The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin.
BACKGROUND
The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts.


RESULTS
The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin 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 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.
Romanowski
P
P
Wellcome/CRC Institute, Cambridge. pr1@mole.bio.cam.ac.uk
Madine
M A
MA
Rowles
A
A
Blow
J J
JJ
Laskey
R A
RA
D000818:Animals; D002467:Cell Nucleus; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051738:Origin Recognition Complex; D011817:Rabbits; D014981:Xenopus / Q000378:metabolism; D029867:Xenopus Proteins
D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D051738:Origin Recognition Complex; C097703:XORC2 protein, Xenopus; C102178:XOrc1 protein, Xenopus; D029867:Xenopus Proteins