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


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 



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


  1. 1.
    Löwe J, Cordell SC, van den Ent F (2001) Crystal structure of the SMC head domain: an ABC ATPase with 900 residues antiparallel coiled-coil. J Mol Biol 306:25–35CrossRefGoogle Scholar
  2. 2.
    Lammens A, Schele A, Hopfner KP (2004) Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases. Curr Biol 14:1778–1782CrossRefGoogle Scholar
  3. 3.
    Schleiffer A, Kaitna S, Maurer-Stroh S et al (2003) Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners. Mol Cell 11:571–575CrossRefGoogle Scholar
  4. 4.
    Palecek JJ, Gruber S (2015) Kite proteins: a superfamily of SMC/kleisin partners conserved across bacteria, archaea, and eukaryotes. Structure 23:2183–2190CrossRefGoogle Scholar
  5. 5.
    Niki H, Jaffé A, Imamura R et al (1991) The new gene mukB codes for a 177 kd protein with coiled-coil domains involved in chromosome partitioning of E. coli. EMBO J 10:183–193CrossRefGoogle Scholar
  6. 6.
    Moriya S, Tsujikawa E, Hassan AKM et al (1998) A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition. Mol Microbiol 29:179–187CrossRefGoogle Scholar
  7. 7.
    Mascarenhas J (2002) Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein. EMBO J 21:3108–3118CrossRefGoogle Scholar
  8. 8.
    Soppa J, Kobayashi K, Noirot-Gros MF et al (2002) Discovery of two novel families of proteins that are proposed to interact with prokaryotic SMC proteins, and characterization of the Bacillus subtilis family members ScpA and ScpB. Mol Microbiol 45:59–71CrossRefGoogle Scholar
  9. 9.
    Hirano T (2016) Condensin-based chromosome organization from bacteria to vertebrates. Cell 164:847–857CrossRefGoogle Scholar
  10. 10.
    Bürmann F, Shin HC, Basquin J et al (2013) An asymmetric SMC–kleisin bridge in prokaryotic condensing. Nat Struct Mol Biol 20:371–379CrossRefGoogle Scholar
  11. 11.
    Zawadzka K, Zawadzki P, Baker R et al (2018) MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin. elife 7:1941CrossRefGoogle Scholar
  12. 12.
    She W, Mordukhova E, Zhao H et al (2012) Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF. Mol Microbiol 87:539–552CrossRefGoogle Scholar
  13. 13.
    Gruber S, Veening JW, Bach J et al (2014) Interlinked sister chromosomes arise in the absence of condensin during fast replication in B. subtilis. Curr Biol 24:293–298CrossRefGoogle Scholar
  14. 14.
    Wang X, Le TBK, Lajoie BR et al (2015) Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis. Genes Dev 29:1661–1675CrossRefGoogle Scholar
  15. 15.
    Hirano M, Hirano T (1998) ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer. EMBO J 17:7139–7148CrossRefGoogle Scholar
  16. 16.
    Niki H, Imamura R, Kitaoka M et al (1992) E. coli MukB protein involved in chromosome partition forms a homodimer with a rod-and-hinge structure having DNA binding and ATP/GTP binding activities. EMBO J 11:5101–5109CrossRefGoogle Scholar
  17. 17.
    Sutani T, Yanagida M (1997) DNA renaturation activity of the SMC complex implicated in chromosome condensation. Nature 388:798–801CrossRefGoogle Scholar
  18. 18.
    Wilhelm L, Bürmann F, Minnen A et al (2015) SMC condensin entraps chromosomal DNA by an ATP hydrolysis dependent loading mechanism in Bacillus subtilis. eLife 4:11202CrossRefGoogle Scholar
  19. 19.
    Niki H, Yano K (2016) In vitro topological loading of bacterial condensin MukB on DNA, preferentially single-stranded DNA rather than double-stranded DNA. Sci Rep 6:595CrossRefGoogle Scholar
  20. 20.
    Britton RA, Lin DC, Grossman AD (1998) Characterization of a prokaryotic SMC protein involved in chromosome partitioning. Genes Dev 12:1254–1259CrossRefGoogle Scholar
  21. 21.
    Ohsumi K, Yamazoe M, Hiraga S (2001) Different localization of SeqA-bound nascent DNA clusters and MukF–MukE–MukB complex in Escherichia coli cells. Mol Microbiol 40:835–845CrossRefGoogle Scholar
  22. 22.
    Sullivan NL, Marquis KA, Rudner DZ (2009) Recruitment of SMC by ParB-parS organizes the origin region and promotes efficient chromosome segregation. Cell 137:697–707CrossRefGoogle Scholar
  23. 23.
    Wang X, Brandão HB, TBK L et al (2017) Bacillus subtilis SMC complexes juxtapose chromosome arms as they travel from origin to terminus. Science 355:524–527CrossRefGoogle Scholar
  24. 24.
    Gruber S, Errington J (2009) Recruitment of condensin to replication origin regions by ParB/SpoOJ promotes chromosome segregation in B. subtilis. Cell 137:685–696CrossRefGoogle Scholar
  25. 25.
    McGhee JD, Von Hippel PH (1977) Formaldehyde as a probe of DNA structure. 3. Equilibrium denaturation of DNA and synthetic polynucleotides. Biochemistry 16:3267–3276CrossRefGoogle Scholar
  26. 26.
    Waldminghaus T, Skarstad K (2010) ChIP on Chip: surprising results are often artifacts. BMC Genomics 11:414CrossRefGoogle Scholar
  27. 27.
    Murayama Y, Uhlmann F (2013) Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505:367–371CrossRefGoogle Scholar
  28. 28.
    Yano K, Niki H (2017) Multiple cis-acting rDNAs contribute to nucleoid separation and recruit the bacterial condensin Smc-ScpAB. Cell Rep 21:1347–1360CrossRefGoogle Scholar

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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