If life is the product of advanced bioengineering, one of the central expectations about living processes is that they will reflect extreme sophistication. Recent studies of the DNA replication of eukaryotes continue to support this prediction[1,2]. And they do so in way that continues to underscore how these sophisticated processes have been designed to promote accuracy and screen out mistakes/errors.

Eukaryotes typically possess large and complex genomes, which require multiple origin points that initiate their replication. These sites are called ORCs (origin recognition complexes). The problem, however, is ensuring they are all activated about the same time and not one is reactivated prior to the completion of chromosomal synthesis. Otherwise, illicit replication initiations will lead ultimately to genomic instability that is likely to trigger cell death or other deleterious conditions. The bottom line is that replication has to be very tightly regulated to ensure daughter cells receive one, and only one, complete copy of the genome.

A process known as licensing ensures that chromosomes are able to be replicated only after they have successfully passed through mitosis and find themselves in a new cell. And the mechanisms of licensing are slowly being teased apart, uncovering a networks of control systems with built in safety features.

To understand the control of the initiation of DNA replication, we first need to consider some of the basic steps in the initiation of eukaryl DNA replication. First, chromosomes contain multiple sets of sequences that bind to a group of proteins to form the ORC. This multi-protein/DNA complex remains intact throughout the cell cycle, yet is completely insufficient to trigger DNA replication. To trigger DNA replication, another hexameric protein complex, known as the MCM (minichromosome maintenance) complex must be recruited to the ORC. It is the MCM which effectively licenses the DNA for replication, as it is composed of several helicases that unwind the DNA to facilitate replication. So how do you get the MCM on the ORC? Two other proteins work in concert to recruit MCM. The first is known as Cdc6. When the cell is ready to replicate its DNA, Cdc6 is recruited by the ORC. Another protein, Cdt1, is then loaded and it potentiates the activity of Cdc6. Yet another protein, known as mcm10 may also be recruited to facilitate the loading of MCM (since MCM does not seem to physically contact Cdc6) [3].

At this point, one might be tempted to argue that this all has the smell of a Rube Goldberg machine, as why not simply allow the ORC to directly recruit the MCM without the intermediaries? The answer here is as in many cases elsewhere - the additional activity allow for more points of regulation to increase accuracy and buffer against mutation.

The design problem that is solved by this level of complexity is this: once you load the MCM onto a replication origin, you don't want to reload another MCM until after synthesis has been completed and mitosis has occurred. And one way this is ensured is through the use of all three components: the ORC proteins, the Cdc6/Cdt1 complex, and the MCM.

It turns out that all three complexes can be independently phosphorylated by a protein kinase known as Clb-Cdc28. This kinase has a dual role. Once the entire initiation complex has formed, it is "turned on" by Clb-Cdc28 (which was just previously activated itself) through phosphorylation. Yet the phosphorlyated complexes have not only been altered to initiate replication, but can no longer reinitiate replication. For example, when phosphates are attached to Cdc6, and it dissociates from the ORC after DNA synthesis begins, it is marked for degradation. And any phosphorylated MCMs are now targeted for transport out of the nucleus. The researchers who uncovered these three distinct pathways for preventing reinitiation summarize their findings as follows:

I've highlighted the last portion of this abstract to suggest that that sophisticated mechanism to prevent reinitiation also appears irreducibly complex. That is, we can now see the logic of the "Rube Goldberg" complexity of this system - we have three fail-safe mechanisms to prevent the reinitiation of any of those thousands of replication origins. Only when all three fail at the same place does reinitiation proceed.

Yet there is another feature to the licensing story that involves the neglected ingredient from the above story - Cdt1. Another nuclear protein, called geminin, has been found which binds to Cdt1 and this binding, in turn, prevents the loading of the MCM [2]. This constitutes another mechanism to prevent re-replication. As cells enter late S phase and G2, Cdt1 expression and levels drop. At the same time, geminin is expressed. Thus, any stray Cdt1 that has not been removed will bind to geminin, thus preventing it from loading in G2.

From: Science 290, p.2272

Geminin may also play an important role coupled to checkpoint surveillance. If the DNA-being-replicated has been previously damaged, pathways feed this information to the protein kinases, whose activity is thus repressed. This repression blocks the cell cycle from continuing until the damage can be repaired. Yet when sitting in this stalled state, the low level of kinase activity might permit a few replication origins to be loaded by the factors discussed above. But with geminin in place to soak up Cdt1, a complete initiation complex will not reform.

Thus, we can see that both Cdc6 and Cdt1 are regulated differently. This buffers against mutation. As the authors of the geminin study note, "This diversity of targets prevents escape from re-replication control through mutation in the gene of a single target gene."

The interesting thing about geminin as that it thus far appears metazoan-specific. It has been found in mammals, frogs, and flies, but is missing from plants and yeast. This makes sense in light of the way yeast and metazoans regulated Cdc6 activity. In yeast, phosphorylayed Cdc6 is degraded (as explained above), but in metazoans, it is shuttled out of the nucleus (like MCM). Since proteins may more likely escape transport than degradation, geminin may be an important back-up control mechanism. On the other hand, as some have suggested, geminin may be a target that helps couple DNA replication regulation to developmental processes.

Included below are some more abstracts for your consideration [4-6].

1. Nguyen VQ, Co C, Li JJ. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 2001 Jun 28;411(6841):1068-73

2. Wohlschlegel JA, Dwyer BT, Dhar SK, Cvetic C, Walter JC, Dutta A. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 2000 Dec 22;290(5500):2271-3

3. Homesley L, Lei M, Kawasaki Y, Sawyer S, Christensen T, Tye BK. Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins. Genes Dev 2000 Apr 15;14(8):913-26.

4. Initiating DNA synthesis: from recruiting to activating the MCM complex. J Cell Sci 2001 Apr;114(Pt 8):1447-54

5. Yanow SK, Lygerou Z, Nurse P. Expression of Cdc18/Cdc6 and Cdt1 during G(2) phase induces initiation of DNA replication. EMBO J 2001 Sep 1;20(17):4648-56

6. Tye BK. MCM proteins in DNA replication. Annu Rev Biochem 1999;68:649-86