17. May I Please See Your License? (1,304)

When the genome is replicated, the cell needs to ensure regions aren't copied multiple times.

If that were to happen, it would be like a novel in which random sentences, parts of sentences, chapters and parts of chapters are randomly repeated one or more times. In a genome, such repetitions would cause serious problems.

In this chapter, we take a look at the tight regulation involved in making sure that the cell replicates the genome once and only once and with no repeated sections.

The focus of this chapter is replication origin licensing. An origin that's licensed is capable of initiating DNA replication in S phase.

Although human genome replication occurs during S-phase, the events I'm about to describe--the attachment of the first initiator protein to a replication origin and the licensing of that origin--take place in G1.

We'll soon appreciate why there must be temporal separation between preparation for replication (licensing) from the execution of replication.

Once an origin is licensed in G1, it's ready to serve as the gathering point for a large array of replication-related proteins that will ultimately form two replisomes and two corresponding replication forks that will move in opposite directions away from the origin copying DNA.

These two terms--replisome and replication fork--are often used to label the site of active DNA replication. But they refer to different things. The replisome is the protein machinery (the workers and machines); the replication fork is the DNA structure it acts on (the work site).

Licensing can't be permitted in S phase. If it could, an origin could be licensed in S phase multiple times, which would cause the DNA near that origin to be replicated multiple times. That would create a mess. Some regions of the genome would be copied once while others would be copied two or more times.

Let me offer a human analogy for replication origin licensing.

A local county fair will be held in June and everyone in your county is emailed one free ticket in May. Each ticket permits one entry. No more tickets can be purchased in June. Given that, everyone will be able to attend the fair once and only once in June.

Similarly, every 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 (resident) will be able to initiate replication (attend the fair) only once in S-phase (June)!

That's what G1 licensing accomplishes: it permits one and only one replication event per segment of the genome in S phase.

Now let's take a look at how the cell licenses replication origins in G1.

A licensed replication origin is one that has two MCM protein complexes loaded around double-stranded DNA in inactive form in a head-to-head configuration. At this stage, MCM is a precursor to a helicase--an enzyme that unwinds double-stranded DNA in S phase at the front of the replication fork--but it needs some additional proteins to become functional.

The trick to avoiding replication duplication is that getting the two MCM's into that position requires at least three other helper proteins: ORC, Cdc6 and Cdt1. These helper proteins are active in G1 phase. But in S phase they're rendered inactive.

Because they are inactive in S phase, no additional MCMs can be loaded onto replication origins in S phase to license them.

Let's take a look at the molecular details behind this.

ORC and Cdc6 arrive

In G1, the first protein complex that lands on a replication origin is ORC (Origin Recognition Complex). ORC is made of six different protein subunits--a hexamer. It's shaped like a partially closed ring-shaped structure.

When ORC lands on a replication origin, the DNA lodges inside its central channel. Then a protein called Cdc6 binds to it.

Cdc6 attaches to ORC and has two effects. It closes its central channel, trapping the DNA inside. It also alters ORC's shape such that a platform for the future arrival of MCM is formed.

We now have a Cdc6 molecule bound to the ORC protein complex which, in turn, encircles and traps replication origin DNA.

Cdt1 and MCM arrive

At the same time, elsewhere in the nucleus, the protein Cdt1 attaches to MCM, the licensing factor. Like ORC, MCM is a large six-protein complex, or hexamer.

Cdt1 binding to MCM stabilizes in its open-ring conformation, which is needed for its attachment to replication origin DNA. Cdt1 binding also alters the shape in MCM to make it compatible for loading onto that ORC landing platform.

Once it arrives at the landing platform, MCM encircles the DNA there. At this point, Cdt1 has finished its job and so is released from MCM.

We now have ORC surrounding replication origin DNA and still bound to Cdc6. Next to the ORC-Cdc6 complex, one MCM protein complex has been loaded onto the DNA.

Now the loading machinery repositions to enable the cell to load a second MCM on the other side of ORC. The cell then uses roughly the same process I just described to load a second MCM on the DNA in a head-to-head configuration with the first MCM.

Loaded around double-stranded DNA in this head-to-head configuration, the two still inactive MCMs are referred to as the MCM-double hexamer, or MCM-DH.

This replication origin is now licensed.

Preventing re-licensing

The activities I just described occur in G1 and licenses tens of thousands of replication origins that become thus capable of initiating replication in S phase.

But what actually stops the system from running again in S phase? How does the cell ensure that new replication origins aren't re-licensed?

Basically, once the cell enters S phase, the conditions that allow for MCM loading will be destroyed.

But let's start from the beginning. We've discussed the cell cycle and I've emphasizing how a family of master regulator proteins called cyclins determine the cell cycle phases.

They do this by activating proteins called CDKs (cyclin-dependent kinases) which then transfer phosphate groups from ATP onto target proteins that, in turn, modify specific proteins to control progression through the cell cycle

Re-licensing in S phase is prevented because the specific cyclins active during S phase (cyclins E and A) turn on a specific new CDK (CDK2).

CDK2 in turn phosphorylates and deactivates all the helper proteins required for licensing: ORC, Cdc6, and Cdt1. With these three helper proteins deactivated, replication origins can no longer be licensed in S phase. That’s how licensing is restricted to G1.

Diving one level deeper, let's look at how exactly are these three proteins are deactivated by their phosphorylations.

ORC remains bound in part, but its ability to initiate new licensing is suppressed.

CDK2 phosphorylation of Cdc6 in S phase marks the protein for export out of the nucleus and for degradation.

Phosphorylation of Cdt1 flags it for the attachment of the protein marker ubiquitin which, in turn, flags it for degradation. Finally, another protein active in S phase--geminin--binds to Cdt1, rendering it inactive. 

Thus, in late G1 and S phases, CDK2 phosphorylates all three proteins required for origin licensing, rendering them inactive--either by export out of the nucleus, by degradation, or by the attachment of another protein that impedes its function.

With the three helper proteins out of the picture, no replication origin licensing can occur in S phase.

By the end of G1, tens of thousands of replication origins will be licensed. Each will be loaded with a pair of inactive MCM helicases--the MCM-DH.

But at this point, nothing has yet been copied. The DNA remains intact, and the helicases are still inactive. Licensing, in other words, prepares the stage--but the replication machinery has not yet begun to fill out or move.

In the next chapter, we’ll see how these loaded components are activated and assembled into replisomes—the molecular machines that unwind DNA and begin copying the genome.