Smc Family Proteins &Associated Factors In Mitotic Chro

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. Most of the current research in the unit is focused on the condensin complex, its function and regulation during cell cycle progression. Studies of condensin function and chromosome condensation in the Unit were focused on regulatory mechanisms ensuring specificity of condensin targeting to the natural chromatin sites in budding yeast and human cells. Three major research directions were followed: (i) investigation of the genome organization role in chromosomal distribution of condensin, (ii) analysis of posttranslational modifications (Smt3 de-conjugation pathway) in condensin regulation and (iii) screening for novel molecular mechanisms determining specificity of mitotic condensin targeting to nucleolus. (i) Whole-genome analysis of condensin-binding sites was based on the previous studies in the Unit that identified nucleolar chromatin (the rDNA genomic locus) as the major binding site for condensin in S. cerevisiae. To find the unique condensin-bound chromosomal sites a whole-genome study was conducted in a microarray format. Immunoprecipitation of chromatin-bound condensin was used to identify condensin-bound DNA fragments (ChIP-on-Chip analysis). It was established that condensin is uniformly distributed over chromosomal arms, with strong binding peaks every 8-9 kb. Thus, numerous unique sites of condensin binding were identified. Comparison of condensin binding modes in the specialized chromatin regions demonstrated that condensin sites have either cell-cycle independent or mitosis-specific binding. (ii) Functional interaction between sumoation machinery and condensin was previously discovered in the Unit. Particularly, mutations in Smt4, the sole nuclear Smt3p-isopeptidase, were shown to be detrimental for proper condensin function in mitosis. In order to determine whether condensin is directly regulated by Smt3 conjugation, a recombinant in vitro SUMO modification system and a comprehensive tests for in-vivo Smt3 analysis were developed. These two approaches demonstrated that condensin subunits are not bona fide Smt3p substrates. Thus, control of condensin binding to chromatin must be mediated by other proteins, probably functionally interacting with condensin. One of such proteins was shown to be Topoisomerase II (Top2). As a result of investigation in the Unit it was demonstrated that Top2 is one of the most potent sumoation substrates in vitro and in vivo. Moreover, the E3 step in SUMO conjugation pathway is essential for Top2 modification. (iii) Cdc14 phospatase pathway was found to be an another control mechanism governing condensin function. cdc14, esp1 and cdc5 mutants were among mutants isolated as a result of a screen for trans-mutations, which impair mitotic condensin localization to the nucleolus. All the corresponding genes are in the same genetic pathway ? the Cdc14 early anaphase release (FEAR). The late-mitosis pathway of Cdc14p activation (MEN) was dispensable for condensin-to-rDNA targeting, however the MEN-network was able to rescue condensin targeting to rDNA and successful segregation of nucleolus in the FEAR mutants. Analysis of biochemical properties of Cdc14p inactivation showed that condensin was physically removed from rDNA in the cdc14 mutant, however it was properly assembled and chromatin-bound elsewhere, suggesting that condensin was specifically mistargeted by the Cdc14p inactivation.