Winding Problems

As the CMG helicase moves along the double helix separating it into a leading and a lagging strand, it creates what is often referred to as the "winding problem." Let me explain what this is with the help of a human-scale analogy.

Consider two ropes wound around each other like a double helix coil. If we attach one end of these two ropes permanently to, say, a table, and then step back and pull the ropes apart to unwind them (in much the same way that CMG helicase unwinds double-stranded DNA), it will generate what's referred to a "torsional stress" in the yet unwound stretch of the rope. This torsional stress creates second level coils, or "supercoils."

For my older readers, think of the second-level supercoiling that used to occur with coiled phone handset cords when they would get twisted up. You'd end up with second level supercoils, as shown in the figure. To remove them, one would typically let the handset hang by the supercoiled cord and then just allow it to untwist itself in the direction opposite the one that created the supercoils in the first place.

As CMG helicase unwinds the double helix, the other end of that double helix is effectively fixed: it can't rotate freely any more than the ropes attached to the table. So CMG helicase unwinding causes the same kind of secondary supercoiling in the double helix in front of the replication fork.

In fact, every time the CMG helicase progresses 10 nucleotides, it introduces one extra twist into the DNA. These twists add up, causing the supercoils. And these supercoils, if unaddressed, bung up the helicase and prevent it from progressing. So they must be removed.

Type I Topoisomerase (TOP1)

Human cells have dedicated molecular machines that remove supercoils as the replication fork moves down the DNA. These machines, which are called topoisomerases, are actually of two types. Type I topoisomerase (TOP1) is a monomeric protein (i.e., made up of one polypeptide chain) that's the primary machine that relaxes the upstream DNA supercoils that we've been discussing. We'll address the role of type II topoisomerase (TOP2) shortly. For now, let's focus on the mechanism by which TOP1 accomplishes the relaxing of a supercoiled double helix in front of the replication fork.

If the problem is overtwisting, then the solution--much like with the phone cord--is to allow the DNA "cord" to untwist. In fact, the over-twisted DNA--much like the supercoiled phone cord--wants to untwist. The built-up torsional stress seeks a lower energy relaxed state, just like the hanging, spinning phone cord.

TOP1 makes this happen. First, it seeks out supercoiled double-stranded DNA in front of the replication fork. TOP1 has a C-clamp shape that encircles about 20 nucleotides of DNA. Inside the clamp, it then attaches to one of the two strands and severs it. This allows the cut strand to rotate in a controlled manner (controlled via friction inside the clamp) in the direction of relaxation, using the uncut strand as the pivot point. No energy from ATP is needed to make this happen. Like the hanging phone headset, the severed strand rotates by itself around the unsevered strand using the torsional stress that has built up.

Unlike the hanging phone set, though, which unravels all the supercoils as it spins around, TOP1 relaxes supercoils in a controlled manner, allowing just one rotation at a time. As I mentioned, every time the CMG helicase progresses 10 nucleotides, it introduces one extra twist into the DNA. Thus, TOP1 needs to effect one free rotation every 10 nucleotides.

So TOP1 relieves torsional strain in duplex DNA by transiently nicking one strand and allowing it to swivel around the uncut strand before sealing the break. In fact, in the 1970s and 1980s TOP1 was called "DNA swivelase." TOP1 is perfectly designed to perform its job. Its hinged C-shaped core opens and then traps DNA securely. Its catalytic lobe nicks one of the two strands while the DNA is trapped in the clamp. And a third domain governs the controlled rotation of the DNA within the clamp.

TOP1 action must be continuous as the replisome progresses. In fact, one TOP1 molecule typically remains on the DNA to facilitate multiple relaxation twists before it leaves--sometimes as many as 20. When the TOP1 molecule does leave, the broken strand reseals to recreate the now more relaxed intact double helix. At this point,another TOP1 protein will attach and continue the untwisting process.

So picture these amazing TOP1 nanomachines attaching to supercoiled DNA one after another in advance of the moving replication fork, cutting one strand of the DNA and allowing it to swivel to remove the supercoils built up from the separation of the DNA into a leading and lagging strand. TOP1 enzymes are like release valves for the supercoiling pressure that builds up in front of a replication fork. Without TOP1, supercoiling pressure would build up in the double helix and cause a literal explosion at the molecular scale.

A final note: it turns out that targeting TOP1 is a highly effective mechanism of cancer chemotherapy action. The chemotherapy drugs camptothecin, topotecan, and irinotecan are all TOP1-targeting agents used in the treatment of colon and ovarian cancer. By crippling TOP1, rapidly growing cancer cells in particular are susceptible to severe DNA damage and cell death. That's how chemotherapy drugs work.

Type II Topoisomerase (TOP2)

There's another DNA topological issue that arises during replication... this time behind, rather than in front of, the moving replication fork. That problem is the ongoing tangling of the two duplex DNA molecules generated by leading and lagging strand synthesis.

Imagine two newly synthesized double strands of DNA being continuously generated at 50 bases per second in the nucleoplasm. Sometimes those two strands will become tangled to create, effectively, double-stranded DNA knots. Eventually these knots will have to be untangled so the two resulting chromosomes can be segregated into the two daughter cells during cell division. They can't remain knotted up.

Once again, human cells have a solution for this problem in the form of a precision nanomachine. It's called type II topoisomerase (TOP2). TOP2 is a much larger protein than TOP1. Whereas TOP1 acted on a single double-stranded molecule, TOP2 acts on two intertwined, or knotted, double-stranded molecules.

TOP2 is a large, symmetric, homodimeric protein complex (i.e., comprised of two identical polypeptides) with three clamping gates that open and close in a strictly enforced sequence, creating an effect like a multi-door airlock. We'll need to keep track of the gates as I explain how the enzyme works. The enzyme has, in order, a top gate, a central gate, and a bottom gate. Here's how TOP2 does its job.

First, the enzyme's open central DNA gate allows entry of about 30-40 nucleotides of the duplex DNA strand that will soon be cleaved (called the "c-segment" where the "c" stands for "cleaved.") The c-segment must be trapped in the central gate firmly and positioned precisely so the enzyme's catalytic site inside its central gate (the part of the enzyme that will cleave the c-segment) is aligned correctly.

Next, the other duplex DNA strand--the one that will not be cleaved--enters the top gate. This strand is called the "t-segment" where the "t" stands for "transported.") At this point, an ATP molecule binds to the enzyme. This causes the top gate to close around the t-segment, trapping it there. The t-segment is now like a guest caught in an entryway waiting for something to happen.

Back in the central gate, once the top gate closes, the enzyme cleaves the c-segment and pulls apart the two pieces of the DNA, creating a double-stranded break. The resulting passage between the newly-formed DNA ends is like an open double door.

Next, with the c-segment passage waiting, the top gate opens, allowing the t-segment to first enter the central gate and then to pass through the break in the c-segment. With the t-segment now positioned on the other side of the c-segment--close to the bottom gate--TOP2 closes the break in the c-segment and religates the strands so there is no longer a double strand break.

Once the break in the c-segment has been repaired, the bottom gate opens to release the two now-untangled DNA strands. The bottom gate won't open until the double stranded break in the c-segment has been repaired, ensuring that this potentially catastrophic insult to the genome doesn't persist. And, finally, when the earlier-bound ATP is cleaved, the top gsaate reopens to all the process to repeat.

Each TOP2 molecule can complete 1-2 strand passages per second.