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Sedimentation Analysis of Bacterial Nucleoid Structure

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DNA Topoisomerase Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 94))

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Abstract

The physiology of bacterial DNA topoisomerases can be studied by examining how perturbation of intracellular enzyme activities affects the structure of extracted nucleoids. Since the few DNA nicks that occur when nucleoids are isolated (1,2) are localized by the presence of 50–100 barriers to strand rotation (2,3), it is possible to recover chromosomal DNA in which most of each molecule is topologically constrained (2,4). Consequently, intracellular changes in topoisomerase activity can be detected as differences in the average supercoiling of nucleoids isolated from cells perturbed in different ways. This general strategy has been used to show that supercoiling is relaxed by inhibition of gyrase (5-7) and that it is increased (becomes more negative) by point mutations in topA (the gene encoding topoisomerase I), by low concentrations of gyrase inhibitors, and by anaerobic conditions (6,8-10). Experiments of this type have contributed to the conclusions that (1) supercoiling is controlled in part by regulated expression of the gyrase and topoisomerase I genes, and (2) the overall level of supercoiling responds to growth environment (reviewed in [11]).

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Author information

Authors and Affiliations

  1. Public Health Research Institute, New York, NY, USA

    Karl Drlica & Samuel Kayman

  2. Department of Biology, New York University, New York, NY, USA

    Chang-Rung Chen

Authors
  1. Karl Drlica
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  2. Chang-Rung Chen
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  3. Samuel Kayman
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Editor information

Editors and Affiliations

  1. St. Jude Children’s Research Hospital, Memphis, TN, USA

    Mary-Ann Bjornsti

  2. Vanderbilt University School of Medicine, Nashville, TN, USA

    Neil Osheroff

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© 1999 Humana Press Inc.

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Drlica, K., Chen, CR., Kayman, S. (1999). Sedimentation Analysis of Bacterial Nucleoid Structure. In: Bjornsti, MA., Osheroff, N. (eds) DNA Topoisomerase Protocols. Methods in Molecular Biology, vol 94. Humana, Totowa, NJ. https://doi.org/10.1385/1-59259-259-7:87

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  • DOI: https://doi.org/10.1385/1-59259-259-7:87

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  • Publisher Name: Humana, Totowa, NJ

  • Print ISBN: 978-0-89603-444-0

  • Online ISBN: 978-1-59259-259-3

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