Origin recognition complex binding to a metazoan replication origin

The initiation of DNA replication in eukaryotic cells at the onset of S pha se requires the origin recognition complex (ORC) [1]. This six-subunit comp lex, first isolated in Saccharomyces cerevisiae [2], is evolutionarily cons erved [1]. ORC participates in the formation of the prereplicative complex [3], which is necessary to establish replication competence. The ORC-DNA in teraction is well established for autonomously replicating sequence (ARS) e lements 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 inte raction in metazoa. For the Drosophila chorion locus, it has been suggested that ORC binding is dispersed [6]. We have analyzed the amplification orig in (ori) II/9A of the fly, Sciara coprophila. We identified a distinct 80-b ase pair (bp) ORC binding site and mapped the replication start site locate d adjacent to it. The binding of ORC to this 80-bp core region is ATP depen dent and is necessary to establish further interaction with an additional 6 5-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 ori gin. Thus, our findings extend the paradigm from yeast ARS1 to multicellula r eukaryotes, implicating ORC as a determinant of the position of replicati on initiation.