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In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA

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SMC Complexes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2004))

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Abstract

Condensins play essential roles in the compaction and segregation of chromosomal DNA in life forms ranging from bacteria to higher organisms. To elucidate the molecular mechanisms underlying these roles, it is crucial to determine how and where condensins are loaded to chromosomal DNA. Here, we describe in vivo and in vitro assays for monitoring the topological loading of two bacterial condensins, Smc-ScpAB and MukBEF. A key step in these assays is washing the samples with a high concentration of salt in order to discriminate between electrostatic and topological binding of the bacterial condensins to DNA. In addition, isolation of bacterial condensin and DNA complexes prevents any undesired interaction between them due to cross-linking reagents. These methodologies provide reproducible and reliable results for the loading of topologically bound proteins such as bacterial condensins.

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Acknowledgments

This work was supported by JSPS KAKENHI Grants JP18H02485 and JP18K14627.

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Authors and Affiliations

  1. Microbial Physiology Laboratory, Department of Gene Function and Phenomics, National Institute of Genetics, Mishima, Shizuoka, Japan

    Koichi Yano, Koichiro Akiyama & Hironori Niki

  2. Department of Genetics, SOKENDAI (The Graduate University for Advanced Studies), Mishima, Shizuoka, Japan

    Hironori Niki

Authors
  1. Koichi Yano
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  2. Koichiro Akiyama
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  3. Hironori Niki
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Corresponding author

Correspondence to Hironori Niki .

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Editors and Affiliations

  1. National Centre for Biological Sciences, Tata Institute of Fundamental Research (TIFR), Bangalore, India

    Anjana Badrinarayanan

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Yano, K., Akiyama, K., Niki, H. (2019). In Vivo and In Vitro Assay for Monitoring the Topological Loading of Bacterial Condensins on DNA. In: Badrinarayanan, A. (eds) SMC Complexes. Methods in Molecular Biology, vol 2004. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9520-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-9520-2_14

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9519-6

  • Online ISBN: 978-1-4939-9520-2

  • eBook Packages: Springer Protocols

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