Replication Origin Licensing in G1

As we kick off the focus of this book—a deep dive into human genome replication—we’ll see that the process involves many different proteins operating in an extremely well-orchestrated and interconnected manner. Tight regulation of the process is necessary to ensure the genome is replicated once and only once and with extremely high accuracy.  

Although the copying of the human genome occurs almost exclusively during S-phase of the cell cycle, the events described in this post--that is, the attachment of the first initiator protein complex (called ORC) to a replication origin and the so-called licensing of that origin--take place in early- and mid-G1. We'll soon see why this must be the case--why there must be temporal separation between licensing and replication.

A licensed replication origin is simply defined as one capable of initiating DNA replication during S-phase. Once an origin is licensed in G1, its ready to serve as the gathering point for the large array of replication-related proteins that will ultimately form two replication forks that will move in opposite directions away from the origin replicating DNA.

In S phase, licensing cannot be permitted. If licensing could occur after G1, then an origin could be licensed multiple times during S-phase and thus the DNA near that origin could be replicated multiple times. That would create, in a word, a mess. Some genomic regions would be replicated once while others would be replicated two, three, or more times. A mess.

Let me offer a human analogy for replication origin licensing. Let's say that the local county fair will be this June, and everyone in your very generous county is emailed one free, non-transferable ticket in May. Each ticket allows for only one entry to the fair. And no additional tickets can be purchased in June. Given that scenario, everyone in the county will be able to attend the fair once and only once in June.

Analogously, every replication origin will be licensed (receive a ticket) in G1 (May). No additional licensing (ticket purchasing) can occur in S-phase (June). Thus, every replication origin (county resident) will be able to initiate replication (attend the fair) once and only once in S-phase (June)!

That's what licensing accomplishes: it permits one and only one replication event per segment of the genome. But how does the cell physically license replication origins in early- and mid-G1? It's time to find out!

ORC and Cdc6 arrive

The initial protein that lands on and thus designates a replication origin is called ORC (for Origin Recognition Complex). ORC is actually a protein complex comprised of six different polypeptide subunits. It's shaped like a not quite fully closed hand that forms a not quite fully closed central channel. When ORC lands on a replication origin the DNA lodges inside the central channel via an opening between two of its subunits.

With ORC now in place, a protein called Cdc6 crashing around the nucleus finds ORC and binds to it. Cdc6 attaches to ORC such that it closes off the central channel, trapping origin DNA inside the ORC-Cdc6 complex. Cdc6 binding to ORC also changes the shape of ORC such that a molecular platform is formed. This platform will be the landing spot for another very important protein complex called MCM2-7 that's involved in licensing.

Cdc6 has an ATP binding site and an ATP molecule must be attached to the Cdc6 for it to attach to ORC and close the gap. This ATP only needs to be attached. It doesn't have to be cleaved (with an associated release of energy). Soon though this ATP will, in fact, have to be cleaved for other energy-requiring licensing steps to occur.

To summarize, we now have the ORC protein complex bound to a Cdc6 molecule that effectively traps replication origin DNA inside ORC's central channel.

Cdt1 and MCM2-7 arrive

At the same time that ORC and Cdc6 are interacting, elsewhere in the nucleus the protein Cdt1--a so-called chaperone protein--attaches to another six-protein complex called MCM2-7. MCM2-7 is the inactive precursor of a type of protein complex called a helicase that's critical in replication. Helicases spearhead replication forks, separating the two strands of the double helix so they can be replicated, or copied.

Before being transformed into a helicase, MCM2-7 first needs to attach to origin DNA. The binding of Cdt1 to MCM2-7 stabilizes the MCM2-7 complex and modestly changes its shape in preparation for loading onto the now-ready ORC-Cdc6 landing platform. Specifically, Cdt1 stabilizes an "open ring" conformation of MCM2-7 that's critical for DNA loading. Cdt1 is referred to as a "chaperone protein." With its help, MCM2-7 will dock onto the ORC-Cdc6 loading platform and then attach to and encircle the DNA there.

Making MCM2-7 encircle DNA requires energy. This comes from cleavage of the ATP that's lodged in Cdc6's ATP binding site. When that ATP is cleaved, the energy released affects the shape of Cdt1 which in turn affects the shape MCM2-7 in a way that it further facilitates DNA binding and encirclement. But this cleavage of the ATP has another effect as well: it causes the release of Cdt1 from MCM2-7. Cdt1 has served its role. It is no longer needed.

To quickly review where we are, ORC is still bound to Cdt6 and to the replication origin. Next to the ORC-Cdc6 complex, one MCM2-7 protein complex has been loaded onto the DNA. But now the cell needs a second MCM2-7 to be loaded.

Now, let's do it again!

Now we repeat the process. A second Cdt1 attaches to a second MCM2-7 complex in the nucleoplasm and chaperones it to the ORC-Cdc6 platform. Once there, the second MCM2-7 will be loaded onto the DNA such that the heads (the fronts) of the two MCM2-7s will face each other. In this "head-to-head" configuration the two MCM2-7s are referred to as the MCM-double hexamer, or MCM-DH.

Now we have the two MCM2-7s loaded, but the cell needs to clean up a few things. This will require a second ATP to be cleaved in the Cdc6 binding site since energy will be required. As before, this second ATP cleavage causes the release the second Cdt1. But now, following the departure of the second Cdt1, the same ATP cleavage also causes Cdc6 to be released from ORC.

The result of all of this activity will be two MCM2-7 complexes attached to the replication origin DNA in a head-to-head configuration with ORC still present.

Preventing re-licensing

The licensing steps I just described happen in early- and mid-G1 to 30,000-50,000 human replication origins. All these licensed origins are, by definition, capable of initiating replication in S-phase. But the question I posed at the outset concerned how the cell ensures that replication origins fire only once during S-phase.

In fact, I'll rephrase that question to make it more actionable: "How does the cell ensure that no replication origins can be relicensed in S-phase?" Earlier I said that it was a matter of the cell environment being "conducive" versus "non-conducive" to licensing. We'll now see what that means.

Recall that in an earlier post, I reviewed the cell cycle, emphasizing how a family of master regulator proteins called cyclins determine the phase the cell cycle is in at a given point in time. They do this by activating proteins called CDKs (cyclin-dependent kinases) which, once activated, use ATP hydrolysis to phosphorylate different proteins in the nucleus to, in turn, effect the appropriate gene expression pattern for that phase.

In early G1, the operative kinase-CDK combinations are CDK4 and CDK6, both of which are activated by cyclin D. These two CDKs perform their jobs as master regulators by defining gene expression patterns in early G1. But neither is capable of phosphorylating Cdc6, Cdt1 or ORC. So conditions are such in early- and mid-G1 that Cdc6, Cdt1, and ORC are all in their un-phosphorylated, active forms and so are busy licensing replication origins.

Everything changes in late G1 and S phases. Cyclin D levels decrease, and cyclin E levels increase. Cyclin E activates its partner CDK2. Soon after, in S phase, cyclin E levels decline and cyclin A is expressed, which activates CDK1 and CDK2. CDK 2 will play the leading role in our story, activated by cyclin E in late G1 and by cyclin A in S phase.

Activated CDK2 proteins target Cdc6, Cdt1 and ORC for phosphorylation. What is the effect of these phosphorylations? I'll go one by one. By phosphorylating Cdc6 in late G1, it is now marked for export out of the nucleus. In S phase, CDK2 (now activated by cyclin A, not cyclin E) maintains the phosphorylated state of Cdc6, which also results in both nuclear export and degradation.

Activated CDK2 proteins also target Cdt1 (exclusively during S phase). By phosphorylating Cdt1, that protein is now flagged for the attachment of another protein degradation marker called ubiquitin. Also in S phase, another non-kinase protein called geminin  binds to Cdt1, rendering it inactive.

Finally, ORC will be phosphorylated in late G1 by CDK2 (activated by cyclin E). This weakens its replication origin licensing capability. Then, in S phase, ORC is phosphorylated again by CDK2 (now activated by cyclin A). This second phosphorylation dissociates ORC from the replication origin DNA and also leads to its destruction.

In summary, in late G1 and S, CDKs phosphorylate all three of the proteins that are central to replication origin licensing. This renders them all inactive, either by export out of the nucleus, by degradation, or by attaching to some other protein that impedes its functioning. Thus, the cell employs multiple redundant methods to ensure that licensing can't occur in late G1 and S phases.