title: High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.
abstract: 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.
authors: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
authors: Timothy
authors: T
authors: Hoggard
authors: Department of Genome Sciences, University of Washington, Seattle, Washington 98105.
authors: Ivan
authors: I
authors: Liachko
authors: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
authors: Cassaundra
authors: C
authors: Burt
authors: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
authors: Troy
authors: T
authors: Meikle
authors: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706.
authors: Katherine
authors: K
authors: Jiang
authors: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706 Department of Mathematics, College of Letters and Science, University of Wisconsin, Madison Wisconsin 53706.
authors: Gheorghe
authors: G
authors: Craciun
authors: Department of Genome Sciences, University of Washington, Seattle, Washington 98105.
authors: Maitreya J
authors: MJ
authors: Dunham
authors: Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin 53706 cfox@wisc.edu.
authors: Catherine A
authors: CA
authors: Fox
mesh_terms: D001483:Base Sequence; D002503:Centromere / Q000235:genetics*; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D019295:Computational Biology / Q000379:methods; D004252:DNA Mutational Analysis; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D063990:Gene Ontology; D059014:High-Throughput Nucleotide Sequencing; D009154:Mutation; D059372:Nucleotide Motifs; D010957:Plasmids / Q000235:genetics*; D011485:Protein Binding; D018741:Replication Origin; D004718:Saccharomycetales / Q000235:genetics* / Q000378:metabolism; D037941:Silencer Elements, Transcriptional; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins
keywords: DNA replication origins; deep mutational scanning; plasmid partitioning; silencers