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.

Role of the Orc6 Protein in Origin Recognition Complex-Dependent DNA Binding and Replication in Drosophila melanogaster

“…In contrast, Drosophila Orc6 is an integral part of the complex and is required for DNA binding by ORC. Moreover, the ORC consisting of subunits 1 to 5 [ORC (1)(2)(3)(4)(5)] and lacking the Orc6 subunit did not complement ORC-depleted extracts for DNA replication (8).…”

Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins

“…In flies, a subset of ORCbinding sites have been identified at loci that promote developmentally regulated DNA amplification through repeated rounds of replication. (23,24) In human cells, certain intergenic regions and CpG islands seem to be enriched in ORC, (25,26) but a consensus binding sequence has not been identified.…”

Duplication of the Genome in Normal and Cancer Cell Cycles

“…A recent elegant report of ORC binding and RIP mapping at the II/9A locus indicates that replication initiates adjacent to the ORC binding site as it does in S. cerevisiae. 52 This encourages the idea that although the regulation is likely more complex in metazoa, the basic mechanism for pre-RC activation and replication initiation is conserved with yeast. To date, the chorion locus of Drosophila and the II/9A locus of Sciara are the two origins in metazoa for which ORC binding has been shown.…”