Browsing by Subject "Chromosome segregation"
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Item Functional interactions of chromosome segregation factors with the 2 micron plasmid : possible evolutionary link between the plasmid portioning locus and the budding yeast centromere(2011-05) Huang, Chu-Chun; Jayaram, Makkuni; Dean, Appling R.; Arlen, Johnson W.; Paull, Tanya T.; Stevens, Scott W.The 2 micron plasmid of Saccharomyces cerevisiae is a multi-copy circular DNA genome that resides in the nucleus and exhibits nearly chromosome-like stability in host populations. Several host factors are required for equal plasmid segregation during cell division. One of them is cohesin (a multi-subunit protein complex) which mediates sister chromatid cohesion, a crucial mechanism for faithful segregation of replicated chromosomes in eukaryotes. The 2 micron plasmid mimics chromosomes in assembling cohesin at its partitioning locus. Studies on minichromosomes (centromere containing plasmids) reveal that cohesin forms a ring that embraces replicated sister centromeres topologically rather than physically. The functional similarities between chromosome and plasmid segregation prompted us to examine whether the topological mechanism proposed for centromere-mediated replicative cohesion is also true in the case of the plasmid. In the present study, we have characterized the nature and stoichiometry of cohesin's association with the 2 micron plasmid. Another host factor required for equal plasmid segregation is the CenH3 histone variant Cse4, so far considered to be uniquely associated with centromeric nucleosomes. Cse4 provides an epigenetic landmark at centromeres, and is required for assembly of the kinetochore complex. Surprisingly, Cse4 also interacts with the 2 micron plasmid partitioning locus. We have now functionally characterized this interaction, which can be preserved even in an ectopic, chromosomal context. The steady state level of Cse4 is highly limiting in yeast due to ubiquitin-mediated proteolysis. Only centromere-associated Cse4 is protected from this regulatory turnover control. We find that, in contrast to the situation with centromeres, association of Cse4 with the 2 micron plasmid is highly sub-stoichiometric but still promotes equal plasmid segregation. We also find that Cse4 induces an unusual right handed DNA writhe at the plasmid partitioning locus, as it does at the centromere. Our findings suggest that the plasmid has designed strategies to minimize the utilization of host factors that are in short supply. They signify the advantage of clustering and group behavior in the evolutionary success of a multi-copy selfish genome. Finally, they also suggest the possible emergence of the yeast centromere and the plasmid partitioning locus from a common ancestral sequence.Item The regulation of chromosome segregation by Aurora kinase, protein phosphatase 1 and nucleolar protein UTp7(2007-08) Jwa, Miri; Chan, Clarence S. M.The Sli15-Ipl1-Bir1 chromosomal passenger complex is essential for proper kinetochore-microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. Subcellular localization of this complex during anaphase is regulated by the Cdc14 protein phosphatase, which is kept inactive in the nucleolus until anaphase onset. I show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. It prevents the abnormal localization of Sli15 on cytoplasmic microtubules, the premature concentration of Sli15 on the pre-anaphase spindle, and the premature nucleolar release of Cdc14 before anaphase onset. Utp7 regulates Sli15 localization not entirely through its effect on Cdc14. Furthermore, the mitotic exit block caused by Cdc14 inactivation is relieved partially by the simultaneous inactivation of Utp7. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation and cell cycle control. Protein phosphatase 1, Glc7 opposes in vivo functions of the Ipl1-Sli15-Bir1 kinase complex in budding yeast. I show here Scd5- a targeting subunit of Glc7 that regulates endocytosis/cortical actin organization and undergoes nuclear-cytoplasmic shuttling- is present at kinetochores. Ipl1 associates with both Glc7 and Scd5. The scd5-PP1[Delta]2 mutation, which disrupts the association between Glc7 and Scd5, also disrupts the association between Ipl1 and Scd5-Glc7 without affecting the kinetochore localization of these proteins. Genetic studies suggest that Scd5 may positively regulate both Glc7 phosphatase and the Ipl1 kinase complex. In accordance, Scd5 stimulates in vitro kinase activity of Ipl1. scd5-PP1[Delta]2 cells missegregate chromosomes severely due to several defects: i) at least one of sister kinetochores appears not attached to microtubule. ii) sister chromatids are persistently cohesed through anaphase. iii) Sli15 is hyperphosphorylated and less abundant on the anaphase spindle resulting in unstable mitotic spindle. These results together suggest that Scd5 functions in diverse processes that are essential for faithful chromosome segregation. How Scd5 coordinately regulates two apparently antagonistic enzymatic activities of Ipl1 and Glc7 remains to be determined.