THE XENOPUS ORIGIN RECOGNITION COMPLEX IS ESSENTIAL FOR DNA-REPLICATION AND MCM BINDING TO CHROMATIN

Background: The origin recognition complex (ORC) and the minichromosom e maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential far DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopu s MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essen tial for DNA replication. Here, we describe the different but interdep endent functions of the ORC and MCM complexes in DNA replication in Xe nopus egg extracts. Results: The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodeplet ion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MC M-depleted and ORC-depleted extracts restores replication capacity. OR C does not co-localize with sites of DNA replication during elongation . However, at initiation the two staining patterns overlap. In contras t to MCMs, which are displaced from chromatin during S phase, XOrc1 an d XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis, Permeable HeLa G1- and G2-phase nuc lei can replicate in ORC-depleted extract, consistent with the presenc e of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding oi ORC to chromatin does not requir e the presence of MCMs; however, the binding of MCM proteins to chroma tin is dependent on the presence of ORC. Conclusions: The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout inte rphase but, in contrast to S. cerevisiae ORC, if appears to be, at lea st partly, displaced from chromatin during mitosis, The binding of MCM proteins requires the presence of ORC. Thus, ?he assembly of replicat ion-competent chromatin involves the sequential binding of ORC and MCM s to DNA.