PURIFICATION OF THE 2-MICRON PLASMID PARTITIONING COMPLEX

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 多拷贝2微米酿酒酵母质粒是一种自私的染色体外元件，表现出近乎染色体样的分离效率。质粒编码蛋白 Rep1 和 Rep2 协调将宿主因子招募到顺式作用基因座 STB，以组装高阶分配复合物。尽管存在明显的区别，STB 和着丝粒仍有相似之处，即 RSC 染色质重塑复合物、核马达 Kip1、组蛋白 H3 变体 Cse4 和粘连蛋白复合物有助于其功能。突变分析和染色质免疫沉淀分析揭示了质粒分区复合物组装和拆卸中的功能和时间层次结构。与遗传和生化数据一致，使用 Rep1 和 Rep2 作为诱饵的 TAP 分析已确定了几种宿主因子作为质粒分配复合物的潜在成分。特别是，它们包含 RSC 染色质重塑复合物的成分。 STB 染色质的功能状态是在分配复合物从头组装的每个细胞周期期间建立的。 2 微米质粒的正常分离因 Rsc2 的缺失以及 Rsc8 和 Rsc58 功能的热失活而受到损害。累积结果表明，芽殖酵母的点着丝粒可能起源于祖先质粒的分配基因座，现存的2微米圆就是从该基因座下降的。

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.