25. DNA Lesions II: Fork Stalling and Protection

In the last post, I introduced the cell's two main DNA lesion repair pathways--base excision repair (BER) and nucleotide excision repair (NER). Both operate well beyond the replication fork during all of the phases of the cell cycle.

But what happens if a lesion escapes BER and NER and finds itself in S phase in front of a moving replication fork? Most DNA lesions get past the CMG helicase but not the DNA polymerase, although some very large lesions can't get past the helicase. Our focus here will be the majority of lesions that stall at the polymerase. We'll consider the cell's response to larger legions in a future post.

Fork Stalling

Picture a replication fork humming along, copying DNA at 50 nucleotides per second. Suddenly, there's a lesion directly in its path on the leading strand. It gets through the helicase but not the polymerase. Most likely it doesn't fit in the polymerase's active site, the domain or region of the protein that does all the work. So let's assume it's a modest lesion: a chemical alteration to a base or an unwanted bond between neighboring bases.

When DNA polymerase epsilon stalls at a lesion like this on the leading strand, the helicase keeps moving forward, unwinding DNA. This generates a stretch of single-stranded DNA (ssDNA) a few hundred to a thousand nucleotides long between the advancing helicase and the stalled polymerase. We'll soon see that this stretch of ssDNA is very informative to the cell. But before getting into that, let's first pause to consider lesions on the lagging strand.

Unlike lesions on the leading strand, lagging strand lesions cause fewer problems because the lagging-strand machinery naturally reprimes downstream. So, even if DNA polymerase delta stalls at a lesion on the lagging strand template, synthesis will continue via repeated primings by DNA polymerase alpha-primase and syntheses of Okazaki fragments by DNA polymerase delta past the lesion.

The lagging strand still has to be repair, though. A lesion on the lagging template strand typically leaves a 20-200 nucleotide gap across from it on the strand being synthesized. And the lesion still sits on the template strand. But these aren't serious problems. The cell will fix them later. The take-away is that lagging strand lesions don't slow down genome replication like leading strand lesions. Thus, our focus on leading strand lesions.

Announcing the Stall

Back to our typical leading strand lesion. As soon as the long stretch of ssDNA appears between the stalled polymerase and the helicase--again, a few hundred to a thousand nucleotides long--Replication Protein A (RPAs) arrive immediately to coat it. Recall that the job of RPAs is to protect ssDNA both against nucleases that would otherwise destroy it and secondary structures that could cause more replication problems.

But the RPA coat on the ssDNA (RPA-ssDNA) plays another role. It is the universal signal to the cell that there is a DNA replication problem. It says, effectively, "There's a lesion on the leading strand. It has stalled the polymerase and it must be dealt with." This distress signal is powerfully agnostic with respect to type of lesion. Any lesion that stalls a polymerase will produce this RPA-ssDNA signal. But how is the signal detected?

By another specialized protein, of course! The detector is one of two subunits of a protein complex called ATR-ATRIP. ATR-ATRIP is always present in the nucleus, flying and crashing around in search of RPA-ssDNA. When it finds some (typically in S-phase, since that's when replication occurs), ATRIP subunits attach in clusters to RPAs along the ssDNA, positioning important but not yet activated kinases--ATR kinases--near the lesion.

ATR kinase is the first molecular messenger that communicates to the cell that there's a replication problem. But first ATR must be activated. To this end, the ring-shaped protein complex 9-1-1 loads onto the lagging strand near the lesion with the help of a specific ring loader. Then 9-1-1 recruits TOPBP1, which binds to the 9-1-1 ring bringing its ATR-activation domain, or AAD, close to the ATR kinases.

TOPBP1's AAD domain reaches over and attaches to an ATR kinase for a few milliseconds to a few seconds. While the AAD is attached, ATR undergoes a conformation change that activates it, giving it the ability to phosphorylate other target proteins. When the AAD detaches, the ATR becomes inactivated. But then, right away, the AAD will attach to and activate another ATR located near the first--also very briefly. In this way, a single TOPBP1 can activate (in succession and transiently) many ATR kinases.

A signalling cascade

The most important protein that these activated ATR kinases will phosphorylate and, in turn activate is another kinase, CHK1. Unlike ATR, which is anchored to the ssDNA via ATRIP and which is dependent on local TOPBP1s for activation, activated CHK1s roam around the nucleus--they are diffusible! So they roam around, phosphorylating dozens of other proteins needed for the response to the RPA-ssDNA signal. These other proteins then go on to do whatever they need to do, including activating other proteins and/or turning on genes.

A bit of an aside: The scenario I just described illustrates a general strategy cells use to amplify messages. It's called a signaling cascade and it's a bit like a pyramid scheme. In this particular signaling cascade, ATR-ATRIP is the founder sitting atop the pyramid. Its ATR subunit phosphorylates a bunch of first-tier recruits: multiple CHK1s. These now-activated CHK1s go on to phosphorylate a bunch of different next level proteins to promote, inhibit or alter their activities. Some of these will go on to activate other proteins.

In this way, information gleaned by one protein (ATR-ATRIP) is amplified by another protein (CHK1) and then further amplified by still other unnamed proteins below it in the pyramid, spreading the message and its effects quickly throughout the cell. A signaling cascade.

The local response

As mentioned, the first and most important protein ATR kinases will activate in this signaling cascade will be CHK1 kinases. Phosphorylations performed by CHK1 of different proteins below it in the signaling cascade will initiate an immediate local response to the lesion and also contribute to a cell-wide global response. We'll focus on the local response first.

I'll refer to the local response as "fork stabilization." Fork stabilization has two phases. The first is fork protection. A paused replisome can easily fall apart and loose DNA strands can break or be damaged. Fork protection freezes and stabilizes the replisome, buying more time to deal with the lesion. Fork protection lasts from minutes to tens of minutes.

If the lesion isn't repaired during fork protection, then the second phase of fork stabilization, fork reversal, will have time to unfold. In fork reversal, the cell literally reverses course, rewinding rather than unwinding the replication fork. This re-positions the problematic lesion back in the context of dsDNA where it will be easier to fix. Fork reversal takes from tens of minutes to hours.

Fork Protection

Fork protection stabilizes and protects two targets creating a fork that can't move forward but that won't fall apart, either. The first is the structure of the replisome... basically, its proteins. These include the CMG helicase, PCNA sliding clamp, the DNA polymerases, and other proteins we've discussed such as TIMELESS and AND-1. The second target is the ssDNA on the leading template strand between DNA polymerase epsilon and the CMG helicase. This stretch of ssDNA will already be protected by RPA proteins. But with a stalled fork, ssDNA requires a greater level of protection.

Protecting the structure of the replisome

To protect the replisome's structure, CHK1 phosphorylates several replisome proteins we've already encountered: the CMG helicase, AND-1, and TIMELESS. Perhaps most importantly, CHK1 phosphorylates the TIMELESS subunit of the TIMELESS-TIPIN complex. During normal leading strand replication, TIMELESS-TIPIN is a component of the fork protection complex (FPC). It travels with the replication fork, coupling CMG helicase unwinding with DNA polymerase epsilon synthesis.

During replication stress (i.e., when RPA-ssDNA appears), TIMELESS is phosphorylated by CHK1 at specific amino acids. These alter its shape with three effects, two of which relate to the CMG helicase and one of which relates to CLASPIN.

• they make TIMELESS bind more strongly to the CMG helicase, stabilizing it in place and preventing its premature disassembly.

• they make the CMG helicase resistant to monoubiquitination at a specific amino acid, which would lead to its unloading off the DNA.

• they keep CLASPIN stably localized at the stalled fork. Without this anchoring, CLASPIN would frequently dissociate.

In addition to phosphorylating TIMELESS, CHK1 also phosphorylates MCM2-7 components of the CMG helicase. These slow it down, avoiding excessive polymerase-helicase uncoupling. Thus, the phosphorylations of these MCM proteins and those on TIMELESS keep the CMG helicase in position and intact.

Last but not least, CHK1 phosphorylates AND-1, the protein that tethers DNA polymerase alpha-primase. This stabilizes the AND-1 protein itself and also maintains DNA polymerase alpha-primase's association with AND-1.

All of these phosphorylations--of TIMELESS, the CMG helicase, and AND-1--combine to create a reinforced, stabilized replisome that remains intact until the lesion is dealt with.

A positive feedback loop

The CHK1 phosphorylation of TIMELESS and a CHK1 phosphorylation of CLASPIN that I haven;t mentioned yet create positive feedback loop. Let me pause to explain what that means. A positive feedback loop is a situation in which a process (in our case, phosphorylations by CHK1) increases or alters the activity of one or more components of that process (in our case, TIMELESS and CLASPIN), that, in turn, enhance the original process (phosphorylations by CHK1). This feedback loop boosts all of the stabilization effects I just mentioned.

This particular positive feedback loop has two parts. Recall that a CLASPIN scaffold is required for ATR phosphorylation and activation of CHK1. Also, as I just mentioned, CHK1 phosphorylations of TIMELESS keep CLASPIN stabilized at the fork. Stabilizing CLASPIN at the fork means that more CHK1 molecules will encounter it there and will be phosphoylated by ATR. So, CHK1 phosphorylation of TIMELESS has, beyond its replisome stabilizing effects, the effect of increasing the number of activated CHK1s!

The second aspect of the feedback loop involves CHK1 phosphorylation of CLASPIN, itself. Phosphorylated, CLASPIN undergoes a conformation change that makes it more efficient at facilitating ATR phosphorylations of CHK1. So, by phosphorylating CLASPIN, CHK1 converts it into a "super-CLASPIN" which is better at helping ATR phosphorylate CHK1!

To summarize, then, the two aspects of the positive feedback loop are: (1) a more present CLASPIN scaffold (due to CHK1's action on TIMELESS) and, (2) a more effective CLASPIN scaffold (due to CHK1's action directly on CLASPIN). Much like signaling cascades, positive (and negative) feedback loops are a common strategy used by cells.

Protecting ssDNA

To better protect the leading strand ssDNA between the DNA polymerase and the helicase, ATR--and to a lesser extent its primary target CHK1--first phosphorylate a protein called FANCD2, a component of the FANCD2–FANCI complex, at two specific amino acids. These change the complex's shape such that it clamps down on the DNA near the lesion. The shape change also permits a chemical addition called "monoubiquitination" of FANCD2. That is, it allows one ubiquitin molecule (chemical structure unimportant) to be attached to FANCD2 at a specific amino acid. This form is called “FANCD2-Ub”.

FANCD2-Ub recruits—and creates a chromatin environment that favors the recruitment of—multiple chromatin remodelers and histone-modifying enzymes. These open chromatin just ahead of and just behind the stalled replication fork, allowing BRCA1 and PALB2 to assemble adjacent to the ssDNA. PALB2 then brings BRCA2 to the ssDNA–chromatin interface, where BRCA2 initiates RAD51 loading onto the RPA-coated ssDNA. BRCA2 enables RAD51 to displace RPA and form a stable, highly protective helical filament around the ssDNA between the polymerase and the helicase.

Also in support of better ssDNA protection, CHK1 phosphorylates RAD51 itself at a specific amino acid. This enhances RAD51’s ability to displace RPAs on the ssDNA and to assemble and form a RAD51 filament that better coats and protects that ssDNA. The CHK1 phosphorylations of FANCD2 (to initiate BRCA1, PALB2 and BRCA2 recruitment) and of RAD51 (to enhance ssDNA coating) are but two of the dozens that CHK1 performs as part of the replication stress response.

Let’s pause to say a few words about the RPA to RAD51 exchange, because I've been saying that RPAs protect ssDNA (and they do, to some extent!). An RPA molecule is a large heterotrimer that covers roughly 30 nucleotides of ssDNA. In contrast, a RAD51 monomer covers only about 3 nucleotides when incorporated into a filament. Unlike RPA, though, which more haphazardly attach to ssDNA, RAD51 monomers polymerize into a dense and continuous helical filament along the ssDNA. Thus, RAD51 is much smaller but achieves a denser and thus more protective coating of the ssDNA.

CLOSING PARAGRAPH