Mechanisms and roles of chromatin organization – decipher the epigenetic code

With this method, the labs of Johannes Stigler, Luis Aragon, and Shixin Liu were able to identify unique binding and mobility behavior of SMC5/6 on ss- vs ds-DNA and could identify DNA fork structures as preferred binding location, emphasizing SMCs’ critical role in DNA replication and repair (see figure).

The real-time single-molecule imaging capabilities of the C-Trap also enabled Frank Uhlmann and his collaborators to directly observe condensin interacting with multiple dsDNA molecules, a mechanism that had earlier been identified for cohesin using a similar C-Trap assay.

In summary, Dynamic Single-Molecule microscopy is key to quantifying

• Binding rates and location of SMC complexes on different DNA structures
• Dynamic movement of SMC complexes on DNA and looping/cross-linking activity
• Influence of additional factors (ATP, small molecules, other DNA-binding proteins) on SMC kinetics and dynamics

By applying tension on cohesin-looped DNA, Luis Aragon and collaborators were able to quantify loop size as well as mechanical stability of the cohesin-mediated cross-links.

Furthermore, tension can also be directly applied to SMC complexes (or subunits) to quantify their conformational dynamics under force. By doing so, Johannes Stigler and colleagues identified structures and intermediate states in the coiled-coil region of SMCs that are critical for dynamic folding of the complex. For more details on the assay, see our protein folding page.

The groups of Taekjip Ha, Carl Wu and Shixin Liu have extensively used LUMICKS’ C-Trap to unravel details of remodeler target search and to discover hitherto unknown nucleosome remodeling capabilities of origin recognition complexes (ORCs), respectively.

The optical traps of the C-Trap allow the application of well-defined mechanical tension on a single DNA molecule, mimicking the case of incomplete sister chromatid segregation or other mechanically challenging scenarios DNA is regularly facing.

By doing so, the groups of Erwin Peterman and Gijs Wuite in collaboration with Ian Hickson discovered an unknown role of the checkpoint helicase PICH: On DNA under tension, PICH shows nucleosome unwrapping and histone transporting activities, as directly observed with correlative dynamic single-molecule microscopy (Figure 1).

Amongst other labs, the groups of Mark Williams and Shixin Liu used this method successfully to unravel the impact of histone chaperones on nucleosome (dis)assembly, and Polycomb complex-mediated nucleosome cross-linking, respectively.