The 2 micron plasmid of Saccharomyces cerevisiae resides in the nucleus as an extra-chromosomal element with a steady state copy number of 40-60 per cell. As a benign but selfish DNA element, the plasmid utilizes a self-encoded partitioning system and an amplification system to ensure its stable, high-copy propagation. The partitioning system consists of the plasmid encoded proteins, Rep1 and Rep2 and a cis-acting partitioning locus STB. The Rep proteins, together with several host factors, assembled at STB couple plasmid segregation to chromosome segregation. A plasmid lacking an active partitioning system is subject to a ‘diffusion barrier’, which causes it to be retained in the mother cell with a strong bias (mother bias). Currently available evidence favors the hitchhiking model for plasmid segregation, in which the tethering of plasmids to chromosome provides the basis for faithful plasmid partitioning. However, direct evidence to support this hypothesis has been difficult to obtain because of the small size of the budding yeast nucleus and the poor resolution of chromosomes in live cells or in chromosome spreads. In this study, we have attempted to verify the hitchhiking model using single copy derivatives of the 2 micron plasmid as reporters. We demonstrate, using two single copy reporters present in the same nucleus, that plasmid association with chromosome spreads is authentic, and is dependent on the partitioning system. By using a strategy that forces all chromosomes to stay in either the mother or the daughter compartment, we show that plasmid segregation can be uncoupled from nuclear envelope segregation. However, plasmid segregation cannot be uncoupled from chromosome segregation under this condition. This tight coupling between plasmid and chromosome segregation is consistent with the hitchhiking model for plasmid segregation. The plasmid partitioning complex is assembled de novo at STB during each cell cycle during the G1-S window. Plasmid replication or cell cycle cues that signal cellular DNA replication appear to trigger this assembly. Furthermore, there is an apparent temporal hierarchy in the association and dissociation of protein factors at STB. When DNA replication is delayed or blocked, the dissociation of factors from STB from the previous portioning cycle and the association of factors for the new partitioning cycle are delayed or blocked, respectively. The precise role of replication in plasmid segregation has not been elucidated. We have addressed this question by blocking either plasmid replication or all cellular DNA replication. We find that replication is not required for plasmid to overcome mother bias. However, replication is critical for the equal segregation of sister plasmid copies. These results provide a refinement of the hitchhiking model by suggesting that sister plasmids tether to sister chromatids in a replication-dependent manner and hitchhike on them during chromosome segregation. Finally, we have attempted to reconstitute the 2 micron plasmid partitioning system in mammalian cells with the goal of exploiting their larger nuclear size and the considerably higher chromosome resolution they provide. In experiments completed so far, we show that Rep2 expressed in COS7 cells localizes to chromosomes, and Rep1 does so in the presence of Rep2. Furthermore, they show co-localization on sister chromatids in a symmetric fashion, implying that plasmids associated with them are likely to follow suit. These observations suggest, by extrapolation, the Rep1-Rep2 assisted association of sister plasmids with sister chromatids in yeast as well, and are consistent with the refined hitchhiking model for plasmid segregation. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/24799 |
| Date | 24 June 2014 |
| Creators | Chang, Keng-Ming |
| Source Sets | University of Texas |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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