High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.
High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.
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DOI：
10.1534/g3.116.027904
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发表时间：
2016-04-07
期刊：
影响因子：
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通讯作者：
Fox CA
中科院分区：
文献类型：
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作者：
Hoggard T;Liachko I;Burt C;Meikle T;Jiang K;Craciun G;Dunham MJ;Fox CA
质粒在酿酒酵母中繁殖的能力有助于确定真核染色体控制元件。稳定繁殖既需要质粒复制，这需要染色体复制起源（即ARS），也需要质粒分布到分裂细胞，这需要染色体着丝粒分离或质粒分裂元件。虽然我们对酵母ars和着丝粒的了解相对较先进，但我们对染色体区域作为质粒分裂元件的了解较少。Rap1蛋白结合位点（Rap1）存在于出芽酵母的转录沉默子和端粒中，是一种已知的质粒分配元件，其功能是将质粒固定在核膜上，从而促进质粒分布到子细胞。这种依赖rap1的inm锚定在高阶染色体结构中也具有重要的染色体作用，可增强转录沉默和端粒稳定性。因此，质粒分配可以反映染色体结构和生物学的基本特征，但对质粒分配元件的系统筛选尚未见报道。在这里，我们将深度测序与含有数千个短ARS片段的质粒库的竞争生长实验相结合，以鉴定新的质粒划分元件。竞争生长实验是用文库进行的，这些文库仅在有或没有着丝粒方面有所不同。在两个实验中比较ARS片段的行为使我们能够确定可能驱动质粒分裂的序列。除了沉默者RAP1位点外，我们还发现了74个新的推测质粒分配基序，这些基序被预测为DNA结合蛋白的结合位点，这些DNA结合蛋白在基因表达的负调控和G2/ m期相关生物学中发挥作用。这些数据扩展了我们对可能在质粒分裂中起作用的染色体元素的认识，并提出了这些元素共享的潜在生物学作用。
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.