title: DNA induces conformational changes in a recombinant human minichromosome maintenance complex.
abstract: ATP-dependent DNA unwinding activity has been demonstrated for recombinant archaeal homohexameric minichromosome maintenance (MCM) complexes and their yeast heterohexameric counterparts, but in higher eukaryotes such as Drosophila, MCM-associated DNA helicase activity has been observed only in the context of a co-purified Cdc45-MCM-GINS complex. Here, we describe the production of the recombinant human MCM (hMCM) complex in Escherichia coli. This protein displays ATP hydrolysis activity and is capable of unwinding duplex DNA. Using single-particle asymmetric EM reconstruction, we demonstrate that recombinant hMCM forms a hexamer that undergoes a conformational change when bound to DNA. Recombinant hMCM produced without post-translational modifications is functional in vitro and provides an important tool for biochemical reconstitution of the human replicative helicase.
authors: From the Department of Biology, University of York, York YO10 5DD and.
authors: Emma L
authors: EL
authors: Hesketh
authors: From the Department of Biology, University of York, York YO10 5DD and.
authors: Richard P
authors: RP
authors: Parker-Manuel
authors: the Department of Crystallography, Birkbeck College London, London WC1E 7HX, United Kingdom.
authors: Yuriy
authors: Y
authors: Chaban
authors: From the Department of Biology, University of York, York YO10 5DD and.
authors: Rabab
authors: R
authors: Satti
authors: From the Department of Biology, University of York, York YO10 5DD and.
authors: Dawn
authors: D
authors: Coverley
authors: the Department of Crystallography, Birkbeck College London, London WC1E 7HX, United Kingdom.
authors: Elena V
authors: EV
authors: Orlova
authors: From the Department of Biology, University of York, York YO10 5DD and james.chong@york.ac.uk.
authors: James P J
authors: JP
authors: Chong
mesh_terms: D001483:Base Sequence; D002877:Chromosomes, Human; D004247:DNA / Q000737:chemistry*; D006801:Humans; D064110:Minichromosome Maintenance Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D004247:DNA; D064110:Minichromosome Maintenance Proteins
keywords: ATPase; DNA Helicase; DNA Replication; Electron Microscopy (EM); MCM; Minichromosome Maintenance; Recombinant Protein Expression
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