A homologous set of plasmids expressing tet, lacY, and melB, genes encoding integral cytoplasmic membrane proteins, and tWlC and ampC, genes encoding proteins for export through the cytoplasmic membrane, was constructed for studying the effects of transcription and translation of such genes on the hypernegative supercoiling of plasmids in Escherichia coli cells deficient in DNA topoisomerase I. The results support the view that intracellular bacterial DNA is anchored to the cytoplasmic membrane at many points through cotranscriptional synthesis of membrane proteins or proteins designated for export across the cytoplasmic membrane; in the latter case, the presence of the signal peptide appears to be unnecessary for cotranscriptional membrane association.The phenomenon of hypernegative supercoiling of plasmids in Escherichia coli topA mutants lacking DNA topoisomerase I was first reported by Pruss (37). The widely used cloning vector pBR322 isolated from topA null mutants was found to exhibit an extremely heterogenous distribution in its linking number, with a large fraction of the topoisomers more than twice as negatively supercoiled as the same plasmid isolated from isogenic topA+ strains. This topAdependent hypernegative supercoiling is plasmid specific: topoisomers of pUC19, a shortened derivative of pBR322, exhibit only minor differences in their linking numbers when isolated from isogenic topA mutant and topA+ strains. Dissection of the pair of plasmids pBR322 and pUC19 led Pruss and Drlica (38) to conclude that transcription of tet, the gene encoding the tetracycline resistance marker in pBR322, is necessary for the hypernegative supercoiling of the plasmid in the absence of DNA topoisomerase I; various deletions within the tet region also show that a functional product of the gene is not necessary for the phenomenon.The findings of Pruss and Drlica (38) provided a key experimental link between transcription and DNA supercoiling. Theoretical considerations on a plausible relation between transcription and DNA mechanics, however, can be traced two decades back. The idea that transcription might require a swivel in the DNA template to facilitate the rotation of the DNA relative to the RNA polymerase was first discussed in the 1960s (29; see also reference 13). With the discovery of E. coli DNA topoisomerase I in 1971, then known as the o protein (52), the possibility that this enzyme is involved in transcription was raised in this connection (53). More recently, it was postulated that a highly negatively supercoiled loop might form in the DNA template when the RNA polymerase is in contact with a templatebound regulatory protein (54).In 1987, Liu and Wang (25) proposed a twin-supercoiled-* Corresponding author.domain model of transcriptional supercoiling to account for all known experimental findings, including both the hypernegative supercoiling phenomenon described above and the observation of Lockshon and Morris (26) that inhibition of DNA gyrase in E. coli leads to the formation of highly positively superc...

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A set of carboxy-terminal deletion mutants of Saccharomyces cerevisiae DNA topoisomerase II were constructed for studying the functions of the carboxyl domain in vitro and in vivo. The wild-type yeast enzyme is a homodimer with 1,429 amino acid residues in each of the two polypeptides; truncation of the C terminus to Ile-1220 has little effect on the function of the enzyme in vitro or in vivo, whereas truncations extending beyond Gln-1138 yield completely inactive proteins. Several mutant enzymes with C termini in between these two residues were found to be catalytically active but unable to complement a top2-4 temperature-sensitive mutation. Immunomicroscopy results suggest that the removal of a nuclear localization signal in the C-terminal domain is likely to contribute to the physiological dysfunction of these proteins; the ability of these mutant proteins to relax supercoiled DNA in vivo shows, however, that at least some of the mutant proteins are present in the nuclei in a catalytically active form. In contrast to the ability of the catalytically active mutant proteins to relax supercoiled intracellular DNA, all mutants that do not complement the temperaturedependent lethality and high frequency of chromosomal nondisjunction of top2-4 were found to lack decatenation activity in vivo. The plausible roles of the DNA topoisomerase II C-terminal domain, in addition to providing a signal for nuclear localization, are discussed in the light of these results. ). In vitro, the DNA transport reaction catalyzed by the yeast enzyme is manifested in interconversions between topological isomers of DNA rings: relaxation of positively and negatively supercoiled DNA, catenation and decatenation, and knot formation and removal. In vivo, the enzyme and its homologs have been shown to unlink multiply intertwined pairs of newly replicated DNA rings (18) and to remove positive and negative supercoils generated by transcription (24). Whereas the supercoiling-relaxation function of the enzyme can be carried out by DNA topoisomerase I, the unlinking of multiply intertwined DNA rings or chromosomal loops without nicks or gaps is a unique function of the type II enzyme; thus, inactivation of the type II enzyme invariably leads to cell inviability (25, 59), which is presumably a consequence of the failure of intertwined pairs of chromosomes to segregate properly during mitosis (18,29,60).In Schizosaccharomyces pombe cells lacking DNA topoisomerase II, chromosome condensation is also blocked (58). The involvement of the enzyme in chromosome condensation is supported by in vitro experiments with cell extracts (3,43) and by results on the time dependence of the cellular localization of the enzyme in Drosophila embryos undergoing synchronized cell division (7, 56). The precise role of the enzyme in chromosomal condensation, namely, whether the enzyme enables or facilitates the process through its catalysis of DNA breakage, passage, and rejoining or participates as a stoichiometric component of the nucleoprotein product, is not known. Th...

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