Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I

The level of DNA supercoiling is crucial for many cellular processes, including gene expression, and is determined, primarily, by the opposing actions of two enzymes: topoisomerase I and DNA gyrase. Escherichia coli strains lacking topoisomerase I (topA mutants) normally fail to grow in the absence of compensatory mutations which are presumed to relax DNA. We have found that, in media of low osmolarity, topA mutants are viable in the absence of any compensatory mutation, consistent with the view that decreased extracellular osmolarity causes a relaxation of cellular DNA. At higher osmolarity most compensatory mutations, as expected, are in the gyrA and gyrB genes. The only other locus at which compensatory mutations arise, designated toc, is shown to involve the amplification of a region of chromosomal DNA which includes the tolC gene. However, amplification of tolC alone is insufficient to explain the phenotypes of toc mutants. tolC insertion mutations alter the distribution of plasmid topoisomers in vivo. This effect is probably indirect, possibly a result of altered membrane structure and an alteration in the cell's osmotic barrier. As tolC is a highly pleiotropic locus, affecting the expression of many genes, it is possible that some of the TolC phenotypes are a direct result of this topological change. The possible relationship between toc and tolC mutations, and the means by which tolC mutations might affect DNA supercoiling, are discussed.

Section: Discussionmentioning

confidence: 99%

Section: Genetie Evidenee That Toe Mutations Involve a Dna Amplifieationmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the tolC locus

Dorman

Lynch

Bhriain

et al. 1989

“…Bacterial strains and growth conditions The E. coli strain HB101 (supE44 hsdS20(r B ¹ m B ¹ ) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1) (Bolivar et al, 1977) and the topA mutant RED31 (Hfr thi-1 rel-1 lac-42 acrA topA20 ::Tn10 toc-1) (Raji et al, 1985) were used in this study. As E. coli cells lacking a functional TopA are not viable without a compensatory mutation (DiNardo et al, 1982;Pruss et al, 1982), the topA strain RED31 also contains a compensatory mutation, toc-1 (topoisomerase one compensatory) which involves an amplification of the parC and parE genes that encode DNA topoisomerase IV (Dorman et al, 1989;Kato et al, 1990).…”

Section: Methodsmentioning

confidence: 99%

Localized domains of DNA supercoiling: topological coupling between promoters

Mojica

Higgins

1996

According to the twin-supercoiled domain model, a local domain with a high level of DNA supercoiling can be generated between two divergently transcribed promoters. We have tested this model directly using the rate of DNA photo-cross-linking by 4,5', 8-trimethylpsoralen to measure local regions of unconstrained supercoiling in vivo. We demonstrate that, in plasmids, a localized domain of highly supercoiled DNA is generated upstream of the tetA promoter. The level of supercoiling of this domain depends on the activity of the tetA promoter and on the extent of translation of the tetA gene product. The highest level of supercoiling within this localized domain was observed in a topA strain, although the generation of a localized supercoiling domain could also be detected in a topA+ background, consistent with potential physiological significance. The level of supercoiling in this localized domain correlated with activation of the supercoiling-sensitive leu-500 promoter located upstream of tetA. These data provide a direct demonstration that localized domains of increased negative supercoiling can be generated upstream of an actively transcribed promoter, and that this can result in supercoiling-mediated transcriptional coupling between two promoters.

“…Because DNA gyrase and Topo I have opposing effects on DNA superhelicity, changes in the expression or activity of either Topo I or DNA gyrase are offset by corresponding changes in the other topoisomerase if the cell is to maintain a level of negative supercoiling that is optimal for growth. Thus, partial or complete loss of Topo I activity by mutations in the topA gene leads to reduced DNA gyrase activity, either by downregulation of the supercoiling-sensitive promoters of gyrA and gyrB (Menzel and Gellert, 1983;Miller and Simons, 1993) or through compensatory mutations in the GyrA and GyrB subunits (DiNardo et al ., 1982;Raji et al ., 1985) respectively.…”

Section: Introductionmentioning

confidence: 99%

Tus‐mediated arrest of DNA replication in Escherichia coli is modulated by DNA supercoiling

Valjavec-Gratian

Henderson

Hill

2005

SummaryIn the absence of RecA, expression of the Tus protein of Escherichia coli is lethal when ectopic Ter sites are inserted into the chromosome in an orientation that blocks completion of chromosome replication. Using this observation as a basis for genetic selection, an extragenic suppressor of Tus-mediated arrest of DNA replication was isolated with diminished ability of Tus to halt DNA replication. Resistance to tus expression mapped to a mutation in the stop codon of the topA gene ( topA869 ), generating an elongated topoisomerase I protein with a marked reduction in activity. Other alleles of topA with mutations in the carboxylterminal domain of topoisomerase I, topA10 and topA66 , also rendered recA strains with blocking Ter sites insensitive to tus expression. Thus, increased negative supercoiling in the DNA of these mutants reduced the ability of Tus-Ter complexes to arrest DNA replication. The increase in superhelical density did not diminish replication arrest by disrupting TusTer interactions, as Tus binding to Ter sites was essentially unaffected by the topA mutations. The topA869 mutation also relieved the requirement for recombination functions other than recA to restart replication, such as recC, ruvA and ruvC , indicating that the primary effect of the increased negative supercoiling was to interfere with Tus blockage of DNA replication. Introduction of gyrB mutations in combination with the topA869 mutation restored supercoiling density to normal values and also restored replication arrest at Ter sites, suggesting that supercoiling alone modulated Tus activity.We propose that increased negative supercoiling enhances DnaB unwinding activity, thereby reducing the duration of the Tus-DnaB interaction and leading to decreased Tus activity.