CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication

DNA replication in eukaryotes is asymmetric, with separate DNA polymerases (Pol) dedicated to bulk synthesis of the leading and lagging strands. Pol alpha/primase initiates primers on both strands that are extended by Pol epsilon on the leading strand and by Pol delta on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol epsilon for leading-strand synthesis, but to date a direct interaction between CMG and Pol epsilon has not been demonstrated. While purifying CMG helicase over-expressed in yeast, we detected a functional complex between CMG and native Pol epsilon. Using pure CMG and Pol epsilon, we reconstituted a stable 15-subunitCMG-Pol epsilon complex and showed that it is a functional polymerase-helicase on a model replication fork in vitro. On its own, the Pol2 catalytic subunit of Pol epsilon is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol epsilon, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol delta function with CMG, and thus we propose that the connection between Dpb2 andCMGhelps to stabilize Pol epsilon on the leading strand as part of a 15-subunit leading- strand holoenzyme we refer to as CMGE. Direct binding between Pol epsilon and CMG provides an explanation for specific targeting of Pol epsilon to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.