The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin

Abstract

The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

Interaction of Xenopus Cdc2·Cyclin A1 with the Origin Recognition Complex

“…Examination of XOrc1 and XOrc2 elution profiles suggests that only a part of XOrc2 is present in a complex with XOrc1, whereas significant amounts of XOrc2 appear to be present in a slightly smaller complex lacking XOrc1. This observation agrees with previously published data demonstrating that immunodepletion of ORC with anti-XOrc1 antibodies failed to immunodeplete XOrc2 completely (15). However, as XOrc1 immunodepletion abolished the ability of egg extract to support DNA replication, the complex lacking XOrc1 cannot perform all the functions required for initiation (14,15).…”

“…1 and 2). In Xenopus, ORC is bound to origins during both the pre-replicative and post-replicative period and the binding of Cdc6 and MCMs to chromatin is dependent on the presence of ORC (14,15,18). Indeed, a hierarchy of binding exists; ORC binds first, followed by ORCdependent Cdc6 binding and Cdc6-dependent MCM binding.…”

Orc1 Binding to Mitotic Chromosomes Precedes Spatial Patterning during G1 Phase and Assembly of the Origin Recognition Complex in Human Cells

“…In U2OS cells, depletion of Orc1 either with siRNA against the coding region of Orc1 or the 3ЈUTR of endogenous Orc1 resulted in loss of Mcm3 from chromatin (Fig. 5B), suggesting loading of Mcm3 protein on chromatin requires Orc1, consistent with earlier findings (26,69).…”

A topoisomerase II-dependent mechanism for resetting replicons at the S–M-phase transition

“…Immunostaining analysis shows that ORC2 was removed from chromatin upon mitotic entry in mock-depleted extracts (Fig. 1B, panel f), in agreement with previous results (Romanowski et al 1996;Cuvier et al 2006). In contrast, chromatin removal of ORC2 was strongly impaired upon topo II depletion (Fig.…”

“…2B, panel g), which does not target ssDNA regions. A checkpoint response might impair the dissociation of ORC, if cdc2 kinase was inhibited, as phosphorylation of RPA and/or ORC by this kinase reduces its binding to chromatin (Romanowski et al 1996;Cuvier et al 2006). However, ICRF did not lead to detectable levels of phosphorylated chk1 or RPA on chromatin (Fig.…”