Stable Maintenance of an Extrachromosomal Selfish DNA Element

描述(申请人提供)：酵母质粒2微米圆为“良性寄生虫基因组”提供了一个简单的模型。在不影响宿主细胞适合性的情况下，为其高拷贝繁殖而对质粒的遗传组织进行了进化优化。负责质粒持续存在的中心分子成分是一个稳定的系统，它确保复制的质粒分子均匀分布到子细胞。两个编码蛋白质的质粒(Replp和Rep2p)和一个顺式作用的DNA基因座(称为STB)共同构成了这个稳定系统。此外，该质粒还进化出一种作为安全装置的扩增系统。只有当罕见的错误分离事件导致拷贝数量下降时，它才会发挥作用。扩增是由FLP重组酶(FLP=‘Flip’，意为翻转或倒转DNA)介导的，它的活性将单个复制起始事件转换为多个复制机制，从而迅速将拷贝数恢复到稳定水平。我们最近发现了几个未被怀疑的质粒分离机制的特征。首先，酵母粘附素复合体以Replp和Rep2p依赖的方式被招募到STB基因座，这是姐妹染色体忠实分离所必需的。第二，与染色体臂上的粘附素募集不同，质粒处的粘附素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。如果在DNA复制的同时，质粒没有获得粘附素，或者如果粘附素的后期拆解受阻，则等量的质粒分离失败。在这个建议中，我们描述了一些实验，试图阐明(1)Rep1和Rep2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割的影响，以及(4)质粒分离和重复的染色体DNA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寄主-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.