PURIFICATION OF THE 2-MICRON PLASMID PARTITIONING COMPLEX

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 多拷贝2微米酿酒酵母质粒，一个自私的染色体外元件，显示出接近染色体的分离效率。质粒编码蛋白Rep 1和Rep 2协调宿主因子到顺式作用位点STB的募集，用于组装高阶分配复合物。尽管有明显的区别，STB和着丝粒有相似之处，RSC染色质重塑复合物，核马达Kip 1，组蛋白H3变体Cse 4和粘着蛋白复合物有助于它们的功能能力。突变分析和染色质免疫沉淀试验揭示了质粒分配复合物组装和拆卸的功能和时间层次。与遗传和生物化学数据一致，TAP分析使用Rep 1和Rep 2作为诱饵已经确定了几个宿主因子作为质粒分配复合物的潜在成分。特别是，它们包括RSC染色质重塑复合物的组分。STB染色质的功能状态在每个细胞周期期间在分配复合物的从头组装期间建立。2微米质粒的正常分离由于Rsc 2的缺失以及Rsc 8和Rsc 58功能的热失活而受到损害。累积的结果表明，点着丝粒的芽殖酵母可能起源于一个祖先质粒的分配位点，从现存的2微米圆已经下降。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.