Restarting Stalled Replication Forks

When a replication fork stalls, DNA polymerase stops synthesizing nascent DNA. However, the helicase continues unwinding the double-stranded DNA for a short distance before dissociating, creating excess single-stranded DNA. This mismatch between polymerase and helicase activity is managed by the fork protection complex, which links their activities together.

Replication protein A (RPA) binds and protects excess single-stranded DNA at the stalled fork, recruiting the 9-1-1 complex and enabling ATR binding. ATR phosphorylates Chk1, which phosphorylates the phosphatase Cdc25, inactivating Cdk1 and pausing the cell cycle. This allows DNA damage can stall the cell cycle while repair mechanisms activate.

Fork regression is a process where Rad51 loads the enzyme SMARCAL1 onto stalled fork DNA. SMARCAL1 acts as an annealing helicase, displacing and sticking the two newly synthesized strands together to form a four-way junction resembling a chicken foot. This remodeled fork structure allows the cell to bypass lesions on the parental DNA strand.

BRCA2 stabilizes the Rad51 nucleofilament between the toes of the chicken foot junction, protecting the remodeled fork from degradation by nucleases. The nascent lagging strand then serves as a template for extending the leading strand, bypassing lesions on the parental strand. SMARCAL1 subsequently reverses the regression by reannealing the parental strands, leaving the lesion intact but allowing replication to continue.

Without BRCA2, the four-way chicken foot junction is cleaved by the structure-specific endonuclease Mus81, complexed with the junction endonuclease Mms4. This cleavage generates double-strand breaks, which may be repaired by homologous recombination and strand invasion pathways to restore replication fork function.

The fork protection complex links DNA polymerase activity with helicase activity. When polymerase stops due to stalled replication, the helicase continues unwinding the double helix briefly before dissociating. This creates an imbalance where helicase activity outpaces polymerase activity, generating excess single-stranded DNA that signals DNA damage to repair mechanisms.

Rad51 replaces replication protein A on the single-stranded DNA at the stalled fork before repair begins. Rad51 then loads SMARCAL1 onto the DNA to initiate fork regression, forming the four-way junction structure. Rad51 nucleofilaments stabilized by BRCA2 protect the remodeled fork and enable template switching to bypass DNA lesions.