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 129 publications

(209 citation statements)

References 61 publications

Supporting

Mentioning

193

Contrasting

Order By: Relevance

“…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.…”

mentioning

confidence: 99%

See 4 more Smart Citations

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

et al. 2014

Self Cite

View full text Add to dashboard Cite

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.

show abstract

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%

mentioning

confidence: 99%

See 3 more Smart Citations

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

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

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

O'Donnell

2018

BioEssays

Self Cite

View full text Add to dashboard Cite

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.

show abstract

SEC‐SAXS/MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded‐Over Conformations

Smith,

Chakravarthy,

Rahi

et al. 2024

Cytoskeleton

View full text Add to dashboard Cite

Cdt1 is a mixed folded protein critical for DNA replication licensing and it also has a “moonlighting” role at the kinetochore via direct binding to microtubules and the Ndc80 complex. However, it is unknown how the structure and conformations of Cdt1 could allow it to participate in these multiple, unique sets of protein complexes. While robust methods exist to study entirely folded or unfolded proteins, structure–function studies of combined, mixed folded/disordered proteins remain challenging. In this work, we employ orthogonal biophysical and computational techniques to provide structural characterization of mitosis‐competent human Cdt1. Thermal stability analyses shows that both folded winged helix domains1 are unstable. CD and NMR show that the N‐terminal and linker regions are intrinsically disordered. DLS shows that Cdt1 is monomeric and polydisperse, while SEC‐MALS confirms that it is monomeric at high concentrations, but without any apparent inter‐molecular self‐association. SEC‐SAXS enabled computational modeling of the protein structures. Using the program SASSIE, we performed rigid body Monte Carlo simulations to generate a conformational ensemble of structures. We observe that neither fully extended nor extremely compact Cdt1 conformations are consistent with SAXS. The best‐fit models have the N‐terminal and linker disordered regions extended into the solution and the two folded domains close to each other in apparent “folded over” conformations. We hypothesize the best‐fit Cdt1 conformations could be consistent with a function as a scaffold protein that may be sterically blocked without binding partners. Our study also provides a template for combining experimental and computational techniques to study mixed‐folded proteins.

show abstract

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

Cited by 129 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

SEC‐SAXS/MC Ensemble Structural Studies of the Microtubule Binding Protein Cdt1 Show Monomeric, Folded‐Over Conformations

Contact Info

Product

Resources

About