Stable Maintenance of an Extrachromosomal Selfish DNA Element

描述（由申请人提供）：酵母质粒2微米环为“良性寄生虫基因组”提供了一个简单的模型。质粒的遗传组织已经过进化优化，使其在不影响宿主细胞适应性的情况下进行高拷贝繁殖。负责质粒持久性的核心分子成分是一个稳定系统，确保复制的质粒分子均匀分布到子细胞。两个质粒编码的蛋白（repp和Rep2p）与一个顺式作用的DNA位点（称为STB）一起构成了这个稳定系统。此外，质粒已经进化出一个扩增系统作为安全装置。只有当罕见的错误分离事件导致拷贝数下降时，它才会发挥作用。扩增是由Flp重组酶（Flp ='Flip'用于翻转或反转DNA）介导的，其活性将单个复制起始事件转化为多个复制机制，从而迅速将拷贝数恢复到稳定状态水平。我们最近发现了质粒分离机制的几个意想不到的特征。首先，姐妹染色体忠实分离所需的酵母内聚蛋白复合物以依赖于repp和Rep2p的方式被招募到STB位点。其次，与染色体臂上的内聚蛋白募集相反，质粒上的内聚蛋白募集完全依赖于有丝分裂纺锤体的完整性。在细胞周期中质粒内聚结合的时间和寿命是至关重要的。如果质粒在DNA复制过程中没有获得黏附蛋白，或者黏附蛋白的后期分解被阻断，质粒分离就会失败。在本提案中，我们描述了试图阐明(1)Rep1和Rep2蛋白帮助质粒获得染色体分离途径的机制，(2)有丝分裂纺锤体在促进质粒内聚蛋白结合中的作用，(3)染色质结构和重塑对质粒分裂的影响，以及(4)质粒分离和重复染色体DNA分离之间潜在的分子联系。从这项研究中收集到的一些原理将对宿主-寄生虫基因组之间的全球共生或评论关系产生影响。

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.