A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

Evrin, Cécile; Clarke, Pippa; Zech, Juergen; Lurz, Rudi; Sun, Jingchuan; Uhle, Stefan; H, Li; Stillman, Bruce; Speck, Christian

doi:10.1073/pnas.0911500106

Cited by 484 publications

(656 citation statements)

References 43 publications

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“…What triggers the subsequent closure of the MCM2-7 ring has not been established. In origin recognition complex-dependent loading reactions, MCM2-7 particles become topologically linked to duplex DNA as double hexamers (8,9), suggesting that the initiation machinery, inter-MCM interactions, and/or nucleic acid binding may seal the Mcm2/5 gate. Biochemical studies of SceMCM2-7 have also indicated that nucleotide alone could promote ring closure (14,15); however, EM analyses of the DmeMCM2-7 complex have not detected this effect (16).…”

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confidence: 99%

“…During the G1-phase of the cell cycle, MCM2-7 heterohexamers are loaded onto DNA by the origin recognition complex together with the Cdc6 and Cdt1 proteins (8,9). As cells enter S-phase, MCM2-7 is activated through phosphorylation of Mcm2, Mcm4, and Mcm6 by the Dbf4-dependent Cdc7 kinase (10,11) and the chaperoned association of two accessory factors, Cdc45 and GINS (11).…”

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confidence: 99%

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ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

Lyubimov

Costa

Bleichert

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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The heterohexameric minichromosome maintenance (MCM2-7) complex is an ATPase that serves as the central replicative helicase in eukaryotes. During initiation, the ring-shaped MCM2-7 particle is thought to open to facilitate loading onto DNA. The conformational state accessed during ring opening, the interplay between ATP binding and MCM2-7 architecture, and the use of these events in the regulation of DNA unwinding are poorly understood. To address these issues in isolation from the regulatory complexity of existing eukaryotic model systems, we investigated the structure/function relationships of a naturally minimized MCM2-7 complex from the microsporidian parasite Encephalitozoon cuniculi. Electron microscopy and small-angle X-ray scattering studies show that, in the absence of ATP, MCM2-7 spontaneously adopts a lefthanded, open-ring structure. Nucleotide binding does not promote ring closure but does cause the particle to constrict in a two-step process that correlates with the filling of high-and low-affinity ATPase sites. Our findings support the idea that an open ring forms the default conformational state of the isolated MCM2-7 complex, and they provide a structural framework for understanding the multiphasic ATPase kinetics observed in different MCM2-7 systems. T he replisome is a large macromolecular machine that coordinates the activity of multiple functional components to ensure the faithful and timely duplication of DNA. Replisome progression is powered, in part, by hexameric helicases, which unwind parental duplexes to provide template strands for DNA synthesis. Because of their ring-shaped structure, the encirclement of single-or double-stranded DNA by replicative helicases is topologically restricted. This restriction can be overcome by either the direct assembly of the motor onto DNA (1-3) or dedicated loading factors that assist ring opening (1, 4, 5). How and why different hexameric helicases use a particular strategy are long-standing questions.The eukaryotic replicative helicase is composed of six distinct but homologous ATPases associated with various cellular activities (AAA+) subunits, collectively termed the minichromosome maintenance (MCM2-7) complex (6, 7). During the G1-phase of the cell cycle, MCM2-7 heterohexamers are loaded onto DNA by the origin recognition complex together with the Cdc6 and Cdt1 proteins (8, 9). As cells enter S-phase, MCM2-7 is activated through phosphorylation of Mcm2, Mcm4, and Mcm6 by the Dbf4-dependent Cdc7 kinase (10, 11) and the chaperoned association of two accessory factors, Cdc45 and GINS (11). The resultant Cdc45-MCM-GINS (CMG) complex then translocates in the 3′→5′ direction along the leading strand, displacing the lagging strand to facilitate DNA unwinding (12, 13).Studies of both Saccharomyces cerevisiae (Sce) and Drosophila melanogaster (Dme) MCM2-7 indicated that the helicase ring spontaneously opens between the Mcm2 and Mcm5 subunits (14-16). This breach has been proposed to act as a gate by which DNA enters into the motor interior. What triggers t...

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confidence: 99%

mentioning

confidence: 99%

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

Lyubimov

Costa

Bleichert

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…A lack of loaded single hexamers and the presence of multiple Cdt1/Mcm2-7 complexes before Mcm2-7 loading supports a model in which both hexamers are loaded in a concerted fashion Takara and Bell 2011). Importantly, both EM and topological linkage studies indicate that loaded Mcm2 -7 complexes encircle and slide nondirectionally on dsDNA (Evrin et al 2009;). Thus, loaded Mcm2 -7 is topologically linked to the DNA but is neither active as a helicase nor tightly engaged with the DNA.…”

Section: Structure Of Loaded Mcm2 -7mentioning

confidence: 79%

“…Unlike the archaea homologs, Mcm2 -7 double hexamers are only detected after loading. Mcm2 -7 double hexamers survive treatment with DNase and gel filtration, indicating that once formed, these complexes are very stable (Evrin et al 2009;Gambus et al 2011). Based on structural studies of the archaea Mcm complex, these interactions likely involve the Zn-finger domains in the amino termini of Mcm2 -7 subunits (Fletcher et al 2003).…”

Section: Structure Of Loaded Mcm2 -7mentioning

confidence: 99%

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Helicase Loading at Chromosomal Origins of Replication

Bell

Kaguni

2013

Cold Spring Harbor Perspectives in Biology

116

109

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Loading of the replicative DNA helicase at origins of replication is of central importance in DNA replication. As the first of the replication fork proteins assemble at chromosomal origins of replication, the loaded helicase is required for the recruitment of the rest of the replication machinery. In this work, we review the current knowledge of helicase loading at Escherichia coli and eukaryotic origins of replication. In each case, this process requires both an origin recognition protein as well as one or more additional proteins. Comparison of these events shows intriguing similarities that suggest a similar underlying mechanism, as well as critical differences that likely reflect the distinct processes that regulate helicase loading in bacterial and eukaryotic cells.

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DNA Replication: Yeast

Smith

Raoof

et al. 2018

Encyclopedia of Life Sciences

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DNA replication is a complex biological process essential to maintaining the fidelity of genetic information passed to subsequent generations, while allowing for the mutations necessary for natural selection. The cellular machinery required for DNA replication must be precisely controlled throughout the cycle. A variety of DNA polymerases have proofreading and repairing capabilities to avoid catastrophic errors, thus maintaining genomic stability. Much of the mechanism involved in this process is evolutionarily conserved. Because yeast is a relatively simple and inexpensive organism to work with, much of our understanding of DNA replication in eukaryotes originates from experiments with yeast. The most thoroughly characterized model yeast systems are Saccharomyces cerevisiae (budding yeast) and Saccharomyces pombe (fission yeast). Key Concepts Yeast is a good model organism for simplifying and characterising cellular processes, such as DNA replication. The two most commonly used yeast models are S. cerevisiae and S. pombe . DNA replication is a complex biological process essential to genomic replication. DNA replication is a process that involves timely organisation of proteins and is regulated during the cell cycle by cyclin‐dependent protein kinases. Termination of DNA replication in yeast is generally not sequence specific, except in special circumstances in the cellular process.

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A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication

Cited by 484 publications

References 43 publications

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

ATP-dependent conformational dynamics underlie the functional asymmetry of the replicative helicase from a minimalist eukaryote

Helicase Loading at Chromosomal Origins of Replication

DNA Replication: Yeast

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