title: High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements.
abstract: In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.
authors.affiliation: Laboratory of Chromosome Replication, Van Andel Research Institute, 333 Bostwick Ave NE Grand Rapids, MI 49503, USA.
authors.forename: Fujung
authors.initials: F
authors.lastname: Chang
authors.forename: Caitlin D
authors.initials: CD
authors.lastname: May
authors.forename: Timothy
authors.initials: T
authors.lastname: Hoggard
authors.forename: Jeremy
authors.initials: J
authors.lastname: Miller
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Fox
authors.forename: Michael
authors.initials: M
authors.lastname: Weinreich
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004265:DNA Helicases / Q000378:metabolism; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D009707:Nucleosomes; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D004265:DNA Helicases