19. Diving Deeper (1,001)

Inside the nucleus of every dividing cell during S phase, massive multi-protein rings race along the DNA. Two of them--both already introduced--are the subject of this chapter.

The first one--the CMG helicase--is long and barrel-shaped and has an internal motor. It leads the replisome's charge. Using its motor, it propels itself and the replisome forward, prying the double helix apart into two single strands to be copied.

The other--the PCNA sliding clamp--is shaped more like a flat ring or thick washer and has no motor. Rather, it moves passively, pulled along by a DNA polymerase. The PCNA's job is to keep the polymerase from slipping off its DNA template as it performs synthesis.

Let's take a higher-resolution look at both of these amazing molecular machines.

CMG Helicase

The CMG helicase--specifically its MCM core--serves as both the motor and the strand separator for the replisome. As it leads the replisome along the double helix, it splits apart the two intertwined strands so they can be copied.

I introduced the MCM protein complex several chapters ago. It's an elongated six protein ring--a hexamer. The subunits are similar but not identical. Each has a top tier and a bottom tier. It's like a two-layered ring with a continuous central channel.

MCM is loaded around double-stranded DNA with the help of Cdt1, but initially in an inactive form. Remember that there are two MCMs aligned head-to-head at the replication origin. So the steps I’m describing here occur at both MCMs roughly simultaneously.

Now two MCMs--facing outward and in opposite directions--encircle the double-stranded DNA at the replication origin like two elongated bracelets.

When Cdc45 and GINS arrive and bind, the MCM ring changes its shape and the complex forms the CMG helicase, which is later activated to become the DNA-unwinding motor.

Several things occur during this transition. First, the shape change promotes local melting of DNA near the helicase. The two strands open and separate from each other. Then the complex transitions to encircle one strand (the leading strand) while excluding the other (the lagging strand).

Once the strands separate, the motor in the bottom tier grabs the leading strand and pulls, causing the two helicases to pull apart from each other and head in opposite directions. The helicases have shifted from “bracelets around rope” to “motors gripping and pulling one strand.”

The motor

How does the motor work? This is an active area of research but I'll describe one widely-accepted model.

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 previous. DNA is negatively charged. The loops are positively charged. The two will attract each other. The motor's mechanism takes advantage of this. 

As the leading strand enters the lower channel, the positively-charged loops transiently attract the negatively-charged DNA strands. ATP-driven conformational changes then ratchet the DNA past a series of interacting loops

In effect, the motor uses a "hand-over-hand" motion. Each loop grabs the strand then pulls and hands off to the next loop. ATP supplies the energy for each pull.

Strand separation

What about strand separation, the other key role of the MCM component of the helicase?

As the helicase translocates, double-stranded DNA approaches the entrance to the channel. But just barely. Immediately upon entering, a different set of six looping protrusions narrow the channel. Now the channel can only fit one strand of DNA. It will be the leading strand.

Thus, the helicase doesn’t tear the strands apart with brute force. It's channel simply can't accommodate more than one strand. It's strand separation by "steric exclusion"--a technical way of saying that the channel is just too narrow!

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, resulting in exclusion of the lagging strand.

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

PCNA Sliding Clamp

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 PCNA sliding clamp encircles DNA. It's placed there by the RFC clamp loader, which we've discussed previously. But PCNA is a trimer rather than a hexamer. And it forms a flat washer-like ring rather than an elongated structure.

Also, unlike the CMG helicase, PCNA has no motor. It moves passively along DNA, dragged along by the DNA polymerase to which it is attached.

PCNA literally clamps a DNA polymerase to its template DNA. Clamping increases DNA polymerase's processivity--or the number of nucleotides, on average, it synthesizes before it falls off the template strand.

Without PCNA, DNA polymerase epsilon synthesizes tens to maybe 100 nucleotides before it falls off. When clamped by PCNA, it synthesizes thousands to tens of thousands of nucleotides before it falls off.

In addition to enhancing DNA polymerase performance, PCNA also serves as a replisome "toolbelt"--a node of attachment for other replisome proteins that I'll introduce shortly. 

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 PCNA sliding clamp.

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.

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.