title: Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase.
abstract: Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs.
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK.
authors.forename: Gideon
authors.initials: G
authors.lastname: Coster
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK.
authors.forename: Jordi
authors.initials: J
authors.lastname: Frigola
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW3 6JB, UK.
authors.forename: Fabienne
authors.initials: F
authors.lastname: Beuron
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, London SW3 6JB, UK.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Morris
authors.affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts. EN6 3LD, UK. Electronic address: John.Diffley@cancer.org.uk.
authors.forename: John F X
authors.initials: JF
authors.lastname: Diffley
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D006868:Hydrolysis; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D016415:Sequence Alignment
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D064110:Minichromosome Maintenance Proteins
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