Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA.
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.
Mesh Terms:
Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, Cloning, Molecular, Cryoelectron Microscopy, DNA Replication, DNA, Fungal, DNA-Binding Proteins, Image Processing, Computer-Assisted, Immunoprecipitation, Models, Molecular, Multiprotein Complexes, Mutagenesis, Site-Directed, Origin Recognition Complex, Protein Conformation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Cell Cycle Proteins, Chromosomal Proteins, Non-Histone, Cloning, Molecular, Cryoelectron Microscopy, DNA Replication, DNA, Fungal, DNA-Binding Proteins, Image Processing, Computer-Assisted, Immunoprecipitation, Models, Molecular, Multiprotein Complexes, Mutagenesis, Site-Directed, Origin Recognition Complex, Protein Conformation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Nat. Struct. Mol. Biol.
Date: Aug. 01, 2013
PubMed ID: 23851460
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