How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases alpha, delta, and epsilon and other proteins to copy the leading- and lagging-strand templates at rates between 1 and 2 kb min(-1). We have now reconstituted this sophisticated machine with purified proteins, beginning with regulated CMG assembly and activation. We show that replisome-associated factors Mrc1 and Csm3/Tof1 are crucial for in vivo rates of replisome progression. Additionally, maximal rates only occur when DNA polymerase epsilon catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase delta can support leading-strand synthesis, but at slower rates. DNA polymerase delta is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase epsilon engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress.