Assembling the Replisome

Our cell of interest is now in S phase and 30,000-50,000 replication origins along its genome have become licensed. The replication origins are licensed based on the presence of two MCM2-7 protein complexes (the licensing factors) lined up head-to-head forming a MCM double hexamer, or MCM-DH.

Assembling a functioning replisome starting from an MCM-DH at a replication origin will involve the following four steps: (1) transformation of each of the MCM2-7s into a complete (but still inactive) CMG helicases, (2) activation of the helicase and initial opening of the double helix, (3) recruitment of a set of proteins called the fork protection complex (FPC), and (4) recruitment of three DNA polymerases and a key protein complex that assists the polymerases, PCNA (Proliferating Cell Nuclear Antigen).

Once these steps are complete, the cell will have constructed two fully functional replisomes led by two CMG helicases pointing in opposite directions away from the replication origin.

Creation of the CMG Helicases

The two MCM-2-7 protein complexes that make up the MCM-DH are, at this point, non-functional. Their job, so to speak, is to serve as the core protein ring of two fully functional helicases.

I mentioned in an earlier post that at the end of G1 and into S phase, a new set of CDKs become active in the nucleus. Recall that CDKs are a type of enzyme called a kinase that attaches a small chemical group called a phosphate to other molecules. The activation of different CDKs by their partner cyclins dictates the progression of the cell cycle. CDK-mediated phosphate addition is also used to render specific proteins active or inactive.

In an earlier post we saw how a new kinase activated at the end of G1 and into S phase (CDK2) inhibits the licensing of any new replication origins in S phase by largely by marking for destruction key proteins required for licensing. But CDK2--and one more kinase called DDK that becomes active around the same time--also play an indispensible role in the transformation of our two MCM2-7s into two CMG helicases.

"CMG" helicase stands for Cdc45-MCM-GINS helicase. Thus, the creation of CMG helicase will require the addition of two other proteins--Cdc45 and GINS--to MCM2-7 (GINS is actually a four polypeptide protein complex). But there are intermediate steps required for the loading of Cdc45 and GINS onto MCM2-7. We'll now take a look at those intermediate steps to get a better sense of how the transformation occurs. For simplicity, I'll focus on just one of the two MCM2-7s in the MCM-DH.

Here are the steps that precede Cdc45 and GINS loading in the nucleus:

1. DDK phosphorylates MCM2-7 at specific locations. These phosphates serve two roles. They neutralize self-inhibitory domains on MCM2-7 that otherwise block helicase function. They also create a future binding site--a kind of "landing pad"--on MCM2-7 for a protein called Treslin that's central to loading Cdc45 and GINS.

2. CDK2 phosphorylates two other proteins--Treslin and RecQL4--to create binding sites for the later attachment (to both proteins) of another protein: TopBP1.

   
   

3. Next, in the nucleoplasm, Treslin pairs up with a protein called MTBP. MTBP stabilizes Treslin and localizes it to the replication origin. The Treslin-MTBP pair then docks onto the MCM2-7 binding site created by DDK phosphorylation.

   
   

4. Elsewhere in the nucleus TopBP1 pairs up with RecQL4 via the RecQL4 binding site created by CDK2. The TopBP1-RecQL4 duo makes its way to the origin. There, it binds to the already-attached Treslin using the TopBP1 binding site on Treslin created by CDK2 phsophorylation.

   
   

5. The Treslin-MTBP pair along withTopBP1 and RecQL4--all now associated with MCM2-7--form a transient scaffold necessary for loading Cdc45 (the "C" in "CMG") onto the MCM2-7 ring.

   
   

6. GINS (the "G" in "CMG") is also recruited and attaches to the MCM2-7-Cdc45 pair. GINS loading also requires the loading scaffold created by Treslin-MTBP, TopBP1, and RecQL4 but the mechanism of GINS attachment is poorly understood.

The two CMG helicases at the replication origin are now fully assembled. All of the scaffolding and loading proteins--that is, Treslin-MTBP, TopBP1 and RecQL4--leave the now-functional helicase. But the new CMG helicase won't be functional or active until the arrival of one more protein, Mcm10.

The Replisome. A simplified version of the replisome that omits the Fork Protection Complex (FPC). The leading strand of the double-stranded DNA (left) enters the CMG helicase ring. The lagging strand (right) is shunted outside of the ring. DNA polymerase epsilon is directly attached to the CMG helicase on the leading strand and the PCNA sliding clamp rides behind it as it synthesizes in the direction of CMG helicase movement. On the lagging strand, note that DNA polymerase alpha-primase has constructed three primers (teal). DNA polymerase delta is synthesizing DNA off of the second primer. The RFC clamp loader is also shown. Finally, note that AND-1 is mislabeled as "Ct43" in this drawing.

CMG Helicase Activation and Initial DNA Unwinding

Mcm10 is another scaffolding protein that provides structure to the replisome and helps initiate DNA unwinding. Mcm10 arrives at the origin and attaches to MCM subunits and Cdc45. Once in place, as mentioned, it performs several functions. Its first role is to serve as a scaffold protein that stabilizes the CMG helicase's grip on the DNA. Without Mcm10, the CMG helicase would quickly fall off and float into the nuceoplasm.

The second task of Mcm10 is to assist the CMG helicase in unwinding the double stranded DNA and to remove one of the strands from inside its ring structure. Yes, Mcm10 literally opens the CMG helicase's ring and extracts one of the DNA strands, leaving only one strand in the helicase. Importantly, it removes a specific strand: the lagging strand, which is oriented in the 5' to 3' direction. If the leading strand (oriented 3' to 5') had been pulled out, replication wouldn't proceed. So Mcm10 has to be choosey!

Now we have the leading strand protected inside the CMG helicase's ring but the lagging strand exposed on the outside of the helicase. But this creates a problem. Inside the cell, single stranded DNA is highly susceptible to destruction; specifically, to cleavage by proteins called nucleases that cleave nucleic acids like DNA and RNA.

Single stranded DNA is also susceptible to the formation of secondary structures: regions where the DNA self-hybridizes based on standard A-T, G-C DNA base pairing rules. Secondary structures in the lagging strand could impede replisome progression.

To address this, the cell calls into action proteins called RPAs (Replication Protein A). These smallish protein complexes coalesce around and coat the lagging strand, protecting it from nuclease digestion and from secondary structure formation.

Let's take stock. We now have a replication origin with two functional CMG helicases pointing in opposite directions. The DNA at the CMG helicases has been partially unwound. The leading strand remains inside the CMG helicase ring. The lagging strand is located outside of the CMG helicase and is protected by numerous RPA proteins.

The next step is to fill out the replisome with proteins that will provide structure and that will make sure that the activities of the CMG helicase and the DNA polymerases (yet to arrive) are coordinated.

Recruitment of the Fork Protection Complex (FPC)

When genome replication begins, CMG helicase will progress down the double stranded DNA, separating it into two single strands. Protein enzymes called polymerases (there are three, each with a specific role) will follow closely behind, synthesizing new strands off the two existing strands (both the leading strand inside the helicase and the lagging strand outside the helicase). These two activities--DNA strand separation and DNA polymerization--must be coordinated.

The next set of proteins to arrive will play structural roles in the replisome and will make sure that DNA strand separation and DNA polymerization by the various DNA polymerases are synchronized. The proteins that serve these structural and coordinating roles are referred to in the aggregate as the fork protection complex, or FPC.

The first proteins of the FPC are arranged as a pair. They are Timeless and Tipin. A third that serves similar roles is Claspin. Timeless-Tipin and Claspin attach to each other and to the CMG helicase. All provide structure to the replisome and keep the helicase harmonized with DNA polymerases. The FPC also stabilizes stalled forks, preventing the CMG helicase from running too far ahead of DNA replication if a fork encounters damage or stress.

Recrutment of the DNA polymerases and PCNA

Three different polymerases, each with different roles, must now be added to the replisome. They are DNA polymerase alpha-primase, epsilon and delta.

DNA polymerase alpha-primase

DNA polymerase alpha-primase arrives first. This polymerase is also referred to as a "primase" because its job is to synthesize the short DNA primers required to for DNA synthesis to begin on both the separated single strands.

DNA polymerase alpha-primase is recruited to the CMG helicase by the AND-1 protein. AND-1 attaches at one end to the CMG helicase and has a long arm that tethers DNA polymerase alpha-primase. Thanks to AND-1's flexible arm, DNA polymerase alpha-primase is able to reach and synthesize short DNA primers on both single strands.

DNA polymerase epsilon

A few blog posts from now we'll learn that DNA replication occurs somewhat differently on the two strands--the leading srtrandand the lagging strand) due to their opposite orientations. The two remaining polymerases, epsilon and delta, will work independently on the leading strand and lagging strand, respectively.

Before discussing DNA polymerase epsilon, though, I have to introduce two other related protein complexes that play crucial roles in the replisome: RFC (Replication Factor C) and PCNA (Proliferating Cell Nuclear Antigen). Once DNA polymerase alpha-primase constructs primers on the two strands, RFC attaches to the ends of each of the primers. It then loads a very large protein complex-- PCNA "sliding clamp"--around the primer and the single stranded DNA to which it is hybridized.

The PCNA sliding clamp is a central player in the replisome. I'll say a few things about it, but its role on the leading strand and lagging strand are somewhat different. We'll get into the difference in detail when we cover leading strand and lagging strand synthesis. But I'll say a few things about the differences here.

On the leading strand, DNA polymerase epsilon tethers directly to the CMG helicase (which is positioned out in front of the replisome). The PCNA sliding clamp encircles and rides along the DNA immediately behind DNA polymerase epsilon. Having DNA polymerase epsilon physically anchored to the CMG helicase ensures that the leading strand is synthesized in tight synchrony with the unwinding action of the CMG helicase.

The PCNA clamp that slides behind the DNA polymerase epsilon increases what is referred to as the enzyme's "processivity." This is, when PCNA is present, DNA polymerase epsilon synthesizes longer stretches of DNA before it falls off and needs to be replaced with another polymerase. Processivity refers to how long a polymerase polymerizes DNA before it falls off. High processivity equals robust DNA replication.

DNA polymerase delta

We're not ready to cover lagging strand synthesis in full, but we can set the stage by placing DNA polymerase delta correctly within the replisome. On the lagging strand, PCNA plays an even more significant role than it does on the leading strand. DNA polymerase delta, the polymerase that synthesizes on the lagging strand, does not attach to the CMG helicase (as does the leading strand). Rather, it attaches directly to PCNA, which itself will initially sit on the DNA primers on the laaggingstrand that were synthesized by DNA polymerase alpha-primase. With that exception, PCNA serves a similar role on the laggging strand as on the leading strand. Most importantly, it increases the DNA polymerase delta's processivity.

If you don't yet have a clear picture of the replisome, it might help to look at the figure above. And if you're not yet clear on leading strand versus lagging strand DNA synthesis, don't worry. I haven't explained it yet. I've only set the stage for its explanation.