CMG helicase and DNA polymerase e 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 α/primase initiates primers on both strands that are extended by Pol e on the leading strand and by Pol δ on the lagging strand. The CMG (Cdc45-MCM-GINS) helicase surrounds the leading strand and is proposed to recruit Pol e for leading-strand synthesis, but to date a direct interaction between CMG and Pol e has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol e. Using pure CMG and Pol e, we reconstituted a stable 15-subunit CMG–Pol e 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 e is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol e, known to bind the Psf1 protein subunit of CMG, allows stable synthesiswith CMG.Dpb2does not affect Pol δ functionwith CMG, and thuswe propose that the connection betweenDpb2 and CMGhelps to stabilize Pol e on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol e andCMGprovides an explanation for specific targeting of Pol e to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.