20. Leading the Way (1,073)

Let's start off with two indisputable facts. One about DNA. The other about DNA polymerases.

Fact one. Recall that the two DNA strands in a double helix are oriented in an antiparallel manner. They run in opposite directions like the lanes of a two-lane highway. Scientists describe strand orientation as either “5’-to-3’” (five prime to three prime) or “3’-to-5’” (three prime to five prime) based on the orientations of the sugars in the strand's backbone.

Fact two: In the same way that you can only drive on one side of a highway, human DNA polymerases can only synthesize DNA in one direction: 5'-to-3' (that is, off a template strand that's oriented 3'-to-5').

These two facts have a major implication for genome synthesis: the two strands must be synthesized in opposite directions!

One strand will be built off the leading strand template toward the advancing helicase and replication fork. The other will be synthesized off the lagging strand template in the direction opposite that of helicase and fork movement.

In this chapter, we focus only on synthesis off the leading strand template--the strand that will be synthesized in the direction that the helicase translocates.

Enter the CMG helicase

Double-stranded DNA approaches the helicase and the leading-strand template is threaded through the central channel. The lagging-strand template is shunted to the outside.

Once the leading strand enters the bottom tier, motors grab it and pull it through the channel. As a result, the helicase is propelled along the leading strand template in the 3′-to-5′ direction.

Leading Strand Replisome.

Among the first proteins to arrive will be DNA polymerase alpha-primase and another protein called AND-1. AND-1 helps tether DNA polymerase alpha–primase to the replisome, linking primer synthesis to the moving helicase. AND-1's arm reaches far enough that the polymerase can build primers on both the leading and lagging strand templates.

Let's pause to revisit this critical notion of a primer.

The primer requirement

DNA polymerase epsilon and DNA polymerase delta--the enzymes that perform leading and lagging strand synthesis, respectively--can't initiate synthesis just anywhere on a stretch of single-stranded DNA.

They both require a short RNA–DNA primer (about 30-40 nucleotides long) hybridized to the template strand. This primer provides the indispensable 3' terminus to which these DNA polymerases can add nucleotides.

So the first step in leading and lagging strand synthesis is building a primer. This will be the task of DNA polymerase alpha-primase.

Let me pause here to say something about enzymes, generally, and about DNA polymerase alpha-primase, specifically.

Many enzymes have more than one activity. DNA polymerase alpha-primase has two activities performed at two different "active sites" on the enzyme. It has a DNA polymerase activity that requires a primer to begin synthesis and an RNA polymerase activity that doesn't require a primer.

To make a primer, DNA polymerase alpha-primase first uses its RNA polymerase active site to build a roughly 8-12 ribonucleotide RNA primer.

With the 3' end of the RNA primer now available, the enzyme shifts its orientation and uses its DNA polymerase active site to add 20-30 more deoxyribonucleotides to the end of the RNA primer. It only adds a limited number of nucleotides before handing the larger synthesis task off to more accurate, processive polymerases

The resulting 30-40 nucleotide hybrid RNA-DNA primer provides the 3' end off of which DNA polymerase epsilon can begin leading strand synthesis.

Once the RNA-DNA primer is built, DNA polymerase alpha-primase has finished its job and detaches from the DNA. It's still tethered to CMG helicase, though. So it remains in the replisome where it can be used for lagging strand synthesis.

Enter DNA polymerase epsilon

Next, the PCNA sliding clamp is loaded at the primer–template junction. This enables recruitment of DNA polymerase epsilon. Recall that PCNA is loaded onto DNA--specifically at the primer-template junction--by its dedicated clamp loader, Replication Factor C (RFC).

DNA polymerase epsilon then arrives and tethers itself to the PCNA sliding clamp via a PIP-box. DNA polymerase epsilon will remain attached to the clamp as it synthesizes a new strand complementary to the leading strand template.

In addition to being attached to PCNA, DNA polymerase epsilon also attaches to the CMG helicase via Cdc45 and GINS. This dual attachment--to PCNA and to the helicase--spatially coordinates DNA unwinding and DNA synthesis.

Finally, some additional proteins arrive, including members of the fork protection complex (FPC)--a group of proteins that supports the attachment of DNA polymerase epsilon to the CMG helicase and that stabilizes the replication fork.

The leading strand replisome is now in place and synthesis can begin in earnest.

After DNA polymerase epsilon adds its first few nucleotides to the primer, it and its sliding clamp follow immediately behind the CMG helicase. As the helicase unwinds the parent strands, the polymerase continuously adds nucleotides at a rate of about 50 nucleotides per second to synthesize the complementary strand.

The choreography of epigenetic inheritance

In the chapter "Eight Challenges of Genome Replication" I described the matter of removing and replacing nucleosomes and their histone protein spools as the replisome progresses along the genome.

But in addition to removing and replacing nucleosomes, the chemical markings both on the histone tails of the nucleosomes and on the DNA nucleotides themselves must be recreated. The two new daughter cells must be marked in the same way as the parent cell.

These markings are critical. They determine a cell's identity, determine gene expression patterns, dictate the level of DNA packaging, and even signal to the cell that a specific repair is needed.

To maintain the histone markings, the cell has a group of specialized proteins called histone chaperones that escort the removed parental histones across the replisome and use them to package the two newly synthesized daughter strands.

Because the DNA content doubles (two daughter strands) but the number of parental strands remains the same, the daughter strands will only be half spooled on these legacy parental histones. The remaining ones are synthesized by the cell.

Then, once they're in place, the cell uses the markings on the parental histones as a template to mark the new ones correctly.

These processes ensure that the epigenetic regulatory patterns are inherited by the new cell withthe same fidelity as the DNA sequence itself.

Leading strand synthesis is elegant in its relative simplicity. Once begun, it is continuous, tightly coordinated with helicase movement, and remarkably accurate.

Lagging strand synthesis is discontinuous, not directly tied to helicase movement, and another story entirely!