Role of the mitotic spindle in the equal segregation of an extrachromosomal element in Saccharomyces cerevisiae
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The Saccharomyces cerevisiae plasmid, 2 micron circle, resides in the yeast nucleus at a high copy number. It provides no apparent growth advantage to its host, nor imposes any significant growth disadvantage. The plasmid is an excellent paradigm for studying mechanisms utilized in the persistence of a eukaryotic selfish DNA element that is selectively neutral. The plasmid achieves stable propagation and copy number maintenance by combining a partitioning system and an amplification system. The partitioning proteins Rep1p and Rep2p promote the recruitment of the histone H3 variant Cse4p and the yeast cohesin complex to the partitioning locus STB during S phase, leading to the formation of a functional partitioning complex which segregates the plasmid equally during mitosis. The integrity of the mitotic spindle is a pre-requisite for the specific nuclear localization of the plasmid as well as for plasmid association with a subset of the partitioning proteins such as Cse4p and the cohesin complex. The work presented in this thesis reveals, using tools of molecular genetics and cell biology, the involvement and possible functions of a microtubule associated nuclear motor protein, Kip1p, in the 2 micron circle partitioning pathway. The plasmid missegregates in kip1[Delta] cells, but not in cells harboring deletions of genes coding for the other nuclear motors. Kip1p interacts with the plasmid partitioning system and promotes the association of Cse4p and the cohesin complex with STB. Lack of Kip1p function delocalizes the plasmid from its characteristic nuclear locale in close proximity to the spindle pole body. The distance between a reporter plasmid and the spindle pole body is nearly doubled in a kip1[Delta] host strain. We propose that, unlike the conventional roles played by nuclear motors in spindle function and chromosome segregation, the Kip1p motor assists the 2 micron circle in associating with the mitotic spindle and translocating to its ‘partitioning center’.