Biochemical characterization of the Structural Maintenance of Chromosomes (SMC) complex from Bacillus subtilis
Structural Maintenance of Chromosomes (SMC) proteins play a key role in the chromosome dynamics throughout the cell cycle in almost all species from bacteria to eukaryotes. Proteins from SMC family are involved in a number of processes, such as chromosomes condensation and segregation, sister-chroma...
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|Summary:||Structural Maintenance of Chromosomes (SMC) proteins play a key role in the chromosome dynamics throughout the cell cycle in almost all species from bacteria to eukaryotes. Proteins from SMC family are involved in a number of processes, such as chromosomes condensation and segregation, sister-chromatid cohesion and DNA double strand break repair. All SMC proteins share a typical structure and consist of N- and C-terminal domains carrying the ATPase motif, the hinge domain and two central coiled-coil domains. Terminal domains come together to form one head domain, while coiled coil domains form a single coiled coil. SMC proteins form an intermolecular dimer via interaction of hinge domains. All eukaryotic SMCs perform their function in complex with a number of other none SMC subunits and recently, two novel prokaryotic proteins, ScpA and ScpB, have been found to interact with bacterial SMC in vivo.
In this work, biochemical studies were performed to understand the properties of B. subtilis SMC, ScpA and ScpB in vitro, and to elucidate the mechanism of their action in vivo. The main state of ScpB in solution was found to be a dimer, while ScpA exists in both monomeric and dimeric forms. Using different approaches, such as size exclusion chromatography, gel shift assay and sucrose gradient ultracentrifugation, I found that SMC, ScpA and ScpB indeed form a ternary complex, which most likely consists of one SMC dimer, two ScpAs and two ScpB dimers. ScpA and ScpB were also able to form two types of complexes in absence of SMC: one formed by one ScpA and a dimer of ScpB, and a larger complex most likely consisting of two ScpAs and two ScpB dimers. ScpB was shown to interact with SMC indirectly only in presence of ScpA, and ScpA interacted stably with the SMC head domains only in the presence of ScpB. In addition, gel filtration assays suggested that the SMC complex is most likely formed by direct binding of the ScpA/ScpB complex to SMC, rather than through binding of individual ScpA and ScpB molecules to SMC. Sucrose gradient analysis also showed that ScpA, ScpB and SMC are present as a complex as well as in non-complexed form, indicating that the SMC complex is in a dynamic state in vivo.
Another aspect investigated here were the DNA binding properties of SMC, ScpA, ScpB as well as of different domains of SMC. I found that neither ScpA, nor ScpB are required for binding of SMC to DNA, and that they have no affinity to DNA in absence of SMC. Isolated hinge and head domains of SMC were also unable to bind DNA, thus, the complete SMC molecule is needed for proper function. SMC bound to dsDNA in a sequence independent manner, and based on data obtained from surface plasmon resonance experiments, binding to DNA occurred via formation of a closed ring-like structure. The data suggest that SMC interacts with DNA via dimerization of its head domains leading to the formation of a ring-like structure with DNA trapped in between the coiled-coil (domains) arms of SMC. Collaborative AFM studies have also shown ring formation by SMC, and large complex structures formed by SMCs were detected in solution that could explain why SMCs in bacterial cells are concentrated in certain regions of the cells (foci) and are not distributed (throughout the inner cellular space) all over the chromosome.
Mutagenesis studies were another part of the project. SMC proteins have a weak ATPase activity and head domains contain conserved motifs that are typical for ABC-type ATPases. In this work I have shown, that ATP binding, but not ATP hydrolysis, is required for DNA binding of SMC. Additionally, none of these activities were required for complex formation with ScpA and ScpB, although formation of the SMC complex was less efficient in the mutant proteins. A model is suggested that ATP binding induces dimerization of head domains causing formation of a ring by SMC with DNA locked in the middle. Data obtained from gel filtration studies suggest that ScpA, in absence of ScpB, causes DNA release from SMC, while in the presence of ScpB, all three proteins form a stable complex. Therefore the condensation state of chromosomes in vivo could possibly be controlled by the levels of ScpB in the cell.|