26. Lesions III: TLS and Re-priming (868) DONE

When a polymerase stalls at a lesion, the fork enters a state called fork protection. Within minutes the cell detects RPA-coated single-stranded DNA, stabilizes the replisome, and protects the exposed DNA while it attempts to repair or bypass the lesion.

Fork protection is the cell’s first response to a stalled DNA polymerase.

The second response, if the lesion isn't successfully bypassed, is fork reversal. This is a more drastic structural rearrangement. Fork reversal repositions a large or stubborn lesion back in the context of double stranded DNA and away from the polymerase where it is more easily fixed.

But that's for later because, in fact, most lesions are successfully dealt with during fork protection by one of two kinds of lesion bypass: (1) translesion synthesis (TLS) or (2) re-priming.

TLS and repriming are lesion tolerance pathways, not repair pathways. They allow replication to continue past a damaged base without fixing the damage itself. The lesion will be fixed later by BER or NER.

The lesion tolerance strategy allows the cell to finish copying its genome during S phase. This is the highest priority, even if it means delaying some repairs.

Translesion Synthesis (TLS)

The first and most common tolerance pathway is translesion synthesis (TLS).

In TLS, the stalled replicative polymerase is replaced by a specialized low-fidelity polymerase primarily from the Y-family. These polymerases are also called "TLS polymerases" or "bypass polymerases."

TLS polymerase active sites are much larger than those of the usual replicative polymerase. Thus, they are more forgiving, allowing passage of most small to medium-sized lesions that would otherwise block replication entirely.

Several TLS polymerases participate in lesion bypass, including DNA polymerases eta, iota, kappa, REV1, and the extender polymerase zeta (which extends the syntheses of the other TLS polymerases). Each accommodates specific kinds of distorted or bulky leasions.

For example, DNA polymerase eta specifically forgives UV-induced pyrimidine dimers (i.e., when two neighboring pyrimidines--Cs and/or Ts--become bound together). In effect, each TLS polymerase specializes in tolerating particular kinds of DNA damage.

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-coated single-stranded DNA.

Like ATR and its partner detector protein, the protein complex RAD6–RAD18 is recruited to sites where RPA-coated single-stranded DNA has accumulated.

Once there, it attaches a chemical group called a ubiquitin onto the PCNA sliding clamp, which itself is located near the stalled DNA polymerase.

This attached ubiquitin molecule increases the affinity of the sliding clamp for a TLS rather than a replicative polymerase. So, a TLS polymerase will replace the stalled replicative polymerase and extend synthesis past the lesion.

The TLS polymerases are considered "low-fidelity" because they are not only more forgiving of lesioned nucleotides, they are also more forgiving of mismatched nucleotides. So they are prone to introduce mutations by inserting the wrong nucleotide across from the lesion.

Let me quantify this. DNA polymerase epsilon has an error rate of one every 10 million to 100 million nucleotides. TLS polymerases vary, but make errors about once every 100-10,000 nucleotides. Thus,TLS polymerases are orders of magnitude more error-prone.

Once the lesion is bypassed, PCNA is de-ubiquitinated. Without the ubiquitin mark, TLS polymerases lose their recruitment advantage and dissociate from the PCNA. A replicative polymerase (e.g., DNA polymerase epsilon) then rebinds the clamp and resumes normal synthesis.

The lesion remains, though, to be repaired later.

Many lesions are bypassed via TLS, but, as mentioned, TLS carries a risk. Because the TLS polymerases have low fidelity, the nucleotides added across from the bypassed lesion may be wrong.

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

Re-priming

The cell's other DNA lesion tolerance pathway is re-priming.

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

PRIMPOL is unusual in that it can synthesize its own DNA primer. Unlike most polymerases, it does not need a pre-existing 3′ end to begin synthesis.

There's one other human DNA polymerase that we've already come across that can do this: DNA polymerase alpha-primase.

PRIMPOL synthesizes a short primer downstream of the lesion—typically 100–1,000 nucleotides ahead of it. This new primer provides a fresh 3′ end. The clamp loader then installs a new PCNA clamp, allowing the replicative polymerase to resume synthesis.

In effect, the replication machinery simply skips past the problem and keeps going.

This leaves in its wake a 100-1,000 nucleotide single-stranded gap between the lesion and the PRIMPOL primer.

This gap must be filled or the cell risks DNA breaks and genome instability. It will be filled later either via TLS or by another more complex repair pathway that we'll cover in a future chapter called homologous recombination (HR).

Both TLS and re-priming pathways address the stalled DNA polymerase but not the lesion, itself. The lesion will be repaired later by BER or NER.

For now, however, the fork has accomplished its immediate mission: the genome keeps being copied.