17. May I Please See Your License? (1,200;7/2)

When the genome is replicated, the cell needs to ensure regions aren't copied multiple times. That 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 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 with no repeated sections. The mechanism is called replication origin licensing. A licensed origin is capable of initiating DNA replication in S phase.

Although genome replication occurs in 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 just prior to S phase in G1. We'll soon understand why there must be temporal separation between the preparation for replication (licensing) and replication itself.

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.

A quick clarification. These two terms--replisome and replication fork--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).

Back to licensing. It 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 multiple times.

Let me offer a human analogy for replication origin licensing.

A 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. Thus, 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 more licensing (ticket purchases) can occur in S-phase (June). Thus, every origin (resident) can 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 accomplishes this-- how it licenses replication origins.

First a definition. Formally, a licensed replication origin is one that has two large multi-protein complexes (MCMs) loaded around double-stranded DNA in a head-to-head configuration. At licensing, these two MCMs are inactive precursors to another very important multi-protein complexes: DNA helicases.

DNA helicase unwinds double-stranded DNA in S-phase at the front of the replication fork. But these outward-facing MCMs each need two additional proteins to become functional DNA helicases.

The trick to avoiding replication duplication is that getting the two MCMs 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 the cell inactivates them. Because these helper proteins are eliminated in S phase, no additional MCMs can be loaded onto replication origins in S phase.

Let's take a look at some of the the molecular details of replication origin licensing.

ORC and Cdc6 arrive

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

When ORC lands on a replication origin, the DNA there lodges inside the ring's open channel. Then another protein called Cdc6 binds to ORC. Cdc6 closes ORC's central channel, trapping the DNA inside. It also alters ORC's shape, creating a platform that will host the future arrival of an MCM-Cdt1 complex.

Cdt1 and MCM arrive

Elsewhere in the nucleus, a protein called Cdt1 attaches to an MCM, which is itself a large protein complex. Cdt1 binding to MCM stabilizes in its open-ring conformation, which is needed for its eventual attachment to replication origin DNA. Cdt1 binding also alters the shape of MCM to make it compatible for loading onto that ORC landing platform.

Once MCM-Cdt1 arrives at its docking platform, it forms a temporary complex with ORC-Cdc6. This complex isn't merely a docking intermediate. It's a partially assembled loading machine in which ORC, Cdc6, Cdt1, and MCM are physically interlocked and guiding the DNA toward the MCM channel and then positioning it there. Now MCM is loaded onto and surrounds the DNA

At this point, Cdt1 has finished its job and is released from MCM. Cdc6 leaves as well. ORC remains in position, ready to load a second MCM facing in the opposite direction.

The cell then uses the same process to load a second MCM on the DNA in what scientists refer to as a "head-to-head configuration" with the first MCM. Loaded around double-stranded DNA like this, the two still inactive MCMs are referred to as the MCM-double hexamer, or MCM-DH.

This replication origin is now licensed.

Once clarification. The term "head-to-head" can be confusing. What scientists first defined as the heads of the two MCM molecules do face each other on either side of ORC. But after these MCMs become DNA helicases, they will travel along the DNA away from the replication origin. So, in a sense, once the MCMs become DNA helicases, they travel tail-first.

Preventing re-licensing

As I mentioned, the activities I just described occur in G1 and results in the licensing of tens of thousands of replication origins. They are now capable of initiating replication in S phase. But what actually disallows licensing in S phase? How does the cell ensure that new replication origins aren't re-licensed?

Essentially, once the cell enters S phase, the conditions that allowed for MCM loading in G1 will be destroyed.

But let's slow down. We've discussed the cell cycle and specifically how a family of master regulator proteins called cyclins determines the cell cycle phases. They do this by activating proteins called CDKs (cyclin-dependent kinases) which transfer phosphate groups from ATP onto target proteins that, in turn, phosphorylate other proteins to control progression through the cell cycle.

Re-licensing in S phase is prevented because the specific cyclins that become active during S phase (cyclins E and A) turn on a new CDK (CDK2). Activated CDK2 then phosphorylates and deactivates all three of the helper proteins required for licensing: ORC, Cdc6, and Cdt1. With these helper proteins deactivated, origins can no longer be licensed. The cell eliminates its own ability to license origins.

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. Licensing prepares the stage, but the replication machinery has not yet begun to fill out.

In the next chapter, we’ll take a look at the assembly of the replication machinery, which isrefered to as the replisome.