We have started this project by studying the overall architecture and atomic structure of the bacterial SMC complex using a multipronged approach including high-throughput site-specific chemical cross-linking, X-ray crystallography, and electron microscopy. The initial results demonstrated that the SMC complex exists in two distinct states: a closed rod-like state and a more open, ATP-bound ring-like state. We showed that ATP binding to SMC shifts the architecture from the rod-like to the ring-like form, while SMC ATP hydrolysis returns the complex to a rod-like configuration.
Next, we investigated how DNA binding to the SMC complex might be regulated and affected by ATP binding and hydrolysis. We determined the position of the DNA within the SMC complex at different stages of the ATP hydrolysis cycle. We showed that DNA binds differently to the ring- and rod-like states thus revealing a dynamic DNA reconfiguration during the ATPase cycle. In the rod-like state, DNA is held in a small DNA compartment, while in the ring-like state a DNA binding sites is exposed in a separate, much larger compartment which leads to binding of a DNA segment (or DNA loop) rather than a simple DNA double helix.
Based on these findings we proposed a hypothesis for how SMC motor complexes may support DNA translocation and promote DNA loop extrusion. A unique feature of this model (called DNA segment capture model or DNA pumping model) is that it involves deformations of the DNA path for the translocation step. Physical modelling supports the feasibility of this model, which made predictions, some of which we have already tested and other will be tested in the future.
Complementary studies on the biochemical reconstitution of a related eukaryotic SMC complex, the Smc5/6 complex from budding yeast, have been initiated. The work revealed striking similarities in DNA loading between the bacterial SMC complex and the eukaryotic one but also significant differences.
While studying the interaction of SMC proteins with the chromosomal loader, ParB, we surprisingly discovered a hitherto hidden enzymatic activity of the DNA-binding protein ParB. We showed that ParB proteins use an unexpected cofactor, the nucleotide cytidine triphosphate, to self-load onto chromosomes. This finding has direct consequences for our study of the chromosomal recruitment of the SMC complex by ParB. It also has much wider implications for multiple other cellular processes involving ParB proteins and ParB-like proteins.
