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SMC Complexes pp 181-196 | Cite as

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

  • Koichi Yano
  • Koichiro Akiyama
  • Hironori NikiEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2004)

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.

Key words

Bacterial condensin Chromosome Smc-ScpAB MukBEF Topological loading rDNA 

Notes

Acknowledgments

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

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Microbial Physiology Laboratory, Department of Gene Function and PhenomicsNational Institute of GeneticsMishima, ShizuokaJapan
  2. 2.Department of GeneticsSOKENDAI (The Graduate University for Advanced Studies)Mishima, ShizuokaJapan

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