How the Eukaryotic Replisome Achieves Rapid and Efficient DNA Replication

Summary

The eukaryotic replisome is a molecular machine that coordinates the Cdc45-MCM-GINS (CMG) replicative DNA helicase with DNA polymerases α, δ, and ε 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 ε catalyzes leading-strand synthesis together with its processivity factor PCNA. DNA polymerase δ can support leading-strand synthesis, but at slower rates. DNA polymerase δ is required for lagging-strand synthesis, but surprisingly also plays a role in establishing leading-strand synthesis, before DNA polymerase ε engagement. We propose that switching between these DNA polymerases also contributes to leading-strand synthesis under conditions of replicative stress.

Graphical Abstract

Highlights

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  Reconstitution of a eukaryotic replisome capable of in vivo replication rates

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  Mrc1 directly stimulates replisome rate and is aided by Csm3/Tof1

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  Maximum rates require leading-strand synthesis by Pol ε together with PCNA

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  Pol δ plays a role in the establishment of leading-strand synthesis

Abstract

By reconstituting a eukaryotic replisome with purified proteins that can synthesize both leading and lagging strands at the in vivo rate, Yeeles et al. reveal the basis for rapid and efficient DNA replication by the eukaryotic replisome. Maximum rates require Mrc1 and Csm3/Tof1, and they are also dependent on leading-strand synthesis by Pol ε in the presence of PCNA. Using this system the authors show that, in addition to functioning on the lagging strand, Pol δ can play an important role in establishing leading-strand synthesis before handing over to Pol ε.