Biophysics of SMC complex function in chromosome organization and DNA repair
Biochemistry
Final Report Abstract
SMC complexes are important architects of structural loops in interphase chromosomes. Higher eukaryotes have three different classes of these complexes: cohesin, condensin and Smc5/6, which all are ATPases that use hydrolysis for motor activity but differ in their molecular composition and in their biological role. This project focused on using single molecule experiments to study molecular aspects of the cohesin and Smc5/6 complexes. DNA curtains are a single molecule method to study protein-DNA interactions at the single molecule level in a high-throughput manner. In one subproject, this approach was used to study how ancillary factors to the cohesin complex like Pds5 or Scc2/4 affect the DNA binding of the complex and how these factors compete with others. In another subproject, force unfolding by single molecule optical tweezers was used to characterize the mechanical stabilization of the ATP binding domain of the Psm3(Smc3) subunit of cohesin. With the same approach, experiments were conducted to test whether nucleotide binding in the ATP domain causes detectable shifts in the coiled-coil domain in a possible mechanism for motor activity. In a third subproject, variants of the Smc5/6 complex were studied to characterize their unique ability to regulate the complex’s motor activity or modify target proteins. The obtained results are important steps toward reconstituting the process of target protein modification in single molecule assays.
