28. Fork Protection (852; 8/6)

BER and NER remove many DNA lesions before they cause trouble. But they don't always get there on time. Sometimes a lesion is still present when a replication fork arrives.

Typically, in such cases, the CMG helicase continues moving and opening the DNA, but the polymerase suddenly stops, impeded by the lesion. The replication fork has entered a dangerous state. Unless the cell responds quickly, the carefully organized replication machinery may come apart and the fork may collapse, potentially leaving a broken chromosome.

This chapter is about the cell's emergency response during these critical moments.

When the Polymerase Hits an Obstacle

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

The CMG helicase, positioned ahead of the DNA 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 DNA polymerase. That gap consists of unwound single-stranded DNA.

This stretch of single-stranded DNA between the two molecular machines can grow to hundreds or--in severe or prolonged cases--even thousands of nucleotides long. Its appearance is not merely a structural problem for the cell. It is also an important signal, as we'll soon see.

The replication fork is now considered stalled, and a stalled fork is unstable. If the replisome comes apart or enzymes attack the exposed DNA, the fork can collapse, sometimes leaving behind a broken chromosome that is much harder for the cell to repair than the original lesion.

The original lesion may have been small. But the situation it creates is not.

Exposed DNA Raises the Alarm

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 them from degradation.

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 find some, ATR becomes activated. Activated ATR then initiates a signaling cascade.

A signaling cascade is a common cellular strategy for amplifying information. One activated protein (in this 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 through the cell.

In this replication stress response signaling cascade, one of ATR's most important targets 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 broadly.

Holding the Fork Together

The first priority after a replication fork stalls is stabilization. ATR-CHK1 signaling modifies numerous proteins at and around the replication fork, including components that help keep the replisome associated with the DNA.

Together, these changes help prevent the replication machinery from disassembling and protect the stalled fork from inappropriate processing. Some of these targets belong to the fork protection complex (FPC), a group of proteins that helps coordinate helicase and polymerase movement and maintain replisome stability.

Another result of this signaling cascade is to slow 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.

Reinforcing the response

The ATR-driven signaling cascade doesn't just trigger the fork-stabilization response once. It also strengthens it through positive feedback.

Many of the phosphorylation events initiated by ATR's main target, 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. The result is a stronger and more sustained response--exactly what the cell needs when a replication fork has stalled.

Buying time for a Decision

Stabilization does not solve the initial problem. The lesion is still there. What the response provides is time--time for the cell to choose what to do next. This protective state can last from minutes to hours.

During that time several routes are possible. Sometimes the lesion can still be repaired by BER or NER. Sometimes the fork itself is remodeled into a different structure that protects the DNA and creates new possibilities for repair or restart. We'll cover this process a couple chapters from now.

But the fastest solution is to temporarily replace the stalled polymerase with a different, specialized DNA polymerase capable of copying directly across lesioned DNA. This allows DNA replication to continue while postponing repair until later in S phase.

This strategy--lesion bypass--is the subject of the next chapter.