The initiation of DNA replication in eukaryotic cells at the onset of S phase requires the origin recognition complex (ORC) . This six-subunit complex, first isolated in Saccharomyces cerevisiae , is evolutionarily conserved . ORC participates in the formation of the prereplicative complex , 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  (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 . 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.
Bibliographical noteFunding Information:
We thank D. Gilbert for antibodies, B. Stillman for poly [d(AG)]-poly [d(CT)] competitor DNA, F. Urnov for preliminary experiments and discussions, K. Veronina for help with RIP mapping, I. Ho for help with Western blots, K. Zaret for protocols, S. Bell and J. Diffley for helpful suggestions, E. A. Hendrickson for reading the manuscript, and S. Lange for help with the figures. Pertinent ori II/9A sequence information is deposited in GenBank with accession number AF332610. A.K.B. is a Special Fellow of the Leukemia and Lymphoma Society, and E.L.B is a Damon Runyon Fellow. This work was supported by the National Institutes of Health grant GM35929 to S.A.G. and the National Institutes of Health grant CA30490 to M.R.B.
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