Concerted Loading of Mcm2–7 Double Hexamers around DNA during DNA Replication Origin Licensing

Clare Hall Laboratories, Cancer Research UK London Research Institute, South Mimms EN6 3LD, UK.
Cell (Impact Factor: 32.24). 11/2009; 139(4):719-30. DOI: 10.1016/j.cell.2009.10.015
Source: PubMed


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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    • "Furthermore, we used super-resolution 3D structured illumination microscopy (SIM) to quantify the chromatin-bound MCM2–7 complexes. SIM reaches 120 nm resolution in the x and y axis and 300 nm in the Z axis (Figure 1E), and a double hexameric MCM2–7 complex on DNA measures 25 3 16 nm (Evrin et al., 2009; Remus et al., 2009). Hence, each focus observed by SIM contains multiple MCM2–7 complexes. "
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    ABSTRACT: Maintaining genomic integrity during DNA replication is essential for stem cells. DNA replication origins are licensed by the MCM2-7 complexes, with most of them remaining dormant. Dormant origins (DOs) rescue replication fork stalling in S phase and ensure genome integrity. However, it is not known whether DOs exist and play important roles in any stem cell type. Here, we show that embryonic stem cells (ESCs) contain more DOs than tissue stem/progenitor cells such as neural stem/progenitor cells (NSPCs). Partial depletion of DOs does not affect ESC self-renewal but impairs their differentiation, including toward the neural lineage. However, reduction of DOs in NSPCs impairs their self-renewal due to accumulation of DNA damage and apoptosis. Furthermore, mice with reduced DOs show abnormal neurogenesis and semi-embryonic lethality. Our results reveal that ESCs are equipped with more DOs to better protect against replicative stress than tissue-specific stem/progenitor cells. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Stem Cell Reports 07/2015; 5(2). DOI:10.1016/j.stemcr.2015.06.002 · 5.37 Impact Factor
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    • "Although high-salt-resistant single hexamers have been detected after artificially closing the Mcm2/5 gate (Samel et al., 2014), previous studies have not detected single loaded (highsalt resistant) Mcm2–7 complexes in unperturbed helicaseloading reactions (Evrin et al., 2009; Kang et al., 2014; Remus et al., 2009). This difference may be due to the higher protein concentrations used in these ensemble reactions. "
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    ABSTRACT: Loading of the ring-shaped Mcm2-7 replicative helicase around DNA licenses eukaryotic origins of replication. During loading, Cdc6, Cdt1, and the origin-recognition complex (ORC) assemble two heterohexameric Mcm2-7 complexes into a head-to-head double hexamer that facilitates bidirectional replication initiation. Using multi-wavelength single-molecule fluorescence to monitor the events of helicase loading, we demonstrate that double-hexamer formation is the result of sequential loading of individual Mcm2-7 complexes. Loading of each Mcm2-7 molecule involves the ordered association and dissociation of distinct Cdc6 and Cdt1 proteins. In contrast, one ORC molecule directs loading of both helicases in each double hexamer. Based on single-molecule FRET, arrival of the second Mcm2-7 results in rapid double-hexamer formation that anticipates Cdc6 and Cdt1 release, suggesting that Mcm-Mcm interactions recruit the second helicase. Our findings reveal the complex protein dynamics that coordinate helicase loading and indicate that distinct mechanisms load the oppositely oriented helicases that are central to bidirectional replication initiation. Copyright © 2015 Elsevier Inc. All rights reserved.
    Cell 04/2015; 161(3):513-525. DOI:10.1016/j.cell.2015.03.012 · 32.24 Impact Factor
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    • "Mcm2–7 loading depends strictly on ORC and Cdc6 and is highly dependent on ARS1, as anticipated (Figure 5B). Previous reports have found that loaded Mcm2–7 double hexamers can diffuse along DNA when challenged with a high-salt wash (Evrin et al., 2009; Remus et al., 2009). We find that a small fraction of Mcm2–7 complexes diffuse along the DNA in the presence of 0.5 M NaCl (1/192), with the vast majority remaining stably bound, without diffusing freely, even when visualized for tens of minutes (Figure S4). "
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    ABSTRACT: Eukaryotic replication initiation is highly regulated and dynamic. It begins with the origin recognition complex (ORC) binding DNA sites called origins of replication. ORC, together with Cdc6 and Cdt1, mediate pre-replicative complex (pre-RC) assembly by loading a double hexamer of Mcm2-7: the core of the replicative helicase. Here, we use single-molecule imaging to directly visualize Saccharomyces cerevisiae pre-RC assembly and replisome firing in real time. We show that ORC can locate and stably bind origins within large tracts of non-origin DNA and that Cdc6 drives ordered pre-RC assembly. We further show that the dynamics of the ORC-Cdc6 interaction dictate Mcm2-7 loading specificity and that Mcm2-7 double hexamers form preferentially at a native origin sequence. Finally, we demonstrate that single Mcm2-7 hexamers propagate bidirectionally, monotonically, and processively as constituents of active replisomes. Copyright © 2015 Elsevier Inc. All rights reserved.
    Molecular Cell 04/2015; 58(3). DOI:10.1016/j.molcel.2015.03.017 · 14.02 Impact Factor
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