26. DNA Lesions III: TLS and Repriming

In the last post, we talked about fork protection, a posture that replication forks take when a lesion stalls a polymerase. Fork protection unfolds in minutes to tens of minutes after the cell detects RPA-dsDNA. Recall that it stabilizes the replisome structure and protects exposed DNA, giving the cell time to deal with the lesion.

But fork protection is only the first part of a two-part process called fork stabilization. Fork stabilization also includes fork reversal. However, it starts with fork protection and most lesions are "dealt with" during fork protection by one of two lesion tolerance pathways: translesion synthesis (TLS) and repriming. If these succeed, fork reversal won't be needed.

But what do I mean when I say that TLS and repriming "deal with" a lesion? What I mean is that they are lesion tolerance pathways rather than lesion repair pathways. They allow synthesis to proceed past the lesion without actually fixing the lesion (the lesion will be fixed later, likely by BER or NER). This enables genome replication to be completed during S phase, which is the highest priority.

But sometimes neither TLS nor repriming succeeds in bypassing the lesion. When that occurs, fork protection persists but, in addition, fork reversal kicks in. Fork reversal starts 30-60 minutes after RPA-ssDNA detection but can take as long as a few hours to complete. The purpose of fork reversal is to place a large or stubborn lesion back in the context of dsDNA where it's more easily fixed. We'll cover fork reversal in this post after we cover TLS and repriming.

Finally, after we discuss fork reversal, I'll discuss the process by which a cell would remove a large lesion following fork reversal. I'll use as an example a type of lesion called a DNA-protein crosslink (DPC), which is quite dangerous to the cell. DPCs arise when a protein becomes chemically glued to a stretch of DNA.

As an aside, I want to mention that I'm going to cite fewer protein names in this post, especially in discussing fork reversal. This is in part because many of the details about their roles are still being worked out. Fork reversal is currently an active area of research. Not only is it fascinating, it also has major implications for cancer treatment.

Translesion Synthesis (TLS)

As lesion tolerance pathways, TLS and repriming both embody the idea that, during the 8-hour S phase of the cell cycle, the cell's highest priority is completing genome replication--even if that means delaying some DNA repairs. TLS and repriming both usually take place while the fork is in fork protection mode, before fork reversal.

In TLS, a new DNA polymerase--not a high-fidelity replicative DNA polymerase like DNA polymerase epsilon or delta, but a lower-fidelity "Y family" DNA polymerase--is recruited to replace DNA polymerase epsilon on the leading strand. Y-family polymerase active sites are larger than those of the replicative polymerase. They are more forgiving, allowing passage of most small to medium-sized lesions. When they let a lesion pass, Y-family polymerases also sometimes add a one or few extra bases across from the lesion. So while they're great at letting lesions pass, they're also more error prone. I'll quantify this shortly.

There are four Y-family DNA polymerases: eta, iota, kappa, and one incongruently called REV1. Each accommodates specific kinds of distorted or bulky nucleotides. For example, DNA polymerase eta deals with UV-induced pyrimidine dimers as we discussed in a previous post (i.e., when two neighboring pyrimidines--Cs and Ts--become bound together). Each Y-family DNA polymerase allows specific kinds of lesions to pass. Y-family polymerases are also sometimes called "TLS polymerases" or "bypass polymerases."

Translesion synthesis (TLS). In TLS a Y-family polymerase replaces the normal replicative polymerase in order to bypass a DNA lesion

How does a cell know when a TLS polymerase is needed? It relies on the universal replication distress signal: RPA-ssDNA! Like ATR-ATRIP, the protein complex RAD6-RAD18 constantly scans the nucleus for RPA-ssDNA and rallies to it once it finds some. Once there, it attaches a specific chemical group called a ubiquitin molecule onto a specific amino acid of the PCNA sliding clamp (K164). The sliding clamp--which is located near the stalled polymerase--is now "monoubiquitinated." A mouthful. But this one ubiquitin attached to lysine 164 of the PCNA screams to the cell "bring on a Y-family polymerase!"

Many lesions are bypassed via TLS, but TLS carries a risk. Because the Y-family polymerases have low fidelity, the nucleotides across from the bypassed lesion may end up being wrong. DNA polymerase epsilon has an error rate of one every 10 million to 100 million nucleotides. Stop and think about that... one error every one hundred million nucleotides! TLS polymerases vary, but make an error about once every 100-10,000 nucleotides. The cell effectively says, "that's ok." It trades a few mutations for completing genome synthesis on time.

Once a Y-family polymerase allows a lesion to pass, it begins synthesizing even further using the template strand. But low-fidelity TLS polymerases aren't good at this. Their active sites don't fit unlesioned template strands as well as DNA polymerase epsilon's active site does. Thus, synthesis slows and DNA polymerase epsilon, with its better fit, supplants the Y-family polymerase. Also, once the lesion is bypassed via TLS, an enzyme complex called USP1-UAF1 arrives to de-monoubiquitinate PCNA ay K164, leaving it with little remaining affinity for Y-family polymerases.

Thus, DNA polymerase epsilon replaces the transiently-needed Y-family polymerase and normal replication restarts. The lesion remains, though. It will be repaired later. And, as I mentioned, the accuracy of the few nucleotides across from the lesion isn't guaranteed.

Repriming

The cell's other DNA lesion tolerance pathway is repriming. This is as simple as it sounds. Once DNA polymerase epsilon reaches a lesion on the leading strand, it stalls. Then, yet another DNA polymerase called PRIMPOL arrives. It, too, is attracted to the universal replication stress signal: RPA-ssDNA.

PRIMPOL is a very special kind of DNA polymerase in that it can synthesize its own primer. It doesn't need a 3' end to begin synthesizing. There's one other human DNA polymerase that can do this and we're already come across it: DNA polymerase alpha-primase.

Once PRIMPOL arrives, it synthesizes an 8-10 nucleotide hybrid RNA-DNA primer 100-1,000 nucleotides beyond the lesion (that is, in the direction of the CMG helicase). Sometimes it also extends the primer a short distance. But then it leaves and RFC loads a PCNA sliding clamp onto the DNA near the new primer. PCNA partners with a DNA polymerase epsilon and leading strand synthesis resumes, leaving that 100-1,000 nucleotide single-stranded gap between the lesion and the PRIMPOL primer in its wake.

This gap must be filled or the cell risks DNA breaks and genome instability. It will be filled later either by TLS or by another more complex repair pathway that we'll cover in the next post called homologous recombination (HR). The TLS and repriming pathways address the gap but not the lesion. The lesion will be repaired later by BER or NER.

Think of TLS and repriming as first response to a lesion that has stalled a DNA polymerase.