20. Lagging Behind (1,132)

DNA replication has a directional problem.

The molecular machines that build DNA — the DNA polymerases — can add new nucleotides in only one direction: 5′ to 3′. That rule is absolute, dictated by the chemistry of the reaction itself.

But the two strands of the DNA double helix run in opposite directions. They're antiparallel. So what happens when the replication fork moves forward?

One strand can be copied smoothly and continuously. The other cannot. The second strand — the lagging strand — must be built in pieces, in short bursts of synthesis that run opposite the direction of fork movement.

These short stretches of DNA, typically 100–200 nucleotides long in human cells, are called Okazaki fragments. Each must be individually initiated, extended, cleaned up, and stitched into place.

Lagging strand synthesis is not smooth. It is stop-and-start, fragmentary, and extraordinarily busy.

Let me slow down and clarify this point about DNA strand orientation.

Which way is up?

Our leading strand exited the MCM ring of the CMG helicase 3′ end first. The DNA strand synthesized from that template must therefore be built 5′ to 3′.

And if the new strand is synthesized 5′ to 3′, that synthesis proceeds toward the CMG helicase — in the same direction that the replisome moves.

The lagging strand, by contrast, was pushed to the outside of the MCM ring with its 5′ end leading. So when DNA is synthesized off that template in the required 5′ to 3′ direction, synthesis necessarily proceeds opposite the direction of replication fork movement.

There is no alternative, given the directional constraint of DNA polymerases.

Lagging strand synthesis

Assembling the machinery

Essentially all the molecular actors who starred in leading strand synthesis will star again in lagging strand synthesis. DNA polymerase alpha-primase will make the primers. A more accurate, replicative polymerase — DNA polymerase delta instead of DNA polymerase epsilon — will extend them. And again, the polymerase will require a PCNA sliding clamp to achieve high speed and processivity.

Because the lagging strand was shunted to the outside of the MCM ring of the helicase, it is exposed to enzymes that degrade single-stranded DNA. It is also susceptible to forming secondary structures that can impede synthesis. So before anything else occurs, RPA proteins arrive to coat and stabilize the exposed lagging-strand template.

After the arrival of the RPAs, the first primer is synthesized by DNA polymerase alpha-primase, which remains tethered to the CMG helicase via its flexible, long-armed protein. As discussed in the previous chapter, this primer is a hybrid of RNA and DNA.

But synthesis by DNA polymerase alpha-primase is more error-prone than synthesis by DNA polymerases epsilon and delta. Alpha-primase lacks the proofreading ability possessed by those enzymes. Thus, the DNA portion of the lagging-strand primer may contain errors. The cell has mechanisms to correct this vulnerability, as we will see.

Next, a PCNA sliding clamp is loaded by the RFC clamp loader onto the lagging strand at the 3′ end of the primer. The clamp recruits DNA polymerase delta, which attaches to PCNA via its PIP-box.

With polymerase delta secured to its sliding clamp, replication can begin — in the direction away from the CMG helicase.

The second primer

As the first primer is being extended, the replication fork continues to move forward. So a second primer is quickly synthesized closer to the advancing helicase, but with its 3′ end — the end from which synthesis proceeds — facing away from it. A second PCNA sliding clamp is loaded, and a second DNA polymerase delta attaches.

Recall the fork protection complex (FPC). It travels with the CMG helicase at the front of the replisome, but it stabilizes lagging strand synthesis as well as leading strand synthesis.

With all of the proteins in place, DNA polymerase delta synthesizes off the second primer toward the first Okazaki fragment.

This illustrates the logic of lagging strand replication. As the helicase advances, an ongoing series of primers is synthesized near the moving CMG helicase but oriented away from it. Each fragment, as it is extended by DNA polymerase delta, eventually runs into the 5′ end of the primer of the preceding Okazaki fragment. After some trimming, the two fragments will be joined.

RNA removal

Before two fragments can be joined, however, the primer must be removed.

The first ~10 nucleotides are RNA. That stretch is followed by 30–40 nucleotides of relatively error-prone DNA. The RNA cannot remain part of the genome. And the more error-prone DNA must eventually be replaced.

Human cells use the enzyme RNase H2 to remove the primer RNA. RNase H2 specifically degrades RNA that is hybridized to DNA — precisely the situation in our RNA–DNA primer. So while the second primer is being extended back toward the first fragment, RNase H2 removes most of the RNA portion of the earlier primer.

One detail: RNase H2 does not completely finish the job. It leaves a very short RNA–DNA junction that must be resolved later.

Once the RNA has been removed (except for that small junctional remnant), DNA polymerase delta fills in the region with new, high-fidelity DNA.

Strand displacement

One might imagine that DNA polymerase delta would then crash into the remaining ribonucleotide at the junction. But it does not. Instead, it displaces it.

As polymerase delta synthesizes forward, it pushes the remaining primer nucleotides aside, creating a short single-stranded “flap.” These flaps are typically very small — often only one or two nucleotides long.

Flap cleavage

Enter FEN1 (Flap Endonuclease 1), an enzyme specialized for cutting such flaps at the junction where single-stranded DNA meets double-stranded DNA.

In what is called the idling cycle, DNA polymerase delta and FEN1 repeatedly coordinate their actions. Polymerase delta performs a small amount of strand displacement, generating a flap. FEN1 cleaves it. Polymerase delta advances again, creating another small flap. FEN1 cleaves again. The cycle repeats.

Through this iterative process, the more error-prone primer DNA is replaced with high-fidelity DNA.

Connecting the fragments

When the idling cycle is complete, a nick remains in the DNA backbone where the last flap was removed. The two Okazaki fragments must be sealed together.

That task falls to DNA ligase 1. It arrives and ligates the adjacent fragments, creating one continuous stretch of newly synthesized DNA hybridized to the lagging-strand template.

That completes the cycle — at least for one pair of fragments. But this cycle is happening over and over again along the length of the chromosome.

Lagging strand synthesis is a frenetic process. A little arithmetic makes that clear.

Given the size of the human genome and the typical size of an Okazaki fragment, a single cell will generate and stitch together tens of millions of Okazaki fragments during the eight-hour S phase of one cell division. The scale alone makes “frenetic” seem almost understated.