Investigation of the dynamics of the SMC chromosome condensation complex at the single molecule level

• Applicant: Professor Dr. Peter Graumann
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Cell Biology
• Term: from 2015 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 274661393

SMC (structural maintenance of chromosomes) proteins perform central roles in various chromosome dynamics in all three domains of life. SMC proteins are ATPases and form the core of several protein complexes essential for chromosome cohesion and compaction in eukaryotes, and for chromosome segregation in bacteria. Loss of SMC or of its complex partners (ScpA and ScpB in bacteria) leads to the inability to proceed through Mitosis, and to severe defects in the compaction and segregation of chromosomes in prokaryotic cells. Thus, SMC proteins are key components of the cell cycle. We are studying the Bacillus subtilis SMC complex as a model for the bacterial SMC complex, which we have recently shown to form several distinct fractions in cells: SMCs not bound to ScpA and ScpB (which form a tight subcomplex) move throughout the entire chromosome, while 20% of SMC molecules are bound to ScpAB and statically localize to distinct sites on the chromosomes (termed condensation centres), one within each cell half, during most of the cell cycle. These specific assemblies are essential for chromosome segregation and are lost upon depletion of any of the SMC complex subunits. Exchange of SMC and ScpA from the condensation centres is different in terms of half time, with SMC exchanging faster than ScpA, and most ScpA molecules (86%) are statically positioned within the centres, while the remaining molecules freely diffuse through the cells. Thus, SMC shows a novel mode of interaction with DNA: the a static ScpAB-bound mode can not bind to DNA de novo in vitro, and the dynamic free mode can interact with and bind to DNA. We will use the visualization and tracking of single SMC-YFP molecules in live cells and advanced biochemical techniques to analyse the mode of formation of condensation centres, binding of SMC to DNA and the function of the ATPase cycle that SMC undergoes. We will analyse the movement of ATP binding, ATPase and many more mutant versions of SMC using single molecule microscopy (SMM) in B. subtilis cells, and analyse the requirement of recruitment of ScpA to condensation centres in a variety of mutant backgrounds. All experiments will be complemented by the analysis of the kinetics of complex formation and DNA binding in vitro. We will also determine if the static ScpA molecules in the condensation centres are associated with any specific region on the chromosome, using CHIP experiments, or if the structures are dynamically interacting with many sites on the chromosome, which we speculate is important for the maintenance of the preferred arrangement and folding of the bacterial chromosome. We will determine binding affinities of SMC, and mutant versions, and its complex partners in vitro using ITC and thermophoresis, to understand the unusual exchange rates in vivo. Visualizing SMC at the single molecule level in live cells will also be informative on the general principle of the interaction with DNA for SMC proteins in eukaryotic cells.

• DFG Programme: Research Grants