title: Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone.
abstract: Genome-scale mapping of pre-replication complex proteins has not been reported in mammalian cells. Poor enrichment of these proteins at specific sites may be due to dispersed binding, poor epitope availability or cell cycle stage-specific binding. Here, we have mapped sites of biotin-tagged ORC and MCM protein binding in G1-synchronized populations of Chinese hamster cells harboring amplified copies of the dihydrofolate reductase (DHFR) locus, using avidin-affinity purification of biotinylated chromatin followed by high-density microarray analysis across the DHFR locus. We have identified several sites of significant enrichment for both complexes distributed throughout the previously identified initiation zone. Analysis of the frequency of initiations across stretched DNA fibers from the DHFR locus confirmed a broad zone of de-localized initiation activity surrounding the sites of ORC and MCM enrichment. Mapping positions of mononucleosomal DNA empirically and computing nucleosome-positioning information in silico revealed that ORC and MCM map to regions of low measured and predicted nucleosome occupancy. Our results demonstrate that specific sites of ORC and MCM enrichment can be detected within a mammalian initiation zone, and suggest that initiation zones may be regions of generally low nucleosome occupancy where flexible nucleosome positioning permits flexible pre-RC assembly sites.
authors.affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306, USA.
authors.forename: Yoav
authors.initials: Y
authors.lastname: Lubelsky
authors.forename: Takayo
authors.initials: T
authors.lastname: Sasaki
authors.forename: Marjorie A
authors.initials: MA
authors.lastname: Kuipers
authors.forename: Isabelle
authors.initials: I
authors.lastname: Lucas
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Le Beau
authors.forename: Sandra
authors.initials: S
authors.lastname: Carignon
authors.forename: Michelle
authors.initials: M
authors.lastname: Debatisse
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Prinz
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Dennis
authors.forename: David M
authors.initials: DM
authors.lastname: Gilbert
mesh_terms: D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D016466:CHO Cells; D019731:Carbon-Nitrogen Ligases / Q000378:metabolism; D002843:Chromatin / Q000737:chemistry; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D016193:G1 Phase; D009707:Nucleosomes / Q000378:metabolism*; D018741:Replication Origin; D012097:Repressor Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009707:Nucleosomes; D012097:Repressor Proteins; D013762:Tetrahydrofolate Dehydrogenase; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli