title: Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA.
abstract: DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.
authors.affiliation: a Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group , c/Melchor Fdez. Almagro 3, Madrid , Spain.
authors.forename: Jasminka
authors.initials: J
authors.lastname: Boskovic
authors.affiliation: b Protein Structure & Function Programme, Novo Nordisk Foundation Centre for Protein Research, Faculty of Heath and Medical Sciences, University of Copenhagen , Denmark.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Bragado-Nilsson
authors.affiliation: b Protein Structure & Function Programme, Novo Nordisk Foundation Centre for Protein Research, Faculty of Heath and Medical Sciences, University of Copenhagen , Denmark.
authors.forename: Bhargrav
authors.initials: B
authors.lastname: Saligram Prabhakar
authors.affiliation: a Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group , c/Melchor Fdez. Almagro 3, Madrid , Spain.
authors.forename: Igor
authors.initials: I
authors.lastname: Yefimenko
authors.affiliation: a Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), Macromolecular Crystallography Group , c/Melchor Fdez. Almagro 3, Madrid , Spain.
authors.forename: Jaime
authors.initials: J
authors.lastname: Martínez-Gago
authors.affiliation: c DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO) , Madrid , Spain.
authors.forename: Sergio
authors.initials: S
authors.lastname: Muñoz
authors.affiliation: c DNA Replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre (CNIO) , Madrid , Spain.
authors.forename: Juan
authors.initials: J
authors.lastname: Méndez
authors.affiliation: b Protein Structure & Function Programme, Novo Nordisk Foundation Centre for Protein Research, Faculty of Heath and Medical Sciences, University of Copenhagen , Denmark.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Montoya
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D021621:Imaging, Three-Dimensional; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D009711:Nucleotides; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D064110:Minichromosome Maintenance Proteins
keywords: DNA helicases; DNA replication; electron microscopy (EM); protein complexes; protein-DNA interaction