Yeast plasmid segregation: investigation of host/plasmid interactions

2微米质粒是一种多拷贝的环状DNA，存在于大多数烘焙酵母的细胞核中。这种2微米的质粒被称为完美的“自私”DNA；该质粒的存在不会对宿主产生不利影响，但也不会带来好处。该质粒通过借用宿主机器进行复制和分离来确保其自身的维护。在宿主细胞分裂过程中，质粒拷贝的平等分割需要将质粒蛋白Rep1和Rep2与质粒STB位点的重复序列相关联。Rep蛋白的联合招募宿主蛋白，帮助在STB建立功能分割复合体。Rep蛋白/STB复合体允许质粒搭乘在分离的染色体或染色体分离装置的某些组件上，但其机制仍有待确定。一种假设认为，酵母染色体的分割序列可能是从祖先的2微米质粒中获得的。在这项研究中，将使用生化、分子遗传学和细胞生物学的方法来鉴定和研究2微米质粒借来的使其能够遗传的宿主蛋白。正在对STB序列促进的RNA表达进行评估，以确定这是否具有功能意义，可能代表了染色体分割序列的共同特征。DNA保护和蛋白质-DNA结合分析将被用来研究我们研究中发现的宿主蛋白的缺失对STB染色质的组织和STB指导的RNA表达的影响。我们的研究将有助于识别对染色体生物学的特定方面做出贡献的宿主蛋白，并揭示其他方面的新功能。我们还在使用分子遗传学方法来研究STB中划分能力所需的序列，以及比较基因组学方法，其中将研究在其他萌芽酵母物种中划分蛋白质和2微米相关质粒序列的方法，以进一步了解这些自私DNA的生物学和进化。这个项目将确定2微米的质粒如何与细胞核的其他成分相互作用，以确保其有效分离。了解酵母质粒如何利用染色体分离的特征，可能会揭示其他遗传元素，如病毒是如何保持的，并提供对进化过程中可能出现的分割功能的洞察。

The 2-micron plasmid is a multi-copy circular DNA found in the nucleus of most strains of the bakers yeast, Saccharomyces cerevisiae. The 2-micron plasmid has been called the perfect “selfish” DNA; presence of the plasmid does not adversely affect the host but nor does it confer a benefit. The plasmid ensures its own maintenance by borrowing host machinery for its replication and segregation. Equal partitioning of plasmid copies during host cell division requires association of plasmid proteins, Rep1 and Rep2, with a repeated sequence at the plasmid STB locus. Association of the Rep proteins recruits host proteins that help establish a functional partitioning complex at STB. The Rep protein/STB complex allows the plasmid to hitchhike on segregating chromosomes or some component of the chromosome segregation apparatus but the mechanism remains to be established. One hypothesis proposes that the partitioning sequences of yeast chromosomes may have been acquired from an ancestral 2-micron plasmid. In this study, biochemical, molecular genetics, and cell biology approaches will be used to identify and investigate host proteins borrowed by the 2-micron plasmid that enable its inheritance. RNA expression promoted by the STB sequence is being assessed to determine if this has functional significance that might represent a feature shared with chromosomal partitioning sequences. DNA protection and protein-DNA association assays will be used to investigate the effect of absence of host proteins identified in our studies on organization of the STB chromatin and RNA expression directed by STB. Our studies will help identify host proteins that contribute to specific aspects of chromosome biology and reveal new functions for others. We are also using molecular genetic approaches to investigate the sequences required at STB for partitioning competence and a comparative genomics approach in which partitioning proteins and sequences of 2-micron-related plasmids in other species of budding yeast will be studied to further our understanding of the biology and evolution of these selfish DNAs. This project will determine how the 2-micron plasmid interacts with other components of the nucleus to ensure its efficient segregation. Understanding how the yeast plasmid exploits features of chromosome segregation may reveal how other genetic elements, such as viruses are maintained and provide insight into how partitioning function may have has arisen during evolution.