title: An ORC/Cdc6/MCM2-7 complex is formed in a multistep reaction to serve as a platform for MCM double-hexamer assembly.
abstract: In Saccharomyces cerevisiae and higher eukaryotes, the loading of the replicative helicase MCM2-7 onto DNA requires the combined activities of ORC, Cdc6, and Cdt1. These proteins load MCM2-7 in an unknown way into a double hexamer around DNA. Here we show that MCM2-7 recruitment by ORC/Cdc6 is blocked by an autoinhibitory domain in the C terminus of Mcm6. Interestingly, Cdt1 can overcome this inhibitory activity, and consequently the Cdt1-MCM2-7 complex activates ORC/Cdc6 ATP-hydrolysis to promote helicase loading. While Cdc6 ATPase activity is known to facilitate Cdt1 release and MCM2-7 loading, we discovered that Orc1 ATP-hydrolysis is equally important in this process. Moreover, we found that Orc1/Cdc6 ATP-hydrolysis promotes the formation of the ORC/Cdc6/MCM2-7 (OCM) complex, which functions in MCM2-7 double-hexamer assembly. Importantly, CDK-dependent phosphorylation of ORC inhibits OCM establishment to ensure once per cell cycle replication. In summary, this work reveals multiple critical mechanisms that redefine our understanding of DNA licensing.
authors.affiliation: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College, London W12 0NN, UK.
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fernández-Cid
authors.forename: Alberto
authors.initials: A
authors.lastname: Riera
authors.forename: Silvia
authors.initials: S
authors.lastname: Tognetti
authors.forename: M Carmen
authors.initials: MC
authors.lastname: Herrera
authors.forename: Stefan
authors.initials: S
authors.lastname: Samel
authors.forename: Cecile
authors.initials: C
authors.lastname: Evrin
authors.forename: Christian
authors.initials: C
authors.lastname: Winkler
authors.forename: Emanuela
authors.initials: E
authors.lastname: Gardenal
authors.forename: Stefan
authors.initials: S
authors.lastname: Uhle
authors.forename: Christian
authors.initials: C
authors.lastname: Speck
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / 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; D000255:Adenosine Triphosphate; D018844:Cyclin-Dependent Kinases; C069199:MCM2 protein, S cerevisiae