28. Lesions IV: Fork Reversal (1,384) DONE

Let's reset the scene. A replication fork has stalled. The polymerase cannot move forward due to a lesion that it has reached that hasn't been repaired.

If the lesion isn't bypassed by TLS or re-priming in the first 20-30 minutes after RPA-single stranded DNA detection, the fork risks collapsing.

The cell's solution to this is surprisingly mechanical: it literally pushes the fork backward.

This maneuver, called fork reversal, moves the lesion away from the polymerase and places it back into double-stranded DNA where repair enzymes can access it more easily.

Fork reversal isn't the easiest cellular process to visualize, so let me use an analogy: a zipper.

During normal DNA replication, the CMG helicase moves along the double helix separating the two strands. In our analogy, the helicase is the zipper handle, pulling the zipper open.

But if a polymerase stalls at a lesion, the cell sometimes performs a surprising maneuver. Instead of trying to force the zipper open past the obstruction, the cell temporarily slides the zipper handle backward, allowing the teeth of the zipper behind it to close again.

That backward motion is fork reversal.

As the fork moves backward, the two template strands re-anneal with each other, while the two newly synthesized strands pair with each other, forming a four-way DNA junction often called a "chicken-foot structure."

With that rough mental picture in mind, we can now look at how the cell actually performs fork reversal.

RAD51 arrives

When a polymerase stalls at a lesion, specialize proteins assemble at the fork to recruit one of the most amazing proteins in genome replication and maintenance: RAD51.

RAD51 is best known for its role in homologous recombination, one of the most accurate repair processes in molecular biology. In homologous recombination, a damaged DNA strand uses RAD51 to help search for an intact copy of the same sequence on the sister chromatid. It uses that other sequence as the template for repair.

RAD51 makes this possible because it has a unique and powerful ability: it can help one DNA strand find and align with another DNA strand that has the same sequence.

Most proteins interact with DNA based on shape or chemical charge. RAD51 is different. It can recognize whether two strands contain the same genetic information.

That ability is truly extrordinary. It's useful not only in homologous recombination, but also at stalled replication forks.

Loading RAD51

At a stalled fork, the proteins BRCA1, PALB2, and BRCA2 collaborate to load RAD51 proteins onto the DNA.

You may recognize the names BRCA1 and BRCA2. Mutations in thse genes dramatically increase the riskk of breast and ovarian cancer. ("BRCA" is an acronym for "breast cancer.") Their importance stems from their normal role in protecting the genome during replication and repair.

In the context of replication, BRCA1 helps determine jwhether RAD51 activity is appropriate at the stalled fork. Once that decision is made, PALB2 connects BRCA1 to BRCA2. BRCA2 then delivers clusters of RAD51 proteins to the DNA.

RAD51 molecules assemble into a filament along the newly synthesized strands near the stalled fork.

This filament performs two critical functions.

First, it stabilizes the fragile DNA structure created by the stalled fork. This RAD51 filament is much more protective of single-stranded DNA than RPA proteins.

Second, it controls the activity of nucleases (enzymes that chew up DNA) such as MRE11. MRE11 chews back the newly synthesized leading strand by about 10-50 nucleotides. This controlled trimming--called resection--helps release the stalled polymerase and reset the fork.

Without RAD51, this trimming would become uncontrolled and thousands of nucleotides could be degraded, severely damaging the replication fork.

RAD51 thus acts as both protector (of single-stranded DNA) and regulator (of MRE11).

Fork reversal

Once the fork has been stabilized and the polymerase disengaged, fork reversal begins.

This step is carried out by specialized motor proteins called translocases. Translocases are molecular transporters that convert chemical energy into directed movement of molecules along DNA, RNA, or across cellular membranes. These enzymes are going to literally push the replication fork backward along the DNA.

As the fork moves backward, the two original template strands re-anneal with each other--just like the teeth of a zipper coming back together.

At the same time, the two newly synthesized strands detatch from these template strands and pair with each other.

The result is a four-way DNA junction known as a reversed fork or a "chicken-foot" structure.

RAD51 coats the paired nascent strands of this new arm, stabilizing the structure and preventing it from collapsing.

At this point the lesion, which originally blocked the polymerase, has been pushed back into a region of normal double-stranded DNA where repair enzymes can now reach it.

Fork reversal. In fork reversal, translocates remodel the replication fork. They push the normal 3-way replication fork backward (i.e., to the right in the figure) which requires that the two strands being synthesized anneal to each other to form the fourth leg of the 4-way junction or "chicken-foot" structure. The reversed fork places the lesion back in the context of double-stranded DNA where it is more easily repaired.

Repairing the DNA around the fork

Before replication can restart, the reversed fork requires some tidying up.

Because MRE11 trimmed the leading strand, the two nascent strands are no longer the same length. In addition, the lagging strand often contains small gaps between Okazaki fragments.

These problems are corrected via repair synthesis, carried out primarily by DNA polymerase delta. This synthesis doesn't have to be extremely accurate. The goal is simply to restore physical continuity to the DNA strands so the fork can safely restart.

Restarting replication

Once repairs are complete, the reversed fork must be restored back to its normal structure. This is essentially the reverse of fork reversal.

The helicase RECQ1 drives this process by pushing the DNA junction forward again, undoing the reversed structure and restoring the familiar three-way replication fork.

Replication can now resume. The zipper begins opening again!

Repairing DNA-Protein Crosslinks (DPCs)

One particularly challenging kind of lesion is a DNA-protein crosslink (DPC)--a protein that has become chemically bonded to DNA. These lesions are large and physically block DNA polymerases.

After fork reversal pushes the lesion into double-stranded DNA, the cell uses a specialized protease called SPRTN.

SPRTN is active only during DNA replicatioon. It recognizes stalled forks and then digests the crosslinked protein into small peptide fragments.

However, SPRTN cannot remove the final amino acid that remains chemically attached to the DNA. This small remnant--called a peptide-DNA adduct--is then removed by the nucleotide excision repair (NER) pathway.

Together SPRTN and NER form a two-step hybrid repair system for DNA-protein crosslinks.

This pathway is essential to humans. Mutations in SPRTN cause a rare disorder called Ruijs-Aalfs syndrome, characterized by chromosomal instability, premature aging, and early-onset cancer.

Why RAD51 matters

RAD51 appears in this chapter because of its role at stalled replication forks, but its importance in the cell extends well beyond fork reversal.

RAD51 is the central protein in homologous recombination (HR), one of the cell’s most accurate DNA repair pathways.

In that process, RAD51 forms filaments on single-stranded DNA and helps a damaged strand locate and pair with an intact copy of the same sequence on the sister chromatid. The intact copy then serves as the template for repair.

At stalled replication forks, RAD51 uses some of the same biochemical abilities for a different purpose. By assembling on the newly synthesized DNA strands, it stabilizes the fragile structures that arise when replication pauses. At the same time, it limits the activity of nucleases such as MRE11, preventing excessive degradation of the nascent DNA.

In this way, RAD51 helps ensure that a stalled replication fork remains stable long enough for repair and restart to occur. Without RAD51, stalled forks are far more likely to collapse, leading to chromosome breaks and genome instability.

Fork reversal is therefore one part of a carefully controlled response to trouble at the replication fork. By stabilizing the fork and repositioning the lesion, it buys the cell time to repair the damage and safely restart replication.

But when replication encounters serious obstacles, the response cannot remain confined to a single fork. Signals must spread outward in order to slow replication elsewhere in the genome and mobilize repair systems across the cell—a coordinated response that we will examine in the next chapter.