PURIFICATION OF THE 2-MICRON PLASMID PARTITIONING COMPLEX

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 多拷贝2微米酿酒酵母质粒是一种自私的染色体外元件，表现出类似染色体的分离效率。Rep1和Rep2是编码蛋白质Rep1和Rep2的编码蛋白，它们协调宿主因子在顺式作用基因STB上募集，以组装一个高阶分配复合体。尽管STB和着丝粒有明显的区别，但它们的相似之处在于RSC染色质重塑复合体、核马达Kip1、组蛋白H3变异体Cse4和粘附素复合体参与了它们的功能活性。突变分析和染色质免疫沉淀分析揭示了质粒分割复合体的组装和拆卸过程中的功能和时间层次。与遗传和生化数据一致的是，以Rep1和Rep2为诱饵的TAP分析已经确定了几个宿主因子作为质粒分割复合体的潜在成分。特别是，它们包括RSC染色质重塑复合体的成分。STB染色质的功能状态是在分割复合体从头组装期间的每个细胞周期中确定的。由于RSC2的缺失以及Rsc8和Rsc58功能的热失活，2微米质粒的正常分离受到影响。这一累积结果表明，发芽酵母的点着丝粒可能起源于一个祖先质粒的分裂位点，现存的2微米圆就是从这个祖先质粒上下来的。

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.