18. Building a DNA Copying Machine (1,319)

A human cell enters S phase of the cell cycle. Across its genome, tens of thousands of replication origins have been prepared in advance. Everything is ready but nothing has started yet.

Each replication origin has been licensed, or approved for use, based on the presence of two head-to-head MCMs. This is the MCM double hexamer, or MCM-DH. The two MCMs are the inactive precursors of the two helicases that will lead the replication machinery down the DNA, unwinding the double helix so that both strands can be copied.

Initially, the two MCMs are inert. Their transformation into CMG helicases (the main human helicase) and the construction of two complete outward-facing DNA copying machines, or replisomes, is the topic of this chapter.

Assembling the replisome involves four precisely timed steps:

(1)  Transformation of both MCMs into complete (but inactive) CMG helicases

(2)  Activation of the CMG helicases.

(3)  Recruitment of proteins that will stabilize and synchronize the replisome

(4)  Recruitment of the DNA polymerases that will synthesize DNA.

Once these steps are complete, the cell will have constructed two fully functional replisomes led by two CMG helicases pointing in opposite directions away from the replication origin. Replisomes are like mobile factories for DNA synthesis.

Building the CMG Helicases

As the cell commits to DNA replication at the G1/S transition and into S phase, a new wave of regulatory enzymes becomes active. These enzymes are CDKs--cyclin-dependent kinases. Their job is to add small chemical tags called phosphates onto specific proteins, flipping their molecular switches.

These phosphate signals accomplish two things at once.

First, CDK phosphorylations prevent re-licensing of replication origins by disabling or removing the helper proteins (e.g.,ORC, Cdc6 and Cdt1) required to load new MCM complexes in S phase.

Second, CDKs, together with additional kinases, trigger the conversion of the two dormant MCM rings at the origin into active DNA-unwinding motors, or helicases--specifically, human CMG helicases.

The transformation of an MCM ring into a CMG helicase involves the addition of two other protein components: Cdc45 and GINS. “CMG” is an acronym for Cdc45-MCM-GINS.

But this transformation doesn’t happen with a snap of the fingers. Temporary scaffold proteins--short-lived assembly factors--gather at the origin, forming a construction platform. Then, multiple intermediary proteins recruit and attach the Cdc45 and GINS proteins to the MCM ring. Once these two proteins are added, the temporary scaffold proteins leave.

What remains are two complete CMG helicase machines facing in opposite directions. But even now, they are not activated or moving. That will require another protein: Mcm10. 

Activate the helicases

CMG helicase activation is defined as its transition to a protein complex that's started to unwind DNA. This involves the MCM double hexamer splitting into two single CMG helicases, separation of the two DNA strands at the replication origin (DNA melting), and each CMG helicase engaging one of the strands and starting to move.

CMG helicase activation establishes two outward-moving replication forks.

Mcm10 is a scaffolding protein that plays an essential role in stabilizing and enabling this transition. It steadies the newly opened DNA and supports the CMG helicase as it begins to move. Mcm10 acts like a stabilizing hand during start-up, ensuring that the motor grips the correct strand and that a stable replication fork is successfully launched.

We now have a replication origin with two activated CMG helicases starting to move in opposite directions.

Now that the double helix has begun to separate, we need to account for each of the two separated strands. One--called the leading strand--is threaded through the helicase ring. The other--the lagging strand--is pushed to the outside of the helicase. Given its vulnerability to nucleases that chew up single-stranded DNA, the lagging strand is also coated and protected by numerous RPA proteins.

As the CMG helicase begins to move, the next steps involve filling out the replisome with proteins that provide more structure to this copying machine and that ensure that the activities of the CMG helicases and the DNA polymerases (yet to arrive) are synchronized.

Stabilize and coordinate the fork

When genome replication begins, the CMG helicase progresses down the double stranded DNA, separating it into two single strands.

Protein enzymes called DNA polymerases (there are three, each with a specific role) will follow closely behind, synthesizing new strands off the two existing template strands. These two activities--DNA strand separation and DNA synthesis, or polymerization--must be coordinated spatially and temporally. 

The next several proteins to arrive play structural and coordination roles in the replisome. They include TIMELESS, TIPIN, and CLASPIN (often referred to as the fork protection complex, or FPC) along with associated factors like AND-1.

These proteins organize, stabilize and coordinate the replication machinery rather than catalyze reactions themselves. They maintain the architecture of the replication fork—the Y-shaped region where DNA is being unwound and copied.

Recruit the DNA polymerases

Finally, the enzymes that will synthesize the DNA--the DNA polymerases--arrive. There are three: DNA polymerases alpha-primase, epsilon and delta. 

The first of these--DNA polymerase alpha-primase--is unique in that it can synthesize short RNA–DNA starter segments called primers. The enzyme first synthesizes an 8-12 nucleotide stretch of RNA. Then it adds about 20-30 nucleotides of DNA using a different part of the protein.

The other two DNA polymerases can’t start synthesizing without this roughly 30- to 40-nucleotide stretch of RNA and DNA laid down by DNA polymerase alpha-primase.

Next, enter the other two DNA polymerases. These will carry out the bulk of DNA synthesis during genome replication.

We'll discuss this in more detail in the coming chapters. But, for now, I'll just note that DNA polymerase epsilon synthesizes continuously on the leading strand and DNA polymerase delta synthesizes discontinuously (generating small fragments that are then stitched together) on the lagging strand.

To keep both DNA polymerases from repeatedly falling off the DNA during synthesis, the cell deploys one of the most elegant machines in the cell: a ring-shaped sliding clamp. Think of a metal washer.

Without the sliding clamp, DNA polymerases have low processivity--they fall off the DNA frequently. With sliding clamps, they stay on the DNA, increasing their processivity.

The sliding clamp is called PCNA (Proliferating Cell Nuclear Antigen). This critical protein complex has a dedicated loader protein called Replication Factor C (RFC). RFC grabs onto the PCNA sliding clamp, opens its ring, inserts the double stranded DNA, then closes the ring around the DNA like a bracelet.

PCNA sliding clamps always accompany DNA polymerases epsilon and delta during synthesis.

Two machines, not one

Because initially two MCM rings were positioned head-to-head at the replication origin, two complete replisomes will form at each origin. I've described them so far as moving away from each other in opposite directions, copying the genome bidirectionally. I'll modify that depiction in a moment.

Replisomes are far from single enzymes. I haven’t come close to mentioning every replisome protein, but a replisome is made up of about 30-40 different protein species all with very specific roles. Building one is a carefully staged molecular construction project--one that takes place tens of thousands of times during one cell division.

Now let me modify the image I've left you with. This replisome--these 30-40 interacting, coordinated protein--don't exactly run down the DNA. No. It's more like the DNA is run through the replisome.

The replisome remains more or less stationary, and the DNA is run through it like a piece of fabric is run through a sewing machine.

So, at this point, two fully assembled, relatively immobile replisomes are rapidly processing double-stranded DNA being run through them.

But we haven't yet discussed how the DNA synthesis actually works. How is the leading strand copied continuously while the lagging strand is built in fragments? How are these two very different processes coordinated at a single moving fork?

Those questions take us to the next chapter of the story: the mechanics of leading- and lagging-strand DNA synthesis.