Smc Family Proteins And Associated Factors In Mitotic Ch

Child Health And Human Development

Investigators

Linked publications & trials

Abstract

The eukaryotic ATP-ases of SMC family (structural maintenance of chromosomes) are in the core of two essential eukaryotic protein complexes: cohesin and condensin, which determine the higher-order chromosome structure in proliferating cells. To understand the molecular mechanisms of these SMC-containing complexes we combine genetic, cytological and biochemical approaches. The studies on the condensin complex, the chief molecular machine of chromosome condensation, were focused on the mechanisms determining the specificity of condensin targeting to the natural chromatin sites in mitosis. This work followed two directions: first, we designed a genetic screen to isolate and identify new mutants that are defective in mitotic targeting of condensin to rDNA; second, we continued investigation of the involvement of Smt3-modification pathway in condensin regulation. We found a link between the Smt3-modification and mitosis-specific condensin targeting to chromatin in the process of characterization of genes in the Smt3 pathway: SMT4, SIZ1 and SIZ2, all discovered in the course of our previous work. We initiated a search for the essential targets of Smt3 conjugation, with a special emphasis on chromatin proteins, using a whole-proteome approach. Our work on cohesin, a four-subunit complex involved in establishment and maintenance of physical association between sister chromatids (sister chromatid cohesion), was directed at the in-vitro characterization of its association with chromatin. We purified the recombinant cohesin holocomplex, its Smc1/Smc3 ATP-ase core and the non-SMC subunits Scc3 and Mcd1. Experiments with the defined chromatin probe established that Smc1p/Smc3p dimer and cohesin holocomplex differ dramatically in their chromatin-binding properties. Only full cohesin is able to form a stoichiometric complex with chromatin. Binding of cohesin to chromatin was independent of linker DNA and histone tails. This work was expanded with investigation of the cohesin-chromatin binding properties in vivo. The studies on eukaryotic cohesin were complemented by a collaborative work on bacterial SMC proteins that resulted in characterization of a cohesin-like complex from Bacillus subtilis.