Stable Maintenance of an Extrachromosomal Selfish DNA Element

描述（由申请人提供）：酵母质粒2微米环提供了“良性寄生虫基因组”的简单模型。质粒的遗传组织已经被进化优化，用于其高拷贝繁殖，而不损害宿主细胞的适应性。负责质粒持久性的中心分子组分是确保复制的质粒分子在子细胞中均匀分布的稳定性系统。两个质粒编码的蛋白质（Replp和Rep 2 p）与顺式作用DNA位点（称为STB）一起构成该稳定性系统。此外，质粒已经进化出扩增系统作为安全装置。只有当一个罕见的错误分离事件导致拷贝数下降时，它才会发挥作用。扩增由Flp重组酶介导（Flp =“Flip”，用于翻转或反转DNA），其活性将单个复制起始事件转化为多拷贝机制，从而快速将拷贝数恢复到稳态水平。我们最近发现了质粒分离机制的几个意料之外的特征。首先，酵母粘附素复合物，所需的忠实分离的姐妹染色体，被招募到STB基因座的Replp和Rep 2 p依赖性的方式。第二，与在染色体臂上的粘着蛋白募集相反，在质粒上的粘着蛋白募集完全依赖于有丝分裂纺锤体的完整性。在细胞周期中，质粒粘附素结合的时机和寿命是至关重要的。如果质粒在DNA复制的同时没有获得粘附素，或者粘附素的后期解体被阻断，则等质粒分离失败。在这个提议中，我们描述了试图阐明（1）Rep 1和Rep 2蛋白帮助质粒进入染色体分离途径的机制，（2）有丝分裂纺锤体在促进质粒-粘附素结合中的作用，（3）染色质结构和重塑对质粒分配的影响，以及（4）质粒分离与染色体DNA重复分离之间的潜在分子联系。从这项研究中收集到的一些原则将对宿主-寄生虫基因组之间的全球共生或共生关系产生影响。

DESCRIPTION (provided by applicant): The yeast plasmid 2 micron circle provides a simple model for a 'benign parasite genome'. The genetic organization of the plasmid has been evolutionarily optimized for its high-copy propagation without compromising the fitness of the host cells. The central molecular component responsible for plasmid persistence is a stability system that ensures equal distribution of replicated plasmid molecules to daughter cells. Two plasmid encoded proteins (Replp and Rep2p), together with a cis-acting DNA locus (called STB), constitute this stability system. In addition, the plasmid has evolved an amplification system as a safety device. It comes into play only when a rare missegregation event causes a drop in copy number. Amplification is mediated by the Flp recombinase (Flp ='Flip' for flipping or inverting DNA), whose activity converts a single replication initiation event into a multiple copying mechanism, thus quickly restoring copy number to steady state levels. We recently discovered several unsuspected features of the plasmid segregation mechanism. First, the yeast cohesin complex, required for faithful segregation of sister chromosomes, is recruited to the STB locus in a Replp and Rep2p dependent manner. Second, in contrast to cohesin recruitment at chromosome arms that at the plasmid is absolutely dependent on the integrity of the mitotic spindle. The timing as well as the life-time of plasmid cohesin-association during the cell cycle is critical. Equal plasmid segregation fails if the plasmid does not acquire cohesin concomitant with DNA replication or if the anaphase disassembly of cohesin is blocked. In this proposal, we describe experiments that attempt to shed light on (1) the mechanisms by which the Rep1 and Rep2 proteins help the plasmid gain access to the chromosome segregation pathway, (2) the role of the mitotic spindle in promoting plasmid-cohesin association, (3) the influence of chromatin architecture and remodeling on plasmid partitioning and (4) the potential molecular connection between plasmid segregation and repeated chromosomal DNA segregation. Some of the principles gleaned from this study will have implications in global symbiotic or commensalist relationships among host-parasite genomes.