The eukaryotic ATP-ases of the SMC (structural maintenance of chromosomes) family are located in the core of two essential eukaryotic protein complexes: cohesin and condensin, which determine the higher-order chromosome structure in proliferating cells. The condensin complex is the chief molecular machine of chromosome condensation and is indispensable for proper separation of chromatids during anaphase. It is also likely to be involved in formation of the higher-order structure of interphase chromosomes. Cohesin is a four-subunit complex essential for establishment and maintenance of a physical association between sister chromatids (sister chromatid cohesion, SCC); it is also implicated in a number of interphase chromatin-mediated processes: control of gene expression, DNA replication, and DNA repair. To understand the molecular mechanisms of the SMC-containing complexes, we combined genetic, cytological, and biochemical approaches. To investigate the mechanisms determining the specificity of condensin’s targeting to the natural chromatin sites, we set up a genetic screen to isolate mutants that are defective in mitotic targeting of condensin to chromatin and investigated the input of the Smt3-modification pathway in condensin regulation. Studies on eukaryotic cohesin were complemented by collaborative work on bacterial SMC proteins that resulted in characterization of a four-subunit SMC complex from bacilli.

Condensin Regulation and Chromatin Targeting
Wang, Yong-Gonzales, Lee
Condensin is a five-subunit, evolutionarily conserved protein complex indispensable for the process of chromosome condensation in mitosis and meiosis. Despite substantial recent progress in understanding the molecular structure and enzymology of condensin, mechanisms regulating its activity remain obscure. Our studies on condensin focus on the budding yeast complex composed of five essential subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4, and Brn1. We designed a genetic screen to isolate and identify new mutants that are defective in mitotic targeting of condensin to rDNA. The genetic studies established that at least two new pathways control condensin localization to the condensing chromatin domains in mitosis and thus the chromosome condensation process. One pathway requires proper establishment of sister chromatid cohesion, as both cohesin mutations and the absence of sister chromatids (due to bypass of DNA replication) impair condensin targeting as well as chromosome condensation. The second pathway is the branch of the SUMO-modification pathway. We established that SUMO (Smt3) itself, Smt3-isopeptidase Smt4, and the E3 Smt3-ligases Siz1 and Siz2 play a crucial role in condensin targeting. The
molecular mechanism of this regulation is under investigation. Using a whole proteome approach, we initiated a search for the essential targets of Smt3 conjugation, with a special emphasis on chromatin proteins.

Cohesin-Chromatin Interaction
Kagansky, Lukyanov
SCC is an essential process that establishes and maintains the link between two sister chromatids from S-phase through anaphase. SCC regulation in mitosis and meiosis is extremely complex but is mostly directed at cohesin, an evolutionarily conserved four-subunit protein complex. In budding yeast, cohesin includes essential proteins Smc1, Smc3, Scc1/Mcd1, and Scc3. The molecular mechanism of cohesin activity in SCC is largely unknown. Thus, we established a purified system to study SCC in vitro. It includes a model chromatin template and the purified cohesin complex. 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 showed that the 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 occurred independently of linker DNA and histone tails. Our experimental approach allowed us to gain insights into the mechanism of the elementary sister chromatid cohesion reaction.

Discovery of a Bacterial SMC Complex
Strunnikov; in collaboration with members of Graumann’s laboratory
SMC proteins forming the core of condensin and cohesin in eukaryotes are also ubiquitous in bacteria. Chromosome segregation is severely impaired in the SMC Bacillus subtilis mutants, yet the molecular role of SMC proteins in bacterial cells is unknown. It was believed that, in bacteria, SMC proteins do not complex with other proteins. However, our biochemical analysis of the SMC complex from bacilli combined with genetic and cell biology data from Dr. Graumann’s group indicates that the bacterial SMC complex does include four subunits: the SMC dimer and two novel proteins ScpA and ScpB. Analysis of the SMC complex composition, localization, and function suggests that it is reminiscent of cohesin and probably drives packaging of bacterial chromatin in vivo.