Single-molecule studies on SMC proteins

When cells divide, they must ensure they carry with them the genetic code of life to be able to carry out their functions. To achieve this, DNA in eukaryotic cells is replicated, compacted, and distributed via mitosis, so each daughter cell has exactly one copy of the genome.

SMC (structural maintenance of chromatin) proteins such as cohesin and condensin play essential roles in these processes. Both protein complexes have a similar hoop-shaped structure.

The condensin complex contains Smc2 and Smc4, and has an important role in condensing the DNA into chromosomes. It is proposed that condensin encircles chromatin fibers, but the mechanism for forming the intra-chromosomal linkages is poorly understood. Information on the structure, dynamics, and flexibility of the condensin complex will likely give important implications for the in vivo mechanism.


Figure 1. SMC Proteins

The cohesin complex contains Smc1 and Smc3, and provides the essential linkage between sister chromatids, likely by embracing the DNA. During cell divisions, cohesin must withstand the forces of the spindle poles. Cohesin rings are therefore expected to be remarkably strong and stable. However, the mechanical strength has not been measured.

With single-molecule assays such as magnetic tweezers and atomic force microscopy, we try to elucidate the structure and mechanical properties of eukaryotic SMC proteins.

Magnetic tweezers assay allows for precise control of the force exerted on individual DNA molecules, hence, helps in elucidating the mechanism of condensin-mediated compaction of DNA in real-time (Figure 2).


Figure 2: A DNA molecule is attached to a surface on one side, and a magnetic bead on the other side. With a pair magnets placed above, a force can be applied to the DNA. At low forces, we observe DNA compaction upon addition of condensin and ATP.

We are studying the structure and molecular mechanism of condensin protein using a new, state of the art single-molecule technique called high speed atomic force microscopy (HS-AFM). HS-AFM is an excellent technique for understanding of protein’s structure and function relationship because it allows to directly visualize a protein’s structural dynamics with high spatio-temporal resolution in a liquid phase (in nearly physiological condition).

We succeeded to obtain a movie of SMC dimers using HS-AFM which shows that SMC dimers are highly flexible and dynamic (Figure 3). Now, we are studying the conformational change of condensin with full subunits via ATP hydrolysis and the condensation of DNA by this conformational change.


Figure 3. HS AFM video images on SMC dimers of condensin. The dynamical conformational changes show that the coiled-coils of SMC dimers are flexible and show extensive fluctuations in time (Eeftens et al., 2016, Cell Reports 14, 1813–1818, 2016).