Mechanism of asymmetric polymerase assembly at the eukaryotic replication fork

Eukaryotes use distinct polymerases for leading- and lagging-strand replication, but how they target their respective strands is uncertain. We reconstituted Saccharomyces cerevisiae replication forks and found that CMG helicase selects polymerase (Pol) epsilon to the exclusion of Pol delta on the leading strand. Even if Pol delta assembles on the leading strand, Pol epsilon rapidly replaces it. Pol delta-PCNA is distributive with CMG, in contrast to its high stability on primed ssDNA. Hence CMG will not stabilize Pol delta instead leaving the leading strand accessible for Pol epsilon and stabilizing Pol epsilon. Comparison of Pol epsilon and Pol delta on a lagging-strand model DNA reveals the opposite. Pol delta dominates over excess Pol epsilon on PCNA-primed ssDNA. Thus, PCNA strongly favors Pol delta over Pol epsilon on the lagging strand, but CMG over-rides and flips this balance in favor of Pol epsilon on the leading strand.