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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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 306:25–35
Lammens A, Schele A, Hopfner KP (2004) Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases. Curr Biol 14:1778–1782
Schleiffer A, Kaitna S, Maurer-Stroh S et al (2003) Kleisins: a superfamily of bacterial and eukaryotic SMC protein partners. Mol Cell 11:571–575
Palecek JJ, Gruber S (2015) Kite proteins: a superfamily of SMC/kleisin partners conserved across bacteria, archaea, and eukaryotes. Structure 23:2183–2190
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 10:183–193
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 29:179–187
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 21:3108–3118
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 45:59–71
Hirano T (2016) Condensin-based chromosome organization from bacteria to vertebrates. Cell 164:847–857
Bürmann F, Shin HC, Basquin J et al (2013) An asymmetric SMC–kleisin bridge in prokaryotic condensing. Nat Struct Mol Biol 20:371–379
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 7:1941
She W, Mordukhova E, Zhao H et al (2012) Mutational analysis of MukE reveals its role in focal subcellular localization of MukBEF. Mol Microbiol 87:539–552
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 24:293–298
Wang X, Le TBK, Lajoie BR et al (2015) Condensin promotes the juxtaposition of DNA flanking its loading site in Bacillus subtilis. Genes Dev 29:1661–1675
Hirano M, Hirano T (1998) ATP-dependent aggregation of single-stranded DNA by a bacterial SMC homodimer. EMBO J 17:7139–7148
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 11:5101–5109
Sutani T, Yanagida M (1997) DNA renaturation activity of the SMC complex implicated in chromosome condensation. Nature 388:798–801
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 4:11202
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 6:595
Britton RA, Lin DC, Grossman AD (1998) Characterization of a prokaryotic SMC protein involved in chromosome partitioning. Genes Dev 12:1254–1259
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 40:835–845
Sullivan NL, Marquis KA, Rudner DZ (2009) Recruitment of SMC by ParB-parS organizes the origin region and promotes efficient chromosome segregation. Cell 137:697–707
Wang X, Brandão HB, TBK L et al (2017) Bacillus subtilis SMC complexes juxtapose chromosome arms as they travel from origin to terminus. Science 355:524–527
Gruber S, Errington J (2009) Recruitment of condensin to replication origin regions by ParB/SpoOJ promotes chromosome segregation in B. subtilis. Cell 137:685–696
McGhee JD, Von Hippel PH (1977) Formaldehyde as a probe of DNA structure. 3. Equilibrium denaturation of DNA and synthetic polynucleotides. Biochemistry 16:3267–3276
Waldminghaus T, Skarstad K (2010) ChIP on Chip: surprising results are often artifacts. BMC Genomics 11:414
Murayama Y, Uhlmann F (2013) Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505:367–371
Yano K, Niki H (2017) Multiple cis-acting rDNAs contribute to nucleoid separation and recruit the bacterial condensin Smc-ScpAB. Cell Rep 21:1347–1360
Acknowledgments
This work was supported by JSPS KAKENHI Grants JP18H02485 and JP18K14627.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9520-2_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9519-6
Online ISBN: 978-1-4939-9520-2
eBook Packages: Springer Protocols