Hoggard Timothy, Liachko Ivan, Burt Cassaundra, Meikle Troy, Jiang Katherine, Craciun Gheorghe, Dunham Maitreya J, Fox Catherine A
Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
Department of Genome Sciences, University of Washington, Seattle, Washington 98105.
G3 (Bethesda). 2016 Apr 7;6(4):993-1012. doi: 10.1534/g3.116.027904.
The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid partitioning and suggest underlying biological roles shared by such elements.
质粒在酿酒酵母中繁殖的能力对于确定真核染色体控制元件起到了重要作用。稳定繁殖既需要质粒复制（这需要一个染色体复制起点，即一个自主复制序列 ，ARS），也需要将质粒分配到分裂细胞中，这需要一个用于分离的染色体着丝粒或一个质粒分配元件。虽然我们对酵母 ARS 和着丝粒的了解相对深入，但我们对可作为质粒分配元件的染色体区域了解较少。存在于芽殖酵母转录沉默子和端粒中的 Rap1 蛋白结合位点（RAP1）是一种已知的质粒分配元件，其作用是将质粒锚定到内核膜（INM）上，进而促进质粒向子细胞的分配。这种依赖 Rap1 的 INM 锚定在增强转录沉默和端粒稳定性的高阶染色体结构中也具有重要的染色体作用。因此，质粒分配可以反映染色体结构和生物学的基本特征，但尚未有关于质粒分配元件的系统筛选报道。在这里，我们将深度测序与包含数千个短 ARS 片段的质粒文库的竞争性生长实验相结合，以鉴定新的质粒分配元件。使用仅在着丝粒存在与否方面有所不同的文库进行竞争性生长实验。通过比较两个实验中 ARS 片段的行为，我们能够鉴定出可能驱动质粒分配的序列。除了沉默子 RAP1 位点外，我们还鉴定出 74 个新的假定质粒分配基序，预计它们可作为 DNA 结合蛋白的结合位点，这些蛋白在基因表达的负调控和 G2/M 期相关生物学中发挥富集作用。这些数据扩展了我们对可能在质粒分配中起作用的染色体元件的认识，并暗示了这些元件共有的潜在生物学作用。
