomerases, Type I