Smc Family Proteins In Mitotic Chromosome Segregation

Child Health And Human Development

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Abstract

The eukaryotic ATP-ases of SMC family (structural maintenance of chromosomes) form several essential eukaryotic protein complexes. Two of them, cohesin and condensin, are the focus of studies by the Unit of Chromosome Structure and Function. These complexes determine the higher-order chromosome structure and dynamics in eukaryotic cells. Condensin complex is the molecular machine of chromosome condensation, a process indispensable for proper individualization of chromatids and their separation during anaphase. The chromosome condensation studies were focused on condensin regulation and specificity of its targeting to the natural chromatin sites in budding yeast and human cells. Particularly, the role of epigenetic makeup of the genome and posttranslational modification pathways in condensin regulation were investigated. In budding yeast and higher eukaryotes condensin is composed of five essential subunits: Smc2, Smc4, Ycs5/Ycg1, Ycs4 and Brn1. Unit's genetic studies established that several pathways determine proper condensin localization to the specific chromatin domains and thus regulate the chromosome condensation process. One such pathway is the SUMO (Smt3) -modification machinery. We continued the whole-proteome screening for the essential targets of Smt3 conjugation in vivo in order to establish the mechanizm, by which Smt3 itself, Smt3-isopeptidase Smt4 and the E3 Smt3-ligases Siz1 and Siz2 play important roles in condensin targeting. In additon, using the purified SUMO-lation system we demonstrated that topoisomerase II modification by Smt3 is in the center of this regulatory pathway. Two condensin subunits were found to be modified in mitosis, both in yeast and mammalian cells, but these modification were not Smt3-conjugations. Molecular analysis of these posttranslational modifications of condensin is under way. Cohesin is a complex essential for establishment and maintenance of transient association between sister chromatids (sister chromatid cohesion, SCC), lasting from S-phase to anaphase. The protein machinery of SCC is extremely complex. In the core of cellular SCC activity is cohesin, an evolutionary-conserved four-subunit protein complex, including essential proteins Smc1, Smc3, Scc1/Mcd1 and Scc3. As no enzymatic activity was found to be associated with cohesin in vitro, the molecular mechanism of cohesin action in SCC is unknown. Unit's work on SCC was centered around characterization of cohesin association with chromatin in vitro. To address this issue we developed a purified in-vitro SCC system, including a model chromatin template (dinucleosome) and recombinant cohesin complex. Analysis of condensin association with DNA and defined chromatin probe demonstrated that cohesin holocomplex forms a stoichiometric complex with chromatin. Binding of cohesin to chromatin was independent of linker DNA and histone tails. In contrast, cohesin was unable to bind naked DNA (even derived from the natural cohesion sites). The properties of cohesin-chromatin interaction agree well with a ring-like model of cohesin structure. The in-vitro studies suggest that cohesin ring is stabilised through interaction with nucleosomal cores. The current development of this experimental system is focused on complementation of the SCC reaction with additional proteins required for establishing and regulation of SCC in vivo.