Fail-safe replication

How does the cell ensure that its DNA is replicated once and only once in every cell cycle? Replication takes place at many sites and must be regulated separately at each one. That's a tough job even in budding yeast, with ∼400 initiation sites, but a Herculean one in human cells, with as many as 100,000. Moreover, not all replication events are initiated at the same time.

Researchers in Joachim Li's lab (University of California, San Francisco, CA) are among those who have implicated three mechanisms in the prevention of reinitiation: phosphorylation of the origin recognition complex (ORC), downregulation of Cdc6 protein levels, and exclusion of the Mcm2–7 complex from the nucleus. Yet the authors were frustrated to find that when they disrupted any one of these mechanisms, the block to reinitiation remained intact.

One possible explanation is that any one of the mechanisms is sufficient to maintain control of reinitiation. “So we had to start knocking out multiple mechanisms. We did that, and the bottom line is that when we perturbed all three of them, when we prevented the ORC from being phosphorylated, and forced the Mcm to stay in the nucleus, and we ectopically expressed Cdc6 protein, now we could see cells reinitiate DNA replication,” Li reports. It makes perfect sense, he points out, that the cell would use multiple overlapping mechanisms as a fail-safe system, one that could all but guarantee that no segment of DNA would replicate more than once per cell cycle. ▪

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