title: Forced binding of the origin of replication complex to chromosomal sites in Drosophila S2 cells creates an origin of replication.
abstract: Origins of replication in higher eukaryotes appear to lack specific sequence characteristics and those mapped often appear to be spread over several kilobases. This has complicated the study of site-specific events at origins of replication in vivo. Here we show that fusion of a Gal4-binding domain to proteins of the origin of replication complex (Orc) is sufficient to direct initiation to Gal4-binding sites inserted in the Drosophila S2 cell chromosome. The activation appears to go via an authentic route, taking place only in the S phase of the cell cycle and involving the formation of a prereplication complex. We have also shown that the origin-associated acetylation of histone H4 at K12 can be directed to the region of Orc binding by the presence of Orc. We expect that this system can provide a useful tool for the study of site-specific events at origins of replication in higher eukaryotes and a means to dissect Orc-dependent and Orc-independent events at origins.
authors.affiliation: Department Basic Medical Sciences, St Georges University London, Cranmer Terrace, London, SW17 0RE,  UK.
authors.forename: Gilles
authors.initials: G
authors.lastname: Crevel
authors.forename: Sue
authors.initials: S
authors.lastname: Cotterill
mesh_terms: D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D059006:Chromosomes, Insect / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics*; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D051738:Origin Recognition Complex / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics*; D016196:S Phase; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C504976:Cdc6 protein, Drosophila; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C052130:GAL4 protein, S cerevisiae; D006657:Histones; D051738:Origin Recognition Complex; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008239:Lysine