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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Cycle Proteins / chemistry*
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Cell Cycle Proteins / metabolism
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Chromosomal Proteins, Non-Histone / chemistry*
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Chromosomal Proteins, Non-Histone / metabolism
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Cloning, Molecular
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Cryoelectron Microscopy
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DNA Replication / physiology
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DNA, Fungal / chemistry
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DNA, Fungal / metabolism
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DNA-Binding Proteins / chemistry*
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DNA-Binding Proteins / metabolism
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Image Processing, Computer-Assisted
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Immunoprecipitation
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Models, Molecular*
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Multiprotein Complexes / chemistry*
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Mutagenesis, Site-Directed
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Origin Recognition Complex / chemistry*
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Origin Recognition Complex / metabolism
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Protein Conformation*
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Saccharomyces cerevisiae / chemistry*
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Saccharomyces cerevisiae Proteins / chemistry*
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Saccharomyces cerevisiae Proteins / metabolism
Substances
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CDC6 protein, S cerevisiae
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Cell Cycle Proteins
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Chromosomal Proteins, Non-Histone
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DNA, Fungal
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DNA-Binding Proteins
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Multiprotein Complexes
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ORC1 protein, S cerevisiae
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Origin Recognition Complex
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Saccharomyces cerevisiae Proteins
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TAH11 protein, S cerevisiae
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MCM2 protein, S cerevisiae