Visualizing Binding of Single SMC Complexes to DNA Substrates Using Combinational Optical Tweezers and Fluorescence...

Structural Maintenance of Chromosomes (SMC) complexes undertake a variety of DNA-centric processes to both organize and maintain genomes throughout the cell cycle. Their multiple DNA binding sites and conformational dynamics have made fully characterizing their interactions with DNA challenging, and indeed, fundamental questions remain around DNA substrate preferences, the stability of their interaction, and requirements of complex loading and unloading. Recent advances in combinational single-molecule techniques now allow for the correlation of force spectroscopy and real-time analysis of protein binding dynamics on DNA at high spatial and temporal resolution with the complementary use of microfluidics for rapid exchange of experimental conditions. This is a powerful toolkit that allows for the direct visualization of individual SMC complexes binding to DNA substrates while probing the contributions of associated factors including ATP turnover and force dependencies of SMC action. Additionally, the single-molecule approach grants the ability to probe complex stoichiometry and subunit kinetics while providing access to heterogeneous and transient behaviors otherwise averaged in ensemble assays. Here, we describe how to recombinantly prepare eukaryotic SMC complexes-cohesin and Smc5/6-with fluorophore labelling and a variety of DNA substrates for single-molecule experimentation using correlative optical tweezers and fluorescence microscopy. We highlight practical considerations for their study using the LUMICKS C-Trap system and provide custom data analysis scripts for automated detection and localization of DNA binding events.