Abstract
Eukaryotic origins of replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM-DNA structure with the structure of CMG-DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of replication.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Cell Cycle Proteins / metabolism*
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Cryoelectron Microscopy
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DNA / metabolism
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DNA / ultrastructure*
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DNA Helicases
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DNA Replication*
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DNA-Binding Proteins / metabolism
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DNA-Binding Proteins / ultrastructure*
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Holoenzymes
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Image Processing, Computer-Assisted
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Minichromosome Maintenance Proteins / metabolism
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Minichromosome Maintenance Proteins / ultrastructure*
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Nuclear Proteins / metabolism
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Nuclear Proteins / ultrastructure*
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Nucleic Acid Conformation*
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Phosphorylation
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Protein Conformation*
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Protein Serine-Threonine Kinases / metabolism*
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Protein Structure, Quaternary
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Saccharomyces cerevisiae
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / ultrastructure*
Substances
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CDC45 protein, S cerevisiae
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Cell Cycle Proteins
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DNA-Binding Proteins
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Holoenzymes
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Nuclear Proteins
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Saccharomyces cerevisiae Proteins
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DNA
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CDC7 protein, S cerevisiae
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Protein Serine-Threonine Kinases
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DNA Helicases
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Minichromosome Maintenance Proteins