PubMed · 6265097
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G R Smith. 1981. DNA supercoiling: another level for regulating gene expression.. https://doi.org/10.1016/0092-8674(81)90085-4
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leu-500 is a chromosomal promoter mutation in Salmonella typhimurium that normally causes the promoter to be inactive in the initiation of RNA synthesis. But in a strain that has mutations in topA, the gene encoding DNA topoisomerase I, the mutant promoter becomes active. We show that the leu-500 promoter can function on a plasmid when it is adjacent to the tetracycline-resistance gene tetA. Activation of the leu-500 promoter requires that the tetA gene is transcribed and translated and that the host cell is topA. We propose that the A----G mutation in the -10 region of the leu-500 promoter is compensated by local negative supercoiling arising from transcription of the tetA gene, which may reach elevated levels in a topA background, provided that diffusional dissipation is reduced due to anchoring of the TetA peptide in the membrane. This is a clear example of the modulation of the activity of a promoter by the activity of another promoter in cis, when they can be coupled through the topology of the template.
Purified human DNA topoisomerase I was assayed quantitatively by enzyme titrations with supercoiled pHC624 DNA in the presence of 0-2.0 microM doxorubicin. Supercoiled and relaxed DNAs were resolved by agarose gel electrophoresis in the presence of ethidium bromide, and the percentage of conversion of supercoiled DNA to relaxed DNA was quantified by scanning microdensitometry. The inhibition of DNA topoisomerase I activity was measured at varying concentrations of doxorubicin. Doxorubicin inhibited enzyme activity at an IC50 value (the concentration required to inhibit 50% of the total activity) of 0.8 microM. Similar inhibition was observed for daunomycin, a structurally related anthracycline antitumor drug. These results indicate that anthracyclines inhibit human DNA topoisomerase I activity at concentrations that cause DNA damage and cytotoxicity in vivo.
The maintenance of partition-defective (Par-) mini-P1 and mini-F plasmids was studied in topA strains of Escherichia coli, which are defective in topoisomerase I activity. The partition defects were substantially but not completely suppressed in broth-grown cultures. This suppression was not due to a large increase in copy number. However, the absolute number of copies of Par- mini-P1 plasmids per average dividing cell is sufficiently high to account for the modest stability observed if a random distribution of the copies to daughter cells is assumed. The similar number of Par- plasmid copies in wild-type cells are distributed in a considerably worse-than-random fashion. Thus, it is unnecessary to propose, as was suggested previously, that an active, par-independent pathway operates in topA strains to ensure proper segregation of the plasmids to daughter cells. Rather, it seems likely that the lack of topoisomerase I activity aids the random distribution of the partition-defective plasmids, perhaps by facilitating their separation after replication. The results of studies carried out at reduced growth rates were consistent with this view; when topA cells containing Par- mini-P1 plasmids were cultured in minimal medium, in which the copy number of the plasmids per average cell is sharply reduced, very little suppression of the partition defect was observed.