In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

Evrin, Cécile; Fernández-Cid, Alejandra; Zech, Juergen; Herrera, Mariano; Riera, Alberto; Clarke, Pippa; Brill, Shlomo; Lurz, Rudi; Speck, Christian

doi:10.1093/nar/gkt043

Cited by 40 publications

(51 citation statements)

References 47 publications

(114 reference statements)

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“…These data provide the evidence that Cdc6, like Mcm4 and Mcm7, is essential for JH-dependent polyploidization, vitellogenesis, and egg production. It has been well established that Cdc6 is a key factor for replication origin licensing late in G 1 phase by regulating the formation of prereplication complexes that initiate DNA synthesis at S phase (37,43,58,59). Polyploidy is known to promote transcriptomic and metabolomic outputs (27,60).…”

Section: Discussionmentioning

confidence: 99%

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis

Guo

Xie

et al. 2016

Journal of Biological Chemistry

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Although juvenile hormone (JH) is known to prevent insect larval metamorphosis and stimulate adult reproduction, the molecular mechanisms of JH action in insect reproduction remain largely unknown. Earlier, we reported that the JH-receptor complex, composed of methoprene-tolerant and steroid receptor co-activator, acts on mini-chromosome maintenance (Mcm) genes Mcm4 and Mcm7 to promote DNA replication and polyploidy for the massive vitellogenin (Vg) synthesis required for egg production in the migratory locust (Guo, W., Wu, Z., Song, J., Jiang, F., Wang, Z., Deng, S., Walker, V. K., and Zhou, S. (2014) PLoS Genet. 10, e1004702). In this study we have investigated the involvement of cell-division-cycle 6 (Cdc6) in JH-dependent vitellogenesis and oogenesis, as Cdc6 is essential for the formation of prereplication complex. We demonstrate here that Cdc6 is expressed in response to JH and methoprene-tolerant, and Cdc6 transcription is directly regulated by the JH-receptor complex. Knockdown of Cdc6 inhibits polyploidization of fat body and follicle cells, resulting in the substantial reduction of Vg expression in the fat body as well as severely impaired oocyte maturation and ovarian growth. Our data indicate the involvement of Cdc6 in JH pathway and a pivotal role of Cdc6 in JHmediated polyploidization, vitellogenesis, and oogenesis.

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Section: Discussionmentioning

confidence: 99%

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis

Guo

Xie

et al. 2016

Journal of Biological Chemistry

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“…DDK was expressed and purified as described (Evrin et al 2014). Most MBP-Mcm2-7 constructs were described in Evrin et al (2013) andFernandez-Cid et al (2013) with the exception of Mcm2-MBP. During its construction using site-directed mutagenesis, a restriction site was inserted after amino acid 712 in Mcm2.…”

Section: Methodsmentioning

confidence: 99%

“…1A, step 2; Speck et al 2005;Randell et al 2006;Speck and Stillman 2007;Tsakraklides and Bell 2010;Evrin et al 2013;Frigola et al 2013;Sun et al 2013). Using an in vitro Mcm2-7 loading system that employs a purified ORC, Cdc6, Cdt1, and Mcm2-7 hexamer and an origin-containing plasmid, we recently observed formation of a loading intermediate termed OCCM in the presence of a slowly hydrolysable ATP analog, ATP-gS, which captured a complex containing ORC-Cdc6 bound to a single Mcm2-7 hexamer and one to two copies of Cdt1 (Takara and Bell 2011;Evrin et al 2013;Sun et al 2013). A cryoelectron microscopy (cryo-EM) structure of this pre-RC intermediate showed that the AAA + ATPase domains of ORC/Cdc6 latch onto the C-terminal AAA + ATPase domains of Mcm2-7, leaving the Mcm2-7 N-terminal domains (NTDs) accessible, detailing the overall architecture of this important complex.…”

mentioning

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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“…ORC/Cdc6 is competent to recruit a Cdt1/ MCM2-7 heptamer ( Fig. 7D; Takara and Bell 2011;Evrin et al 2013;Sun et al 2013). Our data demonstrate that a linkage between Mcm2 and Mcm5 does not prevent OCCM formation but blocks the ATP hydrolysis-dependent establishment of a stable OCM complex, which leads to disassembly of the complex and in turn makes ORC/Cdc6 available again to re-engage Cdt1/MCM2-7 (Fig.…”

Section: Mcm2-mcm5 Linkage Affects Establishment Of Pre-rc Complexes mentioning

confidence: 99%

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

et al. 2014

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The regulated loading of the replicative helicase minichromosome maintenance proteins 2-7 (MCM2-7) onto replication origins is a prerequisite for replication fork establishment and genomic stability. Origin recognition complex (ORC), Cdc6, and Cdt1 assemble two MCM2-7 hexamers into one double hexamer around dsDNA. Although the MCM2-7 hexamer can adopt a ring shape with a gap between Mcm2 and Mcm5, it is unknown which Mcm interface functions as the DNA entry gate during regulated helicase loading. Here, we establish that the Saccharomyces cerevisiae MCM2-7 hexamer assumes a closed ring structure, suggesting that helicase loading requires active ring opening. Using a chemical biology approach, we show that ORC-Cdc6-Cdt1-dependent helicase loading occurs through a unique DNA entry gate comprised of the Mcm2 and Mcm5 subunits. Controlled inhibition of DNA insertion triggers ATPase-driven complex disassembly in vitro, while in vivo analysis establishes that Mcm2/Mcm5 gate opening is essential for both helicase loading onto chromatin and cell cycle progression. Importantly, we demonstrate that the MCM2-7 helicase becomes loaded onto DNA as a single hexamer during ORC/Cdc6/Cdt1/MCM2-7 complex formation prior to MCM2-7 double hexamer formation. Our study establishes the existence of a unique DNA entry gate for regulated helicase loading, revealing key mechanisms in helicase loading, which has important implications for helicase activation.

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In the absence of ATPase activity, pre-RC formation is blocked prior to MCM2-7 hexamer dimerization

Cited by 40 publications

References 47 publications

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis

Juvenile Hormone Activates the Transcription of Cell-division-cycle 6 (Cdc6) for Polyploidy-dependent Insect Vitellogenesis and Oogenesis

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

A unique DNA entry gate serves for regulated loading of the eukaryotic replicative helicase MCM2–7 onto DNA

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