De novo assembly of genuine replication forks on an immobilized circular plasmid in Xenopus egg extracts.

We describe an improved model of DNA replication in Xenopus egg extracts, in which a circular plasmid immobilized on paramagnetic beads is used as a template. DNA synthesis occurred on either circular or linear plasmids coupled to the beads, but only DNA synthesis on the circular plasmid was inhibited by geminin and a CDK inhibitor, p21. DNA synthesis on the circular plasmid occurred after a time lag, during which nuclear formation was probably occurring. Although pre-replicative complexes (pre-RCs) were formed soon after mixing plasmids with egg extracts, binding of CDC45, RPA, Pol alpha, delta and epsilon, and PCNA to the circular plasmid was delayed, but still correlated with DNA synthesis. Moreover, p21 inhibited binding of these replication fork proteins to the circular plasmid. Therefore, the circular plasmid, but not the linear plasmid, assembles bona fide replication forks in egg extracts. We conclude that this improved replication system will be useful for studying the mechanism of formation of replication forks in eukaryotic DNA replication.