8.17 : 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.