25. Lesions II: Fork Stalling and Protection (967)

In the previous chapter, I introduced the two major pathways that repair DNA lesions: base excision repair (BER) and nucleotide excision repair (NER). Both operate throughout the genome and the cell cycle.

But repair does not always occur before a replication fork arrives.

Sometimes a lesion escapes these repair systems and remains in the DNA when S phase begins. When that happens, the replication machinery will eventually encounter it. What happens next depends on the size of the lesion.

Very large lesions can block the helicase itself, halting the replication fork completely. But most lesions allow the helicase to continue unwinding DNA while the DNA polymerase stalls. In this chapter we will focus on this much more common scenario.

Fork stalling

Imagine a replication fork moving smoothly along the DNA, copying about fifty nucleotides per second. Suddenly the polymerase encounters a damaged base in the template strand.

The helicase, positioned ahead of the polymerase, may continue unwinding DNA for a short distance. But the polymerase cannot synthesize past the lesion.

This situation — where helicase movement becomes temporarily uncoupled from DNA synthesis — creates a gap between the advancing helicase and the stalled polymerase. That gap consists of single-stranded DNA.

This stretch of single-stranded DNA can grow to hundreds or even thousands of nucleotides long. Its appearance is not merely a structural problem for the cell. It is also an important signal.

Announcing the stall

Single-stranded DNA is unstable and vulnerable to damage. As we've seen before, almost immediately it becomes coated by Replication Protein A (RPA), a protein that binds tightly to exposed DNA single strands, protecting the DNA from degradation and preventing the single strand from folding into problematic secondary structures.

But RPA does more than protect DNA.

When long stretches of RPA-coated single-stranded DNA appear, the cell interprets this as a distress signal: replication has encountered a problem.

This signal is detected by a surveillance system centered on a protein kinase called ATR. ATR and its attached partner protein constantly scan the nucleus for RPA-coated DNA. When they finds some, ATR becomes activated. Activated ATR then initiates a signaling cascade driven primarily by CHK1.

A signaling cascade is a common cellular strategy for amplifying information. One activated protein (in our case, ATR) modifies several others, usually by phosphorylating them. Those proteins then activate still more proteins. In this way a small local signal can quickly spread throughout the cell.

In the replication stress response, the most important target of ATR is another kinase called CHK1.

Once activated, CHK1 molecules diffuse through the nucleus and phosphorylate many other proteins involved in responding to stalled replication forks. In essence, ATR detects the problem locally, while CHK1 spreads the message more broadly.

Stabilizing the fork

The first priority after a replication fork stalls is stabilization.

A stalled fork is fragile. The replication machinery can fall apart. If that were to happen, the exposed DNA strands could break or be degraded, potentially producing a dangerous chromosome break.

The cell therefore acts quickly to hold the fork in a safe configuration while it decides how to deal with the lesion. Several of the phosphorylations performed by CHK1 target replisome proteins including members of the fork protectin complex (FPC). These phosphorylations tend to strengthen the bonds between those proteins and prevent their degradation.

Also, checkpoint signaling helps keep the replication machinery intact and slows helicase movement so that the gap of single-stranded DNA does not grow uncontrollably.

In effect, the replication fork becomes temporarily frozen: it cannot move forward, but it is also prevented from collapsing.

Protecting the exposed DNA

Stabilizing the replication machinery is only half the problem. The exposed single-stranded DNA itself must also be protected.

Initially, this DNA was coated by RPA proteins. But if the stall persists, another protein replaces them: RAD51.

RAD51 assembles into a filament along the exposed DNA strand. Unlike RPA, which binds in discrete patches, RAD51 forms a more protective continuous helical coating. RAD51 is also phosphorylated by CHK1 and this phosphorylation enhances the protein's ability to displace RPAs on the single-stranded DNA.

The RAD51 filament acts like a protective sleeve around the DNA, stabilizing the strand and shielding it from nucleases and other damaging agents. The transition from RPA to RAD51 also prepares the DNA for later repair or fork remodeling events that may be needed to restart replication.

Reinforcing the response

Checkpoint signaling does not simply trigger the fork-stabilization response once. It also strengthens it through positive feedback.

Many of the phosphorylation events initiated by CHK1 make the signaling system more effective at activating additional CHK1 molecules. This creates a form of positive feedback.

This is another common strategy in cell signaling. An early step in a pathway increases the activity of later steps, which in turn reinforce the original step or signal.

The result is a stronger and more sustained response--exactly what the cell needs when a replication fork has stalled.

Buying time

Together, these mechanisms stabilize the replication fork and protect the exposed DNA while the cell both determines how to resolve the problem and actually resolves it. This protective state can last from minutes to hours.

During that time several outcomes are possible. The lesion may be repaired in place--that is, within the replication fork. Alternatively, the replication machinery might bypass the damage using specialized polymerases. Or, if the lesion is severe, the structure of the fork might be completely remodeled to better allow a repair to occur.

The first of these possibilities occurs within the replication fork, likely by BER or NER. In the next chapter we'll explore the second possibility: lesion bypass by specialized polymerases. The chapter after that will cover the third option: fork remodeling into structures that make repair easier and that then allow for replication fork resumption.

On to lesion bypass!