An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

Fernández-Cid, Alejandra; Riera, Alberto; Tognetti, Silvia; Herrera, Mariano; Samel, Stefan A.; Evrin, Cécile; Winkler, Christian; Gardenal, Emanuela; Uhle, Stefan; Speck, Christian

doi:10.1016/j.molcel.2013.03.026

Cited by 118 publications

(198 citation statements)

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“…Of interest, it was recently shown that loading of the first MCM2-7 hexamer onto DNA occurrs within seconds, whereas the subsequent formation of a MCM2-7 double hexamer is slow and takes several minutes. 35 Consistently, we show here that about 10% of Cdc6-YFP was immobilized on chromatin for more than a minute during telophase suggesting that its engagement in loading MCM2-7 onto replication origins is a time-consuming process.…”

Section: Discussionsupporting

confidence: 86%

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

et al. 2015

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To maintain genome stability, the thousands of replication origins of mammalian genomes must only initiate replication once per cell cycle. This is achieved by a strict temporal separation of ongoing replication in S phase, and the formation of pre-replicative complexes in the preceding G1 phase, which "licenses" each origin competent for replication. The contribution of the loading factor Cdc6 to the timing of the licensing process remained however elusive due to seemingly contradictory findings concerning stabilization, degradation and nuclear export of Cdc6. Using fluorescently tagged Cdc6 (Cdc6-YFP) expressed in living cycling cells, we demonstrate here that Cdc6-YFP is stable and chromatin-associated during mitosis and G1 phase. It undergoes rapid proteasomal degradation during S phase initiation followed by active export to the cytosol during S and G2 phases. Biochemical fractionation abolishes this nuclear exclusion, causing aberrant chromatin association of Cdc6-YFP and, likely, endogenous Cdc6, too. In addition, we demonstrate association of Cdc6 with centrosomes in late G2 and during mitosis. These results show that multiple Cdc6-regulatory mechanisms coexist but are tightly controlled in a cell cycle-specific manner.

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Section: Discussionsupporting

confidence: 86%

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

et al. 2015

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“…The purified Mcm2-7 hexamer can hydrolyze ATP in vitro (Schwacha and Bell 2001;Ilves et al 2010;Fernandez-Cid et al 2013), but the active form of the helicase, when Mcm2-7 is part of a CMG complex, has a significantly higher ATPase activity (Ilves et al 2010). On the other hand, the ATPase rate of the Mcm2-7 double hexamer is not known.…”

Section: Segmentation and Subunit Assignment Of The 3d Em Map Of The mentioning

confidence: 99%

“…Mcm2-7 double-hexamer formation is a multistep reaction during which two Mcm2-7 hexamers are recruited one after another to replication origins Fernandez-Cid et al 2013;Frigola et al 2013). In S. cerevisiae, Ó 2014 Sun et al This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml).…”

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confidence: 99%

“…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. Using biochemical experiments, we observed that Orc1 and Cdc6 ATP hydrolysis promotes rapid Cdt1 release and the formation of an ORC-Cdc6-Mcm2-7 (OCM) complex (Randell et al 2006;Fernandez-Cid et al 2013). One question that arises from that study is whether ATP hydrolysis by ORC-Cdc6 and subsequent Cdt1 dissociation leads to a resetting of ORC-Cdc6 on DNA such that it would be in a position to recruit the next Mcm2-7 hexamer (Fig.…”

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confidence: 99%

“…The details of how these complex reactions occur are not understood (Tanaka and Araki 2013), but it is clear that ATP hydrolysis is central for Mcm2-7 helicase activity and DNA unwinding . The purified Mcm2-7 hexamer hydrolyzes ATP at an intermediate rate (Vijayraghavan and Schwacha 2012;Fernandez-Cid et al 2013), but when integrated in the replisome, the ATPase activity of the helicase becomes strongly activated (Ilves et al 2010 (1) Does ATP hydrolysis and Cdt1 release from OCCM lead to a large gap between ORC-Cdc6 and Mcm2-7 that may allow ORC-Cdc6 to recruit a second Mcm2-7 hexamer (step 3)? (2) How is the second Mcm2-7 hexamer recruited (step 4)?…”

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confidence: 99%

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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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MCM9 deficiency delays primordial germ cell proliferation independent of the ATM pathway

Luo

Schimenti

2015

Genesis

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Maintenance of genome integrity is crucial for the germline, and this is reflected by lower mutation rates in gametes than somatic cells. Germ cells at different stages employ different DNA damage response (DDR) mechanisms. In response to certain DNA repair defects, primordial germ cells (PGCs) either undergo apoptosis or delayed proliferation, although little is known about the underlying mechanisms that govern these outcomes. Here, we report genetic studies of DDR pathways that underlie germ cell depletion in mice mutant for minichromosome maintenance 9 (Mcm9), a gene that plays a role in homologous recombination repair (HRR). Germ cell depletion in these mice is a result of reduced PGC numbers both before and after they arrive in the primitive gonads. This reduction was attributable to reduced proliferation, not apoptosis, and this response was independent of ATM-CHK2-TRP53-P21 signaling. This mechanism of PGC depletion differs from that in Fancm mutants, which also display reduced PGC depletion that is partially orchestrated by the ATM-TRP53-P21 pathway. Germ cell depletion in mice doubly deficient for FANCM and MCM9 was additive, indicating that the damage caused by each mutation triggers different DDR pathways to slow the cell cycle as a means to preserve genomic integrity. genesis 53:678-684, 2015. © 2015 Wiley Periodicals, Inc.

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An ORC/Cdc6/MCM2-7 Complex Is Formed in a Multistep Reaction to Serve as a Platform for MCM Double-Hexamer Assembly

Cited by 118 publications

References 32 publications

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

Spatio-temporal regulation of the human licensing factor Cdc6 in replication and mitosis

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

MCM9 deficiency delays primordial germ cell proliferation independent of the ATM pathway

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