Because eukaryotic cells use thousands of origins, the entire genome can be replicated in a timely manner. The use of multiple origins, however, creates a special problem in the timing of replication: the entire genome must be precisely replicated once, and only once, in each cell cycle so that no genes are left unreplicated and no genes are replicated more than once. How does a cell ensure that replication is initiated at thousands of origins only once per cell cycle?

The precise replication of DNA is accomplished by the separation of the initiation of replication into two distinct steps. In the first step, the origins are licensed—approved for replication. This step takes place early in the cell cycle when a replication licensing factor attaches to an origin. In the second step, the replication machinery initiates replication at each licensed origin. The key is that the replication machinery functions only at licensed origins. As the replication forks move away from the origin, the licensing factor is removed, leaving the origin in an unlicensed state, where replication cannot be initiated again until the license is renewed. To ensure that replication takes place only once per cell cycle, the licensing factor is active only after the cell has completed mitosis and before the next replication is initiated.