With their multiple origins, how does the eukaryotic cell know which origins have been already replicated and which still await replication?
An observation: When a cell in G2 of the cell cycle is fused with a cell in S phase, the DNA of the G2 nucleus does not begin replicating again even though replication is proceeding normally in the S-phase nucleus. Not until mitosis is completed, can freshly-synthesized DNA be replicated again.
Two control mechanisms have been identified — one positive and one negative. This redundancy probably reflects the crucial importance of precise replication to the integrity of the genome.
Licensing: positive control of replication
In order to be replicated, each origin of replication must be bound by:
- an Origin Recognition Complex of proteins (ORC). These remain on the DNA throughout the process.
- Accessory proteins called licensing factors. These accumulate in the nucleus during G1 of the cell cycle. They include:
- Cdc-6 and Cdt-1, which bind to the ORC and are essential for coating the DNA with
- MCM proteins. Only DNA coated with MCM proteins (there are 6 of them) can be replicated.
Once replication begins in S phase,
- Cdc-6 and Cdt-1 leave the ORCs (the latter by ubiquination and destruction in proteasomes).
- The MCM proteins leave in front of the advancing replication fork.