19. Leading the Way

An important fact: The two DNA strands in a double helix are oriented in an antiparallel manner. That is, they run in opposite directions like the lanes of a two-lane highway. Recalling chapter ___, scientists describe the orientation of a given single strand of DNA as running either “3’-to-5’” (three prime to five prime) or “5’-to-3’” (five prime to three prime) based on the orientations of the deoxyribose sugars in the DNA backbone.

But as a hard and fast rule, DNA polymerases can only synthesize in the 5’-to-3’ direction—never in the 3’-to-5’ direction. This has profound implications for genome replication. The two strands will have to be replicated in different directions: one (the leading strand that exited the bottom of the CMG helicase) will be replicated in the same direction that the CMG helicase moves as it unwinds DNA. The other (the lagging strand that gets shunted to the outside of the CMG helicase) will be replicated in the direction opposite that of CMG helicase and replication fork movement.

Let's start from the beginning. After traveling down the TIMELESS ramp, double stranded DNA to be replicated enters the CMG helicase’s N-tier. There, a slimming of the central channel due to protrusions from several MCM proteins allows only one strand (always, somehow, the leading strand) to enter the C-teir. Once there, motors begin pulling the leading strand through the channel and out the bottom of the MCM2-7 complex 3’ end first.

The next thing that happens is that RPA (Replication Protein A) molecules arrive to coat the naked single strand exiting the CMG helicase. Recall that single-stranded DNA is susceptible to both degradation by enzymes in the cell and to the formation of problematic secondary structures. So it must be protected. RPAs coat the DNA, but they don't bind so strongly that other proteins can't displace them when necessary.

Leading Strand Replisome. Note the presence of the CMG helicase (Cdc45-MCM2-7-GINS), DNA polymerase epsilon, PCNA sliding clamp, DNa polymerase alpha-primase, and AND-1 (here called Ctf4). Several of the proteins shown in this diagram are ones we have not discussed (Mrc1, Csm3-Tof1.

Next to arrive at the scene will be both DNA polymerase alpha-primase and the protein that tethers it to the CMG helicase, AND-1. AND-1 attaches to the helicase and has a long, flexible arm that attaches to the DNA polymerase alpha-primase. This arm allows it to reach both the leading strand and the lagging strand to construct the required primers. Let's quickly review this idea of a "required primer."

DNA polymerases (and in the case of the leading strand, DNA polymerase epsilon) can never begin synthesizing off of a naked single strand. They require a small piece of RNA or DNA (or both) to be hybridized to the strand that's being replicated. This short so-called primer provides the 3' end to which a DNA polymerase can begin adding new nucleotides. So, the next step in leading strand synthesis is building this 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, the enzyme can also build short--roughly 10 base pair (bp)--RNA primers on single strands. It uses RNA nucleotides (ribonucleotides) rather than DNA nucleotides (deoxyribonucleotides) to do this. In the cell, ribonucleotides are readily available because they fuel a constant flow of new mRNAs.

Let me pause here to say something about enzymes. Many enzymes have more than one activity, or function. That is, in many cases, different domains of an enzyme perform different activities. DNA polymerase alpha-primase, for example, has two activities. It has both a DNA polymerase activity that requires a primer to begin synthesis and it has an RNA polymerase activity that does not require a primer to initiate synthesis.

DNA polymerase alpha-primase first uses its RNA polymerase activity. It displaces the RPA molecules on the leading strand and builds an 8-12 ribonucleotide RNA primer. Given that there is now an available 3' end on this RNA primer, it then adjusts its orientation and uses its DNA polymerase activity to add 20-30 more deoxyribonucleotides to the RNA primer. The resulting roughly 40-bp hybrid RNA-DNA primer provides a new 3' end off of which DNA polymerase epsilon can begin leading strand replication.

Once the hybrid RNA-DNA primer is built on the leading strand, DNA polymerase alpha-primase has completed its job and detaches from the DNA. It is still tethered to CMG helicase, though, through the AND-1 protein. So it remains near the front of the replication fork where it will be needed for lagging strand synthesis.

The next replisome protein (or, rather, protein complex) to arrive at the leading strand is the PCNA sliding clamp. The RFC clamp loader loads it onto the DNA at the exact point where the hybrid RNA-DNA primer ends--that is, at the primer's 3' end. DNA polymerase epsilon then tethers to PCNA using its own PIP-box. DNA polymerases require sliding clamps to achieve acceptable performance in terms of both speed and processivity. So DNA polymerase epsilon will remain attached to the sliding clamp as it synthesizes a new strand complementary to the leading strand template.

In addition to being attached to the sliding clamp, DNA polymerase epsilon also becomes attached directly to the CMG helicase via the helicase's Cdc45 and GINS proteins. Given its attachment to the helicase, synthesis by DNA polymerase epsilon will now occur in lockstep with the unwinding of the double stranded DNA. The two processes--DNA unwinding and DNA synthesis--are now synchronized.

The final replisome proteins to arrive will be members of the fork protection complex, or FPC. The fork protection complex is an amalgam of proteins--most notably Claspin, Timeless and Tipin--that support the attachment of DNA polymerase epsilon to the CMG helicase and that more generally stabilizes the replication fork if it runs into problems, such as conditions of replicative stress.

The leading strand replisome is now complete. After DNA polymerase adds its first few bases to the primer, it and the PCNA sliding clamp follow the CMG helicase as it unwinds the parents strands of DNA, the polymerase continuously adding DNA nucleotides at a rate of about 50 per second to the 3’ end of the growing new strand.

In a previous post, I brought up the cell’s first of several proofreading abilities. This is simply a DNA polymerase enzyme’s ability to sense a mis-paired nucleotide about to be added to a growing daughter strand and to avoid attaching it. It does this based on the shape of the mis-paired nucleotide, which prevents the enzyme from effecting its own normal conformational change—a shape change that occurs readily when a correct nucleotide is being added to the chain. So DNA polymerases sense mismatched nucleotides and refrain from adding them to the growing strand.

We now move to the cell’s second proofreading ability, which is performed by a second activity of DNA polymerase epsilon. Remember, enzymes can have multiple activities: they can play more than one role in the cell. This second kind of error correction only occurs in the rare event that the first proofreading effort fails and immediately after that mis-paired nucleotide is added to the growing chain. This proofreading function is performed by DNA polymerase epsilon, but this time using the enzyme’s 3’-to-5' exonuclease activity.

If a mis-paired nucleotide is added to the end of a growing chain, then the 3’ end of the chain will be mis-shaped, making the next nucleotide difficult to add to the chain’s 3’ end. DNA polymerase epsilon senses this situation and reacts. It physically shifts itself in order to use its 3’-to-5’ exonuclease catalytic site to chew back as many nucleotides as necessary to remove the mis-paired nucleotide. Once it has removed the bad nucleotide and created a normal 3’ end, it uses its DNA polymerase activity to add the correct nucleotide then it retakes its place behind the helicase synthesizing the daughter strand.      

Replicating the leading strand is straightforward compared to replicating the lagging strand. Leading strand synthesis is called "continuous." Once the first primer is laid down, synthesis continues more or less uninterrupted until the replisome reaches an already synthesized strand or until the DNA polymerase falls off. Lagging strand synthesis is called "discontinuous" because it must be performed in steps. It requires repeated priming and fragment joining. We'll take a closer look at lagging strand synthesis next.

A brief note. I haven’t covered one detail: how the cell removes the RNA portion of the primer from the daughter strand and replaces it with DNA. I will do that in the context of discussing lagging strand synthesis.