PURIFICATION OF THE 2-MICRON PLASMID PARTITIONING COMPLEX

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The multi-copy 2 micron Saccharomyces cerevisiae plasmid, a selfish extra-chromosomal element, displays nearly chromosome-like segregation efficiency. The plasmid coded proteins Rep1 and Rep2 orchestrate the recruitment of host factors to the cis-acting locus STB for assembly of a high-order partitioning complex. Despite obvious distinctions, STB and centromeres share similarities in that the RSC chromatin remodeling complex, the nuclear motor Kip1, the histone H3 variant Cse4, and the cohesin complex contribute to their functional competence. Mutational analyses and chromatin immunoprecipitation assays reveal functional and temporal hierarchies in the assembly and disassembly of the plasmid partitioning complex. Consistent with genetic and biochemical data, TAP-analyses using Rep1 and Rep2 as baits have identified several host factors as potential constituents of the plasmid partitioning complex. In particular, they include components of the RSC chromatin remodeling complex. The functional status of the STB chromatin is established during each cell cycle during the de novo assembly of the partitioning complex. The normal segregation of the 2 micron plasmid is compromised by the absence of Rsc2 as well as thermal inactivation of Rsc8 and Rsc58 functions. The cumulative results suggest that the point centromere of budding yeast may have originated from the partitioning locus of an ancestral plasmid from which the extant 2 micron circle has descended.