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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Available from: John F X Diffley
    • "To ensure that DNA replication occurs exactly once during the cell cycle the two steps of replication initiation need to be temporarily separated. First, the replicative DNA helicase minichromosome maintenance complex 2–7 (MCM2–7) is loaded at replication origins exclusively during late mitosis and G1 phase, when CDK activity is low121314. This process is termed origin licensing or Pre-RC formation and requires the six-subunit origin recognition complex (ORC) and the activities of the cell division cycle 6 (CDC6) ATPase and the chromatin licensing and DNA replication factor 1 (CDT1). "
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    ABSTRACT: A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy.
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    • "Archaeal and eukaryal MCM double-hexamerize via their N-terminal domains (Costa and Onesti, 2009; Fletcher et al., 2003; Remus et al., 2009). The dependence of MCM loading on the inverted repeats of ORB2 and ORB3, combined with our previous high-resolution mapping of the start site of replication (Robinson et al., 2004), is compatible with a model in which a double hexamer of MCM is loaded on the intervening AT-rich DNA, with the C-terminal domains of MCM being oriented toward the ORB-bound Orc1-1 proteins. "
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    ABSTRACT: Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins.
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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.
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