Cryo-EM structure of a helicase loading intermediate containing ORC–Cdc6–Cdt1–MCM2-7 bound to DNA

Sun, Jingchuan; Evrin, Cécile; Samel, Stefan A.; Fernández-Cid, Alejandra; Riera, Alberto; Kawakami, Hironori; Stillman, Bruce; Speck, Christian; H, Li

doi:10.1038/nsmb.2629

Cited by 126 publications

(209 citation statements)

References 61 publications

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“…The Mcm3 CTE was shown to activate the ATPase activities of ORC-Cdc6 (Frigola et al 2013). The Mcm3 CTE partially protrudes from the end of the MCM hexamer, providing a structural basis for its function in the initial binding to ORC-Cdc6 (Frigola et al 2013;Sun et al 2013). Furthermore, its prominent position suggested that it could alter Mcm2-7 subunit interactions or influence Mcm2-7 ATP hydrolysis.…”

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

confidence: 89%

“…3D, 4). This is in stark contrast to the relative arrangement in the single Mcm2-7 hexamer and the Cdc45-Mcm2-7-GINS (CMG) helicase, where the NTD and CTD rings are vertically aligned (see the Discussion; Sun et al 2013;Costa et al 2014). …”

Section: Mapping the Architecture Of The Mcm2-7 Double Hexamermentioning

confidence: 87%

“…Therefore, we employed a 3D mapping strategy employing MBP insertions into the N-terminal or C-terminal region of individual MCM protein subunits, similar to the strategy used for the OCCM (Sun et al 2013), and assembled the MBP-inserted Mcm2-7 double hexamers one at a time (Supplemental Fig. 2 (Randell et al 2010;Tanaka and Araki 2013).…”

Section: Mapping the Architecture Of The Mcm2-7 Double Hexamermentioning

confidence: 99%

“…Note that archaeal MCMs have both the NTD and CTD but lack any N-terminal extensions (NTEs) like the eukaryotic NSDs. Because the order of the subunits within the Mcm2-7 hexamer has been well established as Mcm5, Mcm3, Mcm7, Mcm4, Mcm6, and Mcm2 (Bochman et al 2008;Costa et al 2011;Lyubimov et al 2012;Vijayraghavan and Schwacha 2012;Sun et al 2013), mapping one Mcm protein would have been sufficient for assigning the remaining five MCM proteins if the handedness of the EM structure was determined, and mapping two Mcm proteins was sufficient to assign both the handedness and the identity for all proteins. We experimentally mapped the NTD of four Mcm protein subunits, which is more than sufficient to identify all NTDs as well as the handedness of the structure (Fig.…”

Section: Mapping the Architecture Of The Mcm2-7 Double Hexamermentioning

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.…”

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

confidence: 89%

Section: Mapping the Architecture Of The Mcm2-7 Double Hexamermentioning

confidence: 87%

Section: Mapping the Architecture Of The Mcm2-7 Double Hexamermentioning

confidence: 99%

Section: Mapping the Architecture Of The Mcm2-7 Double Hexamermentioning

confidence: 99%

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confidence: 99%

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

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The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

O'Donnell

2018

BioEssays

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The eukaryotic helicase is an 11-subunit machine containing an Mcm2-7 motor ring that encircles DNA, Cdc45 and the GINS tetramer, referred to as CMG (Cdc45, Mcm2-7, GINS). CMG is "built" on DNA at origins in two steps. First, two Mcm2-7 rings are assembled around duplex DNA at origins in G1 phase, forming the Mcm2-7 "double hexamer." In a second step, in S phase Cdc45 and GINS are assembled onto each Mcm2-7 ring, hence producing two CMGs that ultimately form two replication forks that travel in opposite directions. Here, we review recent findings about CMG structure and function. The CMG unwinds the parental duplex and is also the organizing center of the replisome: it binds DNA polymerases and other factors. EM studies reveal a 20-subunit core replisome with the leading Pol ϵ and lagging Pol α-primase on opposite faces of CMG, forming a fundamentally asymmetric architecture. Structural studies of CMG at a replication fork reveal unexpected details of how CMG engages the DNA fork. The structures of CMG and the Mcm2-7 double hexamer on DNA suggest a completely unanticipated process for formation of bidirectional replication forks at origins.

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Cryo‐EM of dynamic protein complexes in eukaryotic DNA replication

et al. 2016

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DNA replication in Eukaryotes is a highly dynamic process that involves several dozens of proteins. Some of these proteins form stable complexes that are amenable to high-resolution structure determination by cryo-EM, thanks to the recent advent of the direct electron detector and powerful image analysis algorithm. But many of these proteins associate only transiently and flexibly, precluding traditional biochemical purification. We found that direct mixing of the component proteins followed by 2D and 3D image sorting can capture some very weakly interacting complexes. Even at 2D average level and at low resolution, EM images of these flexible complexes can provide important biological insights. It is often necessary to positively identify the feature-of-interest in a low resolution EM structure. We found that systematically fusing or inserting maltose binding protein (MBP) to selected proteins is highly effective in these situations. In this chapter, we describe the EM studies of several protein complexes involved in the eukaryotic DNA replication over the past decade or so. We suggest that some of the approaches used in these studies may be applicable to structural analysis of other biological systems.

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Cryo-EM structure of a helicase loading intermediate containing ORC–Cdc6–Cdt1–MCM2-7 bound to DNA

Cited by 126 publications

References 61 publications

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

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

The Eukaryotic CMG Helicase at the Replication Fork: Emerging Architecture Reveals an Unexpected Mechanism

Cryo‐EM of dynamic protein complexes in eukaryotic DNA replication

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