title: Structure of eukaryotic CMG helicase at a replication fork and implications to replisome architecture and origin initiation.
abstract: The eukaryotic CMG (Cdc45, Mcm2-7, GINS) helicase consists of the Mcm2-7 hexameric ring along with five accessory factors. The Mcm2-7 heterohexamer, like other hexameric helicases, is shaped like a ring with two tiers, an N-tier ring composed of the N-terminal domains, and a C-tier of C-terminal domains; the C-tier contains the motor. In principle, either tier could translocate ahead of the other during movement on DNA. We have used cryo-EM single-particle 3D reconstruction to solve the structure of CMG in complex with a DNA fork. The duplex stem penetrates into the central channel of the N-tier and the unwound leading single-strand DNA traverses the channel through the N-tier into the C-tier motor, 5'-3' through CMG. Therefore, the N-tier ring is pushed ahead by the C-tier ring during CMG translocation, opposite the currently accepted polarity. The polarity of the N-tier ahead of the C-tier places the leading Pol ε below CMG and Pol α-primase at the top of CMG at the replication fork. Surprisingly, the new N-tier to C-tier polarity of translocation reveals an unforeseen quality-control mechanism at the origin. Thus, upon assembly of head-to-head CMGs that encircle double-stranded DNA at the origin, the two CMGs must pass one another to leave the origin and both must remodel onto opposite strands of single-stranded DNA to do so. We propose that head-to-head motors may generate energy that underlies initial melting at the origin.
authors.affiliation: Department of DNA Replication, The Rockefeller University, New York, NY 10065.|Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065.
authors.forename: Roxana
authors.initials: R
authors.lastname: Georgescu
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503.|Biochemistry and Structural Biology Graduate Program, Stony Brook University, Stony Brook, NY 11794.
authors.forename: Zuanning
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503.
authors.forename: Lin
authors.initials: L
authors.lastname: Bai
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503.|Biochemistry and Structural Biology Graduate Program, Stony Brook University, Stony Brook, NY 11794.
authors.forename: Ruda
authors.initials: R
authors.lastname: de Luna Almeida Santos
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503.
authors.forename: Jingchuan
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of DNA Replication, The Rockefeller University, New York, NY 10065.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of DNA Replication, The Rockefeller University, New York, NY 10065.|Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065.
authors.forename: Olga
authors.initials: O
authors.lastname: Yurieva
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503; Huilin.Li@VAI.org odonnel@rockefeller.edu.|Biochemistry and Structural Biology Graduate Program, Stony Brook University, Stony Brook, NY 11794.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
authors.affiliation: Department of DNA Replication, The Rockefeller University, New York, NY 10065; Huilin.Li@VAI.org odonnel@rockefeller.edu.|Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065.
authors.forename: Michael E
authors.initials: ME
authors.lastname: O'Donnell
mesh_terms: D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry*; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry*; D011487:Protein Conformation; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
chemical_list: C102209:CDC45 protein, S cerevisiae; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D064110:Minichromosome Maintenance Proteins
keywords: CMG helicase; DNA polymerase; DNA replication; origin initiation; replisome