Multiple Functions for Mcm2–7 ATPase Motifs during Replication Initiation

Kang, Sung-Myung; Warner, Megan D.; Bell, Stephen P.

doi:10.1016/j.molcel.2014.06.033

Cited by 95 publications

(157 citation statements)

References 42 publications

(89 reference statements)

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“…Thus, the roles for ORC and Cdc6 in MCM loading would involve helping to release Cdt1 from an ori-tethered hexamer, after which the complex would revert to an open gate state competent to subsequently engage duplex DNA (18). ATPase activity has been shown to be important for helicase loading (41,42). Elegant studies of the licensing reaction by Speck and colleagues have substantially validated the role of the Mcm2/5 gate in this step toward initiation (43).…”

Section: Discussionmentioning

confidence: 99%

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

Petojevic

Pesavento

Costa³

et al. 2015

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

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DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/ Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.hromosomal DNA replication begins with the separation of the complementary strands of the duplex. Following this melting step, helicases continuously separate the paired strands, exposing the template for enzymatic synthesis. For eukaryotic DNA replication, a growing body of work suggests that the initial DNA melting step involves an enzymatic conversion of a double hexamer of the minichromosome maintenance (Mcm2-7) complex into an active helicase, with hexamer separation forming two forks moving in opposite directions (1). The molecular mechanisms and a complete list of the factors that work to achieve melting and the topological conversion of double to single strand are still unknown, but both Cdc45 and the GINS complex are present at the time of melting (2) and are critical components of an activated Mcm2-7 helicase (3-6). Understanding the initiation process requires studies focused on the various transitions accessed by Mcm2-7 proteins and defining what roles the GINS and Cdc45 may play in the melting and unwinding processes.The CMG helicase characterized in vitro in Drosophila and humans contains a single Cdc45 protein, a single hetero-hexameric Mcm2-7, and a tetrameric GINS complex (3-7). Mcm2-7 acts as the motor that drives helicase activity; however, for metazoans, only when the Mcm2-7 complex is associated with Cdc45 and GINS do significant helicase, ATPase, and DNA binding activities follow (4). The two Mcm2-7 complexes are first loaded to DNA by mechanisms that appear to retain certain parallels to the processes used for loading sliding processivity clamps onto DNA. The endpoint of loading results in the deposition of a duplex DNA that runs through the central channel of an MCM double hexamer. Although to...

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Section: Discussionmentioning

confidence: 99%

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

Petojevic

Pesavento

Costa³

et al. 2015

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

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“…Consequently, mutations affecting the intersubunit interface geometry could block the ring closure, which in turn could affect (1) ATP hydrolysis-dependent Cdt1 release, (2) enclosure of DNA by the MCM2-7 ring, or (3) the ability of the complex to establish the correct hexamer-hexamer interface. Interestingly, MCM2-7 ATP-binding mutants are known to impair complex stability in the presence of low salt and elevated temperatures and display strong defects in the OCCM-to-DH transition (Coster et al 2014;Kang et al 2014). We suggest that the underlying reason for the pre-RC formation defect of Mcm ATPbinding mutants is the reduced complex stability, particular during the spiral-to-ring transition-a concept that could be tested using cryo-EM.…”

Section: Mcm2-7 Atp Hydrolysis Activities Of the Occm Dh And Cmgmentioning

confidence: 90%

“…Crucially, the MCM2-7 recruitment process depends on an Mcm6-Cdt1 interaction, which alleviates an autoinhibitory activity of the Mcm6 C terminus . Upon ORC/Cdc6/Cdt1/MCM2-7 (OCCM) complex formation, ATP hydrolysis results in sequential Cdc6 and Cdt1 release and formation of an ORC/MCM2-7 (OM) intermediate Coster et al 2014;Kang et al 2014;Ticau et al 2015). Interestingly, Cdt1 release is associated with a structural change, as it promotes the closure of the MCM2-7 ring (Ticau et al 2017).…”

Section: Initiation Of Dna Replicationmentioning

confidence: 99%

“…MCM2-7 ATP hydrolysis during helicase loading has been analyzed by in vitro reconstituted reactions using a battery of ATP-binding and ATP hydrolysis mutants and a limited number of direct ATP hydrolysis assays Coster et al 2014;Kang et al 2014). Here we discuss these data in the context of recent structural insights.…”

Section: Mcm2-7 Atp Hydrolysis Activities Of the Occm Dh And Cmgmentioning

confidence: 99%

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From structure to mechanism—understanding initiation of DNA replication

et al. 2017

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DNA replication results in the doubling of the genome prior to cell division. This process requires the assembly of 50 or more protein factors into a replication fork. Here, we review recent structural and biochemical insights that start to explain how specific proteins recognize DNA replication origins, load the replicative helicase on DNA, unwind DNA, synthesize new DNA strands, and reassemble chromatin. We focus on the minichromosome maintenance (MCM2-7) proteins, which form the core of the eukaryotic replication fork, as this complex undergoes major structural rearrangements in order to engage with DNA, regulate its DNA-unwinding activity, and maintain genome stability.

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“…3); it appears possible that the Mcm3 C terminus allosterically regulates the ATPase activity of the first loaded Mcm2-7 hexamer as well as that of the second hexamer yet to be loaded. Since Mcm2-7 ATPase is required for double-hexamer assembly (Coster et al 2014;Kang et al 2014), the potential allosteric function may regulate double-hexamer assembly.…”

Section: The Implication Of the Ocmm Structure In Pre-rc Assemblymentioning

confidence: 99%

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

et al. 2014

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Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

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Multiple Functions for Mcm2–7 ATPase Motifs during Replication Initiation

Cited by 95 publications

References 42 publications

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement

From structure to mechanism—understanding initiation of DNA replication

Structural and mechanistic insights into Mcm2–7 double-hexamer assembly and function

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