title: Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.
abstract: In eukaryotes, the Cdt1-bound replicative helicase core MCM2-7 is loaded onto DNA by the ORC-Cdc6 ATPase to form a prereplicative complex (pre-RC) with an MCM2-7 double hexamer encircling DNA. Using purified components in the presence of ATP-γS, we have captured in vitro an intermediate in pre-RC assembly that contains a complex between the ORC-Cdc6 and Cdt1-MCM2-7 heteroheptamers called the OCCM. Cryo-EM studies of this 14-subunit complex reveal that the two separate heptameric complexes are engaged extensively, with the ORC-Cdc6 N-terminal AAA+ domains latching onto the C-terminal AAA+ motor domains of the MCM2-7 hexamer. The conformation of ORC-Cdc6 undergoes a concerted change into a right-handed spiral with helical symmetry that is identical to that of the DNA double helix. The resulting ORC-Cdc6 helicase loader shows a notable structural similarity to the replication factor C clamp loader, suggesting a conserved mechanism of action.
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York, USA.
authors.forename: Jingchuan
authors.initials: J
authors.lastname: Sun
authors.forename: Cecile
authors.initials: C
authors.lastname: Evrin
authors.forename: Stefan A
authors.initials: SA
authors.lastname: Samel
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fernández-Cid
authors.forename: Alberto
authors.initials: A
authors.lastname: Riera
authors.forename: Hironori
authors.initials: H
authors.lastname: Kawakami
authors.forename: Bruce
authors.initials: B
authors.lastname: Stillman
authors.forename: Christian
authors.initials: C
authors.lastname: Speck
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
mesh_terms: D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D020285:Cryoelectron Microscopy; D004261:DNA Replication / Q000502:physiology; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D047468:Immunoprecipitation; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D051738:Origin Recognition Complex / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C497214:ORC1 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; C069199:MCM2 protein, S cerevisiae