Yeast plasmid segregation: investigation of host/plasmid interactions

酵母质粒分离：宿主/质粒相互作用的研究

• 批准号: RGPIN-2016-05516
• 负责人: Dobson, Melanie
• 金额: $ 2.26万
• 依托单位: Dalhousie University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=615743
• 关键词: Yeast; plasmid; segregation; investigation; host

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.

2微米质粒是在大多数面包酵母（酿酒酵母）菌株的细胞核中发现的多拷贝环状DNA。2微米的质粒被称为完美的“自私”DNA;质粒的存在不会对宿主产生不利影响，但也不会带来益处。质粒通过借用宿主机器进行复制和分离来确保自身的维持。在宿主细胞分裂过程中质粒拷贝的均等分配需要质粒蛋白Rep 1和Rep 2与质粒STB基因座处的重复序列相关联。Rep蛋白的结合招募宿主蛋白，帮助在STB建立功能性分配复合物。Rep蛋白/STB复合物允许质粒在分离的染色体或染色体分离装置的某些组分上搭便车，但其机制仍有待建立。一种假设认为酵母染色体的分配序列可能是从祖先的2微米质粒中获得的。在这项研究中，生物化学，分子遗传学和细胞生物学的方法将被用来确定和调查宿主蛋白质借用的2微米质粒，使其遗传。正在评估STB序列促进的RNA表达，以确定其是否具有可能代表与染色体分配序列共享的特征的功能意义。将使用DNA保护和蛋白质-DNA缔合测定来研究在我们的研究中鉴定的宿主蛋白质的不存在对STB染色质的组织和由STB指导的RNA表达的影响。我们的研究将有助于确定有助于染色体生物学特定方面的宿主蛋白质，并揭示其他蛋白质的新功能。我们还使用分子遗传学的方法来调查所需的序列在STB分区能力和比较基因组学的方法，其中分区蛋白质和序列的2微米相关的质粒在其他物种的芽殖酵母将进行研究，以进一步了解这些自私的DNA的生物学和进化。该项目将确定2微米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件（如病毒）是如何维持的，并提供有关进化过程中如何产生分配功能的见解。