title: Origin recognition complex binding to a metazoan replication origin.
abstract: The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) [1]. This six-subunit complex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily conserved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA interaction is well established for autonomously replicating sequence (ARS) elements in yeast in which the ARS consensus sequence [4] (ACS) constitutes part of the ORC binding site [2, 5]. Little is known about the ORC-DNA interaction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification origin (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-base pair (bp) ORC binding site and mapped the replication start site located adjacent to it. The binding of ORC to this 80-bp core region is ATP dependent and is necessary to establish further interaction with an additional 65-bp of DNA. This is the first time that both the ORC binding site and the replication start site have been identified in a metazoan amplification origin. Thus, our findings extend the paradigm from yeast ARS1 to multicellular eukaryotes, implicating ORC as a determinant of the position of replication initiation.
authors.affiliation: Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA.
authors.forename: A K
authors.initials: AK
authors.lastname: Bielinsky
authors.forename: H
authors.initials: H
authors.lastname: Blitzblau
authors.forename: E L
authors.initials: EL
authors.lastname: Beall
authors.forename: M
authors.initials: M
authors.lastname: Ezrokhi
authors.forename: H S
authors.initials: HS
authors.lastname: Smith
authors.forename: M R
authors.initials: MR
authors.lastname: Botchan
authors.forename: S A
authors.initials: SA
authors.lastname: Gerbi
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004261:DNA Replication; D018076:DNA, Complementary; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004175:Diptera / Q000235:genetics / Q000378:metabolism; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex; D018741:Replication Origin
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D051738:Origin Recognition Complex; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases