title: Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1.
abstract: To initiate DNA replication, the origin recognition complex (ORC) and Cdc6 load an Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, thus forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase-loading intermediate. Here we report a 3.9-Å structure of Saccharomyces cerevisiae OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHDs) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, thus comprising nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds the Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix within Orc4 and positively charged loops within Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by an Mcm5 loop that embraces Mcm2, but the N-tier-ring Mcm2-Mcm5 interface remains open. This structure suggests a loading mechanism of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, USA.
authors.forename: Zuanning
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: MRC London Institute of Medical Sciences (LMS), London, UK.|DNA Replication Group, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.
authors.forename: Alberto
authors.initials: A
authors.lastname: Riera
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Bai
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, USA.
authors.forename: Jingchuan
authors.initials: J
authors.lastname: Sun
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
authors.forename: Saikat
authors.identifier: 0000-0002-5693-6129
authors.initials: S
authors.lastname: Nandi
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Christos
authors.initials: C
authors.lastname: Spanos
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.
authors.forename: Zhuo Angel
authors.initials: ZA
authors.lastname: Chen
authors.affiliation: MRC London Institute of Medical Sciences (LMS), London, UK.|DNA Replication Group, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.
authors.forename: Marta
authors.initials: M
authors.lastname: Barbon
authors.affiliation: Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK.|Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
authors.forename: Juri
authors.identifier: 0000-0001-5999-1310
authors.initials: J
authors.lastname: Rappsilber
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA.
authors.forename: Bruce
authors.identifier: 0000-0002-9453-4091
authors.initials: B
authors.lastname: Stillman
authors.affiliation: MRC London Institute of Medical Sciences (LMS), London, UK.|DNA Replication Group, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, UK.
authors.forename: Christian
authors.identifier: 0000-0001-6646-1692
authors.initials: C
authors.lastname: Speck
authors.affiliation: Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, Michigan, USA.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
mesh_terms: D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D004261:DNA Replication; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D013058:Mass Spectrometry; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009711:Nucleotides; D029701:Saccharomyces cerevisiae Proteins; C499592:TAH11 protein, S cerevisiae; D064110:Minichromosome Maintenance Proteins