18. Diving Deeper (1,593)

Inside every dividing cell, rings of protein race along DNA. Some pry apart the double helix, strand by strand. Others snap into place to keep the copying machinery from slipping off as it works. Two of the most important of these molecular machines are the CMG helicase and the PCNA sliding clamp.

The helicase acts as both engine and strand separator. It leads the replisome forward along the double helix while simultaneously splitting apart the two intertwined strands so they can be copied. PCNA plays a very different role. It forms a movable clamp that fastens a DNA polymerase to its template DNA strand, preventing the enzyme from falling off as it synthesizes new DNA. At the same time, the clamp serves as a docking platform that allows other proteins involved in replication to attach to it.

Structurally, the two machines could hardly look more different. The CMG helicase—specifically its MCM core—is long and barrel-shaped, built to house a motor within its central channel. The PCNA sliding clamp, by contrast, is a flat ring, more like a thick washer. The helicase actively drives itself along DNA using chemical energy. The sliding clamp has no motor at all; it simply glides along, pulled forward by the polymerase it secures.

CMG Helicase

Let's take a closer look at how the CMG helicase accomplishes both propulsion and DNA strand separation. It is the MCM component of the CMG helicase that contains both the motor that propels the helicase along the DNA, and the mechanism that splits the double helix into two single strands. The Cdc45 and GINS components of the CMG play critical but supporting roles. 

I introduced the MCM protein complex in Chapter __. Recall that it's an elongated six protein ring. The six subunits are similar but not identical. Each has a top portion, or tier, and a bottom tier. The six subunits are arranged such that the top tiers form a ring that creates an internal channel, and the bottom tiers continue that channel all the way through the protein complex.

We’ll soon see that the bottom tier of MCM contains a "motor" inside the channel that drives helicase motion. The top tier of MCM is separates the two strands as the CMG helicase moves along the DNA.

When first delivered to the origin of replication, MCM does not encircle the DNA. But the protein complex has a “side gate” between two of its six subunits that cracks open and allows double stranded DNA enter the central channel. The gate then closes, trapping the DNA inside. MCM now encircles the double-stranded DNA at the replication origin like a bracelet.

As a side note, remember that there are two MCMs aligned head-to-head at the origin. Thus, the steps I’m describing here occur at both MCMs roughly simultaneously.

Next, Cdc45 and GINS arrive and bind to the MCM ring. Their addition turns the MCM into a true CMG helicase. If the transition from the MCM passive ring to a CMG helicase misfires, replication stalls—and stalled replication is one of the most dangerous events a cell can face.

When Cdc45 and GINS bind, the MCM ring changes shape. The quiet bracelet becomes an active machine. Several things occur. First, the shape change promotes local melting of the duplex DNA within the top tier of the channel. The two strands of the double helix open and separate from each other.

Once the strands separate, the motor inside the bottom portion of the channel grabs one of the two strands—specifically, the leading strand. The conformation change also causes the other strand, the lagging strand, to be pushed to the outside of the MCM channel.

Now the leading strand is captured. The lagging strand expelled.  The motor engages. The two helicases pull apart and head in opposite directions. At this point, the two helicases have shifted from “bracelets around rope” to “motors gripping and pulling one strand.”

At the heart of the MCM lies the motor I’ve been referring to. It’s located on the inside of the channel in the lower tier of MCM. If we could peer down into the channel, we would see six positively charged loops arranged like a spiral staircase, each one slightly lower than the last.

DNA molecules are negatively charged. The loops of the motor are positively charged. The mechanism of translocation takes advantage of this. 

As the single leading strand enters the bottom portion of the channel, the positive-charged loops transiently attract the negatively charged DNA strand one after another to ratchet the single strand through the channel in a "hand-over-hand" motion. Each loop grabs the strand, pulls, and hands it to the next. ATP supplies the energy for each step.

During active human genome replication in the S phase of just one cell in your body, tens of thousands of these molecular motors are unzipping DNA with astonishing precision at a clip of about 50 nucleotides per second. At this speed, a single CMG helicase would need nearly two years to unravel the entire human genome on its own. Instead, as we’ve seen, thousands work in parallel, turning what could be years into hours.

If even a small fraction stall or slip, mutations accumulate. Chromosomes break. Checkpoints halt division. The margin for error is small.

But movement is only half the story. Strand separation is the other.

As the CMG helicase translocates, double stranded DNA enters the MCM channel at the top tier of the barrel. But just barely. Immediately upon entering, a different set of six looping protrusions narrow the channel. Now it can only fit one strand of DNA, not two—and it will be the leading strand that enters the channel. The lagging strand will be blocked, diverted, and shunted to the outside.

Strand separation is thus based the idea of “steric exclusion." This just means that space runs out. The channel narrows such that only one strand can fit. Thus, the helicase doesn’t tear the strands apart with brute force. It simply makes space for one strand and not the other. But, of course, strand separation by steric exclusion only works if the leading strand is being physically pulled through the central channel by the bottom tier motor.

Every time you heal a cut, grow hair, or replace skin cells, the CMG helicase machinery within the replisome runs flawlessly billions of times over, driving the replisome forward along genomic DNA and opening the double helix as it does.

PCNA Sliding Clamp and the Clamp Loader

If the CMG helicase is the engine that pulls the replication machinery forward, the PCNA sliding clamp is the seatbelt that keeps DNA polymerases from falling off.

Like the CMG helicase, the sliding clamp encircles DNA. But unlike helicase, it’s a flat ring—more like a thick washer. A simple shape performing an indispensable task. Also, unlike the CMG helicase, the sliding clamp has no motor. It moves passively along DNA.

How? The answer lies in its partnership with DNA polymerase. On both the leading and lagging strands, the clamp is attached to a DNA polymerase. As the polymerase synthesizes DNA, it actively moves along the strand being replicated. The PCNA sliding clamp is tethered to the polymerase, so it moves, too. 

At any given moment there are at least two active PCNA clamps at a replisome—one on the leading strand supporting DNA polymerase epsilon, and one or more on the lagging strand supporting one or more DNA polymerase deltas. As we’ll see two chapters from now, the lagging strand will require repeated loading of new clamps.

PCNA is called a clamp because it literally clamps a DNA polymerase to DNA. Clamping is required for high DNA polymerase processivity, which is the number of nucleotides, on average, a DNA polymerase synthesizes before it falls off the strand it's replicating.

To provide a sense of its impact, without PCNA, DNA polymerase epsilon will synthesize tens to about 100 nucleotides of the leading strand before it falls off. But when clamped by PCNA, the processivity of DNA polymerase epsilon increases to thousands to tens of thousands of nucleotides—a 100-1,000-fold increase!

In addition to enhancing DNA polymerase performance, PCNA also serves as a kind of replisome "toolbelt." It is a central node of attachment for other replisome proteins, some of which I'll introduce when we discuss leading and lagging strand synthesis. 

To make the toolbelt work, each of PCNA's three ring proteins contains a binding pocket for the attachment of other proteins. The proteins that interact with PCNA have what's called a “PIP-box” (for PCNA Interacting Protein box). The PIP box is like a custom hook that can attach any protein to the sliding clamp.

At this point you might be wondering how the PCNA closed ring comes to encircle a long strand of DNA. A separate highly specialized protein machine does the job. It’s called the clamp loader. It uses energy from ATP to pry open the PCNA ring, snap it around DNA, and then release it. On the lagging strand (as we will see), the clamp loader will repeat this process over and over.

All of this—the gates, the melting, the ratcheting loops, the sliding clamps snapping into place—happens invisibly inside your cells every day. Genome replication is one of the most complex coordinated mechanical operations in biology—yet you never feel it. Life depends on it proceeding with quiet, astonishing reliability.

Let's leave it there for now. Over the past few chapters, I've been pre-emptively referring to leading strand synthesis and lagging strand synthesis. It's time to look at both, starting with the leading strand.