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      Structural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1

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          Abstract

          To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

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          Most cited references59

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          DNA replication in eukaryotic cells.

          The maintenance of the eukaryotic genome requires precisely coordinated replication of the entire genome each time a cell divides. To achieve this coordination, eukaryotic cells use an ordered series of steps to form several key protein assemblies at origins of replication. Recent studies have identified many of the protein components of these complexes and the time during the cell cycle they assemble at the origin. Interestingly, despite distinct differences in origin structure, the identity and order of assembly of eukaryotic replication factors is highly conserved across all species. This review describes our current understanding of these events and how they are coordinated with cell cycle progression. We focus on bringing together the results from different organisms to provide a coherent model of the events of initiation. We emphasize recent progress in determining the function of the different replication factors once they have been assembled at the origin.
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            AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes.

            Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously. Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase. Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class. Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life. These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes. The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes.
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              Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing.

              The licensing of eukaryotic DNA replication origins, which ensures once-per-cell-cycle replication, involves the loading of six related minichromosome maintenance proteins (Mcm2-7) into prereplicative complexes (pre-RCs). Mcm2-7 forms the core of the replicative DNA helicase, which is inactive in the pre-RC. The loading of Mcm2-7 onto DNA requires the origin recognition complex (ORC), Cdc6, and Cdt1, and depends on ATP. We have reconstituted Mcm2-7 loading with purified budding yeast proteins. Using biochemical approaches and electron microscopy, we show that single heptamers of Cdt1*Mcm2-7 are loaded cooperatively and result in association of stable, head-to-head Mcm2-7 double hexamers connected via their N-terminal rings. DNA runs through a central channel in the double hexamer, and, once loaded, Mcm2-7 can slide passively along double-stranded DNA. Our work has significant implications for understanding how eukaryotic DNA replication origins are chosen and licensed, how replisomes assemble during initiation, and how unwinding occurs during DNA replication.
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                Author and article information

                Journal
                101186374
                31761
                Nat Struct Mol Biol
                Nat. Struct. Mol. Biol.
                Nature structural & molecular biology
                1545-9993
                1545-9985
                6 July 2017
                13 February 2017
                March 2017
                13 August 2017
                : 24
                : 3
                : 316-324
                Affiliations
                [1 ]Cryo-EM Structural Biology Laboratory, Van Andel Research Institute, Grand Rapids, MI 49503, USA
                [2 ]DNA Replication Group, Institute of Clinical Sciences (ICS), Faculty of Medicine, Imperial College London, London, W12 0NN, United Kingdom
                [3 ]Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
                [4 ]Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom
                [5 ]Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany
                [6 ]MRC London Institute of Medical Sciences (LMS), Du Cane Road, London W12 0NN
                Author notes
                Article
                EMS71067
                10.1038/nsmb.3372
                5503505
                28191893
                aea65a2d-e34b-4b24-aa6b-547e12eaa7df

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                Molecular biology
                Molecular biology

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