Visualizing the Assembly of DNA Condensation Clusters by SMC using Single-Molecule Microscopy

Abstract

SMC (structural maintenance of chromosomes) family members play essential roles in chromosome condensation, sister chromatid cohesion, and DNA repair. Despite intensive efforts, it is still unclear how SMCs are loaded onto DNA and structure chromosomes. We employed single-molecule fluorescence microscopy to visualize how individual Bacillus subtilis SMC (BsSMC) dimers interact with flow-stretched DNAs. We find that BsSMC can vary its initial interaction with DNA, switching between static binding and sliding. Its diffusive properties are insensitive to the presence of ATP, suggesting that the initial loading of BsSMC onto DNA is ATP-independent. At higher concentrations, BsSMCs form distinct clusters that condense DNA by both wrapping and bridging distal DNA segments. SMC-mediated DNA compaction occurs in a weakly ATP-dependent manner. However, ATP increases the apparent cooperativity of DNA condensation, demonstrating that multiple BsSMCs can interact cooperatively through their ATPase head domains. Consistent with these results, BsSMC mutants that alter the ATPase cycle compact DNA more slowly than wild-type BsSMC in the presence of ATP, and an ATPase headless construct compacts DNA non-cooperatively with substantially lower compaction rate. Our results suggest that transiently static BsSMC can nucleate the formation of functionally active clusters that locally condense the chromosome while forming long-range DNA bridges.