7.9: Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a fork protection complex (FPC) travels with the growing fork. This conserved protein complex can be found in eukaryotes and is composed of proteins like Tim, Tipin, And1, and Claspin.

In the laboratory, replication forks can be stalled by the action of hydroxyurea. Hydroxyurea depletes the cellular pools of dNTPs, which are needed by DNA polymerase for DNA synthesis. When dNTPs are unavailable, DNA synthesis slows down and ultimately stops completely. Thus, the stalling of replication forks in living cells is linked to the inactivity of DNA polymerase.

FPC links the activity of the polymerase with that of the helicase. So even when the polymerase stops, the helicase keeps unwinding the DNA to produce an excess of single-stranded DNA (ssDNA) before coming to a halt. This excess ssDNA resembles resected overhangs from double-stranded break repair. To stabilize the structure, RPA proteins bind to the ssDNA and recruits the ATR proteins. ATR binding activates the cell cycle regulator protein Chk1 to block the firing of replication origins and stall the cell cycle for DNA repair. Thus, ssDNA serves as a potent signal that connects DNA damage to repair.

While halting a replication fork, the DNA polymerase stops synthesizing nascent DNA, but the helicase continues to unwind the double-stranded DNA for a short way before dissociating. Next, replication protein A, or RPA, binds and protects this excess single-stranded DNA at the stalled fork. 

The RPA-coated single-stranded DNA then recruits the Rad9-Rad1-Hus1, or “9-1-1” complex, which in turn enables the binding of ATR. The ATR binding triggers the phosphorylation of Chk1 and Chk1 in turn phosphorylates the phosphatase Cdc25. The phosphorylation means that Cdc25 cannot accept further phosphates from the cell cycle regulator protein Cdk1 - so Cdk1 remains inactive, and the cell cycle is paused.

Next, before repair starts, a recombination protein called Rad51 replaces RPA on the single-stranded DNA. Then, to initiate fork reversal, Rad51 loads an enzyme called SMARCAL1 on the DNA, which acts like an annealing helicase to displace and stick the two newly synthesized strands together and form a four-way junction that resembles a chicken foot. This process is called the fork regression.

There are two ways to resolve a fork regression. In the first, BRCA2 stabilizes the Rad51 nucleofilament between the toes of the chicken foot and protects the remodeled fork from degradation by nucleases. Now the nascent lagging strand can serve as a template for extending the leading strand, thus bypassing the lesions on the parental strand. 

Finally, SMARCAL1 reverses the regression fork by reannealing the parental strands. Here the lesion remains in the parent strand but the template switch allows the replicated DNA to be intact.

The second way of resolving the fork regression occurs in the absence of BRCA2, and here the four-way ‘chicken foot’ junction is cleaved by the structure-specific endonuclease Mus81, complexed with a junction endonuclease, Mms4. The cleavage generates double-stranded breaks, which may be repaired by homologous recombination.