title: Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function.
abstract: Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA;
authors.forename: Jingchuan
authors.initials: J
authors.lastname: Sun
authors.affiliation: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London W12 0NN, United Kingdom;
authors.forename: Alejandra
authors.initials: A
authors.lastname: Fernandez-Cid
authors.affiliation: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London W12 0NN, United Kingdom;
authors.forename: Alberto
authors.initials: A
authors.lastname: Riera
authors.affiliation: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London W12 0NN, United Kingdom;
authors.forename: Silvia
authors.initials: S
authors.lastname: Tognetti
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA.
authors.forename: Zuanning
authors.initials: Z
authors.lastname: Yuan
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA hli@bnl.gov stillman@cshl.edu chris.speck@csc.mrc.ac.uk.
authors.forename: Bruce
authors.initials: B
authors.lastname: Stillman
authors.affiliation: DNA Replication Group, MRC Clinical Sciences Centre, Imperial College Faculty of Medicine, London W12 0NN, United Kingdom; hli@bnl.gov stillman@cshl.edu chris.speck@csc.mrc.ac.uk.
authors.forename: Christian
authors.initials: C
authors.lastname: Speck
authors.affiliation: Biosciences Department, Brookhaven National Laboratory, Upton, New York 11973, USA; Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794, USA hli@bnl.gov stillman@cshl.edu chris.speck@csc.mrc.ac.uk.
authors.forename: Huilin
authors.initials: H
authors.lastname: Li
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004789:Enzyme Activation; D006868:Hydrolysis; D008854:Microscopy, Electron; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008968:Molecular Conformation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
chemical_list: D000255:Adenosine Triphosphate; D064110:Minichromosome Maintenance Proteins
keywords: DNA replication initiation; electron microscopy; origin recognition complex; prereplication complex; replicative helicase