Single-Molecule Studies of Chromosome Organization by SMC

Abstract

The structural maintenance of chromosomes complex (SMC) plays an essential role in chromosome condensation, sister chromatid cohesion, and recombinational repair. SMCs possess a unique structural architecture: SMC monomers dimerize through interactions within their hinge domains while long coiled-coil arms bring together the N- and C-termini which form an ATPase head domain. Despite its cellular importance and extensive efforts, the role of the ATPase domain of SMC still remains mysterious. How ATP binding and hydrolysis is coupled to DNA remodeling or how the mechanochemical cycle of SMC modulates the SMC-DNA interaction is unclear. Using Bacillus Subtilis SMC (BsSMC) as a model we are utilizing single-molecule microscopy to visualize how SMCs interact with flow stretched DNAs. In an approach we have termed DNA motion capture, we site-specifically label DNAs with quantum dots which allow us to track how DNA segments under differing tensions are condensed by SMC. We find that BsSMC is able to compact individual DNAs even in the absence of ATP although ATP greatly enhances the rate of DNA compaction. This supports a picture in which ATP does not provide a powerstroke for DNA organization but instead acts to bias BsSMC conformations that facilitate chromosome remodeling. To better understand the role of ATP in the mechanochemistry of BsSMC we examined how two mutants, E1118Q which slows the rate of ATP hydrolysis and S1090R which blocks head-head engagement, alter the kinetics of BsSMC-mediated DNA condensation. Our single-molecule assay shows that the E1118Q mutant compacts DNA more slowly than the wild-type in the presence of ATP, and significant compaction rate reduction was observed with S1090R mutant. These results suggest a role for both ATP hydrolysis and SMC head reengagement in SMC-mediated DNA condensation.