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Quality control mechanisms exclude incorrect polymerases from the eukaryotic replication fork

G. Schauer,M. O’Donnell

Published 2017 in Proceedings of the National Academy of Sciences of the United States of America

ABSTRACT

Significance DNA replication is a central life process and is performed by numerous proteins that orchestrate their actions to separate the strands of duplex DNA and produce two new copies of the genome for cell division. While the antiparallel architecture of DNA is elegant in its simplicity, replication of DNA still holds many mysteries. For example, many essential replication proteins still have unknown functions. In eukaryotes the two DNA strands are duplicated by different DNA polymerases. The mechanism by which these different polymerases target to their respective strands is understood. This report examines the mechanisms that eject incorrect polymerases when they associate with the wrong strand. The eukaryotic genome is primarily replicated by two DNA polymerases, Pol ε and Pol δ, that function on the leading and lagging strands, respectively. Previous studies have established recruitment mechanisms whereby Cdc45-Mcm2-7-GINS (CMG) helicase binds Pol ε and tethers it to the leading strand, and PCNA (proliferating cell nuclear antigen) binds tightly to Pol δ and recruits it to the lagging strand. The current report identifies quality control mechanisms that exclude the improper polymerase from a particular strand. We find that the replication factor C (RFC) clamp loader specifically inhibits Pol ε on the lagging strand, and CMG protects Pol ε against RFC inhibition on the leading strand. Previous studies show that Pol δ is slow and distributive with CMG on the leading strand. However, Saccharomyces cerevisiae Pol δ–PCNA is a rapid and processive enzyme, suggesting that CMG may bind and alter Pol δ activity or position it on the lagging strand. Measurements of polymerase binding to CMG demonstrate Pol ε binds CMG with a Kd value of 12 nM, but Pol δ binding CMG is undetectable. Pol δ, like bacterial replicases, undergoes collision release upon completing replication, and we propose Pol δ–PCNA collides with the slower CMG, and in the absence of a stabilizing Pol δ–CMG interaction, the collision release process is triggered, ejecting Pol δ on the leading strand. Hence, by eviction of incorrect polymerases at the fork, the clamp machinery directs quality control on the lagging strand and CMG enforces quality control on the leading strand.

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