19. Leading the Way (1,150)

Let's start this chapter off with two fundamental facts: one about DNA and the other about DNA polymerases.

Fact one. Recall that the two DNA strands in a double helix are oriented in an antiparallel fashion. They run in opposite directions like the lanes of a two-lane highway. Scientists describe the orientation of a strand as either “5’-to-3’” (five prime to three prime) or “3’-to-5’” (three prime to five prime) based on the position of the sugars in the DNA 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'. This has major implications for genome synthesis. It means the strands must be synthesized in opposite directions: one toward the advancing CMG helicase and the other away from it.

Here we focus on the leading strand.

Enter the CMG helicase

Let me set the stage. Double-stranded DNA has entered the top tier of the MCM subunit of the CMG helicase where a slimming of the central channel allows only the leading strand to enter the bottom tier. 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 central channel. As a result, the helicase is propelled along the leading strand in the 3′-to-5′ direction.

The leading strand exiting the MCM is at risk. Single-stranded DNA is susceptible to both degradation by enzymes and to the formation of problematic secondary structures. So RPAs (Replication Protein A) arrive immediately to protect it. RPAs coat the DNA, but they don't bind so strongly that other proteins can't displace them when necessary.

Leading Strand Replisome.

The first two proteins to arrive are DNA polymerase alpha-primase and a second protein with a long, flexible arm that will tether the polymerase to the helicase. The arm allows DNA alpha-primase to reach far enough that it can construct primers on both the leading strand and the lagging strand. Let's pause to review this critical notion of a primer.

The primer problem

DNA polymerases epsilon and delta--the enzymes tasked with leading and lagging strand synthesis, respectively--can't start synthesis just anywhere along bare, single-stranded DNA. They require a small piece of RNA or DNA (or both) to be hybridized to the template strand. This short primer provides the indispensable 3' terminus to which these DNA polymerases can begin adding 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. This enzyme—as the name suggests—has two activities. In addition to its DNA polymerase activity, it can also construct short primers made of RNA or DNA or both on single strands. It initially uses RNA nucleotides rather than DNA nucleotides because, in the cell, ribonucleotides are much more readily available since they fuel a constant flow of new mRNAs.

Let me pause here to say something about enzymes, generally. Many enzymes have more than one activity, or function. Usually, different domains of an enzyme perform the different roles. DNA polymerase alpha-primase, for example, has two activities. It has a standard DNA polymerase activity--one that requires a primer to begin synthesis--and it has an RNA polymerase activity that doesn't require a primer to initiate synthesis.

DNA polymerase alpha-primase uses its RNA polymerase activity first. It displaces the protective RPAs on the leading strand and builds a roughly 10 ribonucleotide RNA primer.

Given the now available 3' end of this RNA primer, the enzyme then adjusts its orientation and uses its DNA polymerase activity to add 20-30 more deoxyribonucleotides to the end of the RNA primer. The resulting 30-40 nucleotide hybrid RNA-DNA primer provides the 3' end off which DNA polymerase epsilon can begin leading strand synthesis.

Once the hybrid RNA-DNA primer is built, DNA polymerase alpha-primase has completed its job and detaches from the DNA. It's still tethered to CMG helicase, though. So it remains near the front of the replication fork where it will be needed for lagging strand synthesis.

Enter DNA polymerase epsilon and others

The next protein complex to arrive at the leading strand will be the PCNA sliding clamp. The clamp loader complex, RFC (Replication Factor C), loads the sliding clamp onto the primer–template junction--that is, onto the primer's 3' end.

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

In addition to being attached to the sliding clamp, DNA polymerase epsilon also attaches directly to the CMG helicase via the helicase's Cdc45 and GINS proteins. This attachment makes the two processes--DNA unwinding and DNA synthesis--spatially coordinated.

The final replisome proteins to arrive are members of the fork protection complex, or FPC--an amalgam of proteins that support the attachment of DNA polymerase epsilon to the CMG helicase and that more generally stabilizes the replication fork.

The leading strand replisome is now complete. After DNA polymerase epsilon adds its first few bases to the primer, it and its PCNA sliding clamp follow immediately behind the CMG helicase as it unwinds the parents strands and continuously adds DNA nucleotides in order to synthesize the second strand at a rate of about 50 per second.

Error avoidance and proofreading

Genome replication is not only an enormous job, it's one that must be performed with incredible accuracy. Non-complementary nucleotides are not allowed.

DNA polymerases reduce nucleotide misincorporations and errors in two ways. First, they are intrinsically selective: their active sites strongly favor correct base pairing. The aberrant shape of a mis-paired nucleotide prevents the enzyme from undergoing a conformation change required to attach that nucleotide to the growing strand. So mis-incorporations are rare... but do occur.

But because misincorporations can occur, DNA polymerases epsilon and delta have what is referred to as a "proofreading" ability. Proofreading is performed by a different part of the enzyme that contains a 3’-to-5' exonuclease activity.

Here's how it works. If a mis-paired nucleotide is added to the end of a growing chain, then the 3’ end of the chain will be mis-shapen, making the next nucleotide difficult to add. DNA polymerase epsilon senses this situation and reacts. It shifts itself such that it can use its 3’-to-5’ exonuclease activity to chew back as many nucleotides as necessary to remove the mis-paired one.

Once it removes the bad nucleotide and creates a normal 3’ end, it then shifts back again to use its DNA polymerase activity to add the correct nucleotide. Then it assumes its usual place trailing behind the CMG helicase synthesizing DNA. 

Leading strand synthesis, then, is elegant in its 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!