27. Lesions III: Lesion Bypass (1,035;8/7)

As we saw in the last chapter, when a DNA polymerase stalls at an unrepaired lesion, the replication fork becomes vulnerable. Within minutes, the cell detects the RPA-coated single-stranded DNA, stabilizes the replisome, and protects the exposed DNA—a collection of responses known as fork protection.

From there, molecular pathways compete to determine what happens next: repair the lesion immediately via BER or NER, bypass it and perfrom BER or NER later in S phase, or completely reconfigure the replication fork to make a more difficult repair possible.

Of these three options, lesion bypass is the most likely. This chapter focuses on two lesion bypass options: (1) translesion synthesis (TLS) or (2) re-priming.

Think of TLS and re-priming not as repair pathways, but as lesion tolerance pathways. They allow genome replication to continue past a lesion without fixing it. The lesion will be repaired later by BER or NER. Lesion tolerance pathways allow the cell to finish copying its genome during S phase, which is the highest priority, even if it delays lesion repair.

Translesion Synthesis (TLS)

The first tolerance pathway we'll consider is translesion synthesis (TLS). In TLS, the stalled leading- or lagging-strand DNA polymerase is replaced by one of several specialized low-accuracy DNA polymerases. These polymerases are referred to as "TLS polymerases" or "bypass polymerases."

Bypass polymerases have active sites—the regions where the template base and incoming nucleotide come together—that are larger and more flexible than those of the leading- and lagging-strand polymerases. This allows them to accommodate many damaged or distorted bases on the template strand that would not fit properly into the tightly constrained active site of the two polymerases that perform the majority of genome replication. They can therefore insert a nucleotide opposite the lesion and allow DNA synthesis to continue past it.

There is not just one bypass polymerase. The cell uses several. Their names aren't important here, but they include polymerases eta, iota, kappa, and zeta, as well as one called REV1. Each is particularly well suited to bypass certain kinds of damaged or distorted DNA.

For example, DNA polymerase eta is particularly adept at bypassing UV-induced pyrimidine dimers, in which two neighboring pyrimidine bases—Cs and/or Ts—become strongly bound to each other. Each bypass polymerase is especially well suited to particular kinds of DNA damage, although there is some overlap in the lesions they can handle.

How does a cell know when a TLS polymerase is needed?

It relies on the universal replication distress signal: RPA-coated single-stranded DNA.

Like ATR and its partner detector protein, a different protein complex is recruited to sites where RPA-coated single-stranded DNA has accumulated. Once there, it attaches a small protein called a ubiquitin to the PCNA sliding clamp, which is itself located near the stalled DNA polymerase.

This attached ubiquitin molecule acts as a recruitment signal. Bypass polymerases contain regions that recognize ubiquitin, giving them an advantage in binding to the modified PCNA clamp. A bypass polymerase can then replace the stalled leading- or lagging-strand polymerase and synthesize DNA across the lesion.

Earlier I referred to bypass polymerases as "low-accuracy." This is because they are not only more forgiving of lesioned template bases, they are also more forgiving of non-complementary nucleotides. Thus, they are prone to introduce mutations by inserting a mismatched nucleotide across from the lesioned nucleotide.

The difference in accuracy between bypass polymerases and standard leading- and lagging-strand polymerases can be enormous. The two replicative polymerases, aided by proofreading, make errors only rarely, whereas some bypass polymerases can make errors as often as once every hundred to several thousand nucleotides. But the accuracy of bypass polymerases depends greatly on both the specific polymerase and the lesion. DNA polymerase eta, for example, can copy across the UV-induced lesions it specializes in with very high accuracy.

Once the lesion has been bypassed, the cell removes the ubiquitin molecule from the PCNA clamp. Without the ubiquitin, bypass polymerases lose their recruitment advantage and dissociate from the sliding clamp. A leading- or lagging-strand DNA polymerase then rebinds the clamp and resumes normal synthesis.

The lesion remains in the template strand, however. Replication continues, but the damage has not been repaired. It is left to BER or NER, or another repair pathway to deal with later.

The cell’s strategy is clear, though: complete genome replication first and repair the damage later.

Re-priming

The cell's other DNA lesion tolerance pathway is re-priming. As we'll see, whereas TLS goes through the obstacle, re-priming's strategy is to go around it.

Again, we start with a situation in which leading- or lagging-strand DNA polymerase reaches a lesion and stalls. Now, another unusual enzyme called PRIMPOL is recruited. It, too, is attracted to the universal replication stress signal: RPA-coated single-stranded DNA.

PRIMPOL is an unusual polymerase because it can synthesize its own short DNA primer from scratch. Unlike an ordinary DNA polymerase, it does not need a pre-existing 3′ end to begin synthesis.

We've already encountered one other human DNA polymerase that can do this: DNA polymerase alpha-primase. But this enzyme makes an RNA primer that it then extends with DNA whereas PRIMPOL constructs a DNA primer.

So, PRIMPOL simply synthesizes a short primer a short distance past the lesion. This new primer provides a fresh 3′ end from which normal DNA synthesis can resume The clamp loader then installs a new PCNA clamp, allowing the leading- or lagging-strand polymerase to resume synthesis. In effect, the replication machinery skips over the problem and keeps going.

But skipping the lesion leaves something behind: a stretch of single-stranded template DNA single-stranded gap between the lesion and the PRIMPOL primer and newly synthesized DNA. This gap must be filled or the cell risks DNA breaks and genome instability. One option is TLS. Another uses the newly made sister chromatid as a template to reconstruct the missing DNA—a powerful repair strategy called homologous recombination, which we will return to later.

Both lesion bypass pathways--TLS and re-priming--solve the immediate problem of a stalled DNA polymerase without removing the lesion, itself. TLS copies across the damaged site. Re-priming skips over it and leaves a gap to be dealt with later.

For the moment, however, the replication fork has accomplished its most urgent task: DNA synthesis can continue.