title: CMG helicase and DNA polymerase ε form a functional 15-subunit holoenzyme for eukaryotic leading-strand DNA replication.
abstract: 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 ε 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 ε for leading-strand synthesis, but to date a direct interaction between CMG and Pol ε has not been demonstrated. While purifying CMG helicase overexpressed in yeast, we detected a functional complex between CMG and native Pol ε. Using pure CMG and Pol ε, we reconstituted a stable 15-subunit CMG-Pol ε 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 ε is inefficient in CMG-dependent replication, but addition of the Dpb2 protein subunit of Pol ε, known to bind the Psf1 protein subunit of CMG, allows stable synthesis with CMG. Dpb2 does not affect Pol δ function with CMG, and thus we propose that the connection between Dpb2 and CMG helps to stabilize Pol ε on the leading strand as part of a 15-subunit leading-strand holoenzyme we refer to as CMGE. Direct binding between Pol ε and CMG provides an explanation for specific targeting of Pol ε to the leading strand and provides clear mechanistic evidence for how strand asymmetry is maintained in eukaryotes.
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and.
authors.forename: Lance D
authors.initials: LD
authors.lastname: Langston
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and.
authors.forename: Olga
authors.initials: O
authors.lastname: Yurieva
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and.
authors.forename: Roxana E
authors.initials: RE
authors.lastname: Georgescu
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and.
authors.forename: Jeff
authors.initials: J
authors.lastname: Finkelstein
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and.
authors.forename: Nina Y
authors.initials: NY
authors.lastname: Yao
authors.affiliation: Manhattan College, Riverdale, NY 10471.
authors.forename: Chiara
authors.initials: C
authors.lastname: Indiani
authors.affiliation: The Rockefeller University, Howard Hughes Medical Institute, New York, NY 10065; and odonnel@rockefeller.edu.
authors.forename: Mike E
authors.initials: ME
authors.lastname: O'Donnell
mesh_terms: D002850:Chromatography, Gel; D004265:DNA Helicases / Q000302:isolation & purification / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism*; D004261:DNA Replication; D004270:DNA, Circular / Q000378:metabolism; D020035:Holoenzymes / Q000378:metabolism*; D008954:Models, Biological; D021122:Protein Subunits / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D004270:DNA, Circular; D020035:Holoenzymes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D004257:DNA Polymerase II; D004265:DNA Helicases
keywords: CMG; DNA replication; helicase; polymerase; replication fork