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W4240271709 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. https://doi.org/10.7554/eLife.03273.001 eLife digest Before a cell divides, it must duplicate its DNA so that each new cell inherits its own copy of the genome. To do this, the DNA double helix must be unwound so that the two individual strands of DNA can serve as templates for making new DNA molecules. Unwinding begins when two helicase complexes, termed the Mcm2-7 rings, are loaded together onto the DNA. At first, the two Mcm2-7 rings encircle the double-stranded DNA and remain bound together in an inactive form. Activating the Mcm2-7 rings requires the binding of five other proteins to each ring, which forms two larger complexes called CMG helicases. When the CMG helicases form, the two DNA strands separate and an individual Mcm2-7 ring ends up encircling each of the single DNA strands. However, how an activated CMG complex is assembled, and how it binds to and unwinds DNA, is not fully understood. Now, Costa et al. have determined the three-dimensional structure of the fruit fly CMG helicase bound to a DNA double helix with a single-stranded overhang at one end. The activated Mcm2-7 ring binds to the overhang, which confirms previous findings indicating that the activated helicase prefers single-stranded over double-stranded DNA. The structure also shows that, as a CMG helicase slides along the single-stranded DNA towards the double-stranded DNA, it is the ring complex's ‘motor domains’ that lead the way, while its DNA-binding domains trail behind. Costa et al. also found that disrupting some of the interactions between two of the five proteins that bind to the Mcm2-7 ring either prevented the replicative helicase from forming or made it unstable. Furthermore, it was revealed that one of these two proteins—called Cdc45—was ideally placed to capture the strand of DNA that might be accidentally released from the Mcm2-7 ring. It was also discovered that when the complex is bound to DNA, the motor domains of the Mcm2-7 complex change shape from a flat ring to a spiral structure; the DNA-binding domains, however, remain in a flat ring. Costa et al. note that this structure is similar to that adopted by many viral and bacterial helicases, and that it even shares many features with the molecular machinery that breaks down unneeded or damaged proteins inside cells. Finally, Costa et al. were able to image a structure composed of two CMG complexes bound together. This reveals the relative orientation of the two Mcm2-7 rings before they separate and move in opposite directions to unravel the DNA. The findings of Costa et al., combined with previous structural work in this field, demonstrate that the Mcm2-7 helicase complex can adopt many different shapes as it is assembled on DNA and activated to support DNA replication. https://doi.org/10.7554/eLife.03273.002 Introduction The faithful copying of DNA requires the correct spatial and temporal assembly of replication machineries at specific chromosomal loci known as origins. In eukaryotes, origins are licensed for replication by recruitment of the Mcm2-7 complex, a ring-shaped helicase that serves as the principal unwinding activity for separating parental DNA strands (Blow, 1993; Bochman and Schwacha, 2008; Costa et al., 2011; Lyubimov et al., 2012). Mcm2-7 is initially loaded around duplex DNA as an inactive double hexamer by the origin recognition complex (ORC), Cdc6, and Cdt1 in the G1 phase of the cell cycle (Evrin et al., 2009; Remus et al., 2009), forming a stable intermediate known as the pre-replicative complex (pre-RC, Diffley et al., 1994; Donovan et al., 1997; Maiorano et al., 2000; Sun et al., 2013; Yanagi et al., 2002). Upon entry into S phase, Mcm2-7 associates with the GINS complex and Cdc45 generating an 11-member assembly termed the CMG (Kanemaki et al., 2003; Gambus et al., 2006; Moyer et al., 2006; Pacek et al., 2006; Ilves et al., 2010). GINS/Cdc45 assembly is dependent on the CDK kinase (Zegerman and Diffley, 2007), while post-translational modification of Mcm subunits 2, 4, and 6 by the Cdc7/Dbf4 kinase (DDK) further contributes to CMG activation (Labib, 2010; Sheu and Stillman, 2010). Following the assembly of replicative polymerases and replisomal scaffolding factors (Gambus et al., 2009; Muramatsu et al., 2010), the two CMG particles split apart into discrete complexes that have been proposed to each encircle a single DNA strand during translocation (Yardimci et al., 2010; Boos et al., 2012). At present, multiple aspects of the Mcm2-7 loading and activation cycle remain poorly understood. Although the six homologous subunits of one Mcm2-7 complex are known to pair with a second Mcm2-7 complex through their N-terminal domains in the context of a double-hexamer (Evrin et al., 2009; Remus et al., 2009), the precise register by which these subunits interact with each other across the two rings is not known. How DDK phosphorylation of the Mcm2, Mcm6, and Mcm4 N-termini (Labib, 2010), or how a DDK-bypass mutation in the N-terminus of either Mcm4 (Sheu and Stillman, 2010) or Mcm5 (Jackson et al., 1993), might aid in the switch from an inactive Mcm2-7 double hexamer state to a functional CMG is similarly unclear, particularly as Mcm4 is spatially segregated from Mcm2 and Mcm5 (Costa et al., 2011). During unwinding and fork progression, the CMG translocates 3′→5′ along DNA. How the various components of the CMG engage nucleic acid strands during this process has remained ill-defined. Cdc45 has recently been shown to contain a RecJ exonuclease domain that can bind DNA but that is catalytically inactive (Petojevic et al., unpublished data, as well as Sanchez-Pulido and Ponting, 2011; Krastanova et al., 2012; Szambowska et al., 2014). Whether or how the Cdc45 RecJ fold might bind single DNA strands formed in the context of the CMG has not been established. Conflicting models likewise exist for how Mcm2-7 engages substrate DNAs as it moves 3′→5′ during strand separation, with biochemical data from archaeal MCMs and phylogenetic relationships to superfamily III (SFIII) helicases (such as the SV40 Large T antigen and the papillomavirus E1 protein) predicting mutually exclusive binding orientations (McGeoch et al., 2005; Enemark and Joshua-Tor, 2006; Rothenberg et al., 2007; Lee et al., 2014). To begin to understand several extant questions surrounding how the CMG is formed and operates at molecular level, we have determined structure of the full-length complex from Drosophila melanogaster in the presence of a 3′-tailed DNA duplex and the non-hydrolyzable ATP analog, ATPγS, using negative-stain electron microscopy and single-particle reconstruction methods. The structure establishes that: 1) the CMG preferentially associates with single-stranded DNAs over double-stranded substrates, 2) the C-terminal ATPase domains of Mcm2-7 form the leading edge of the motor as it advances on a duplex, and 3) the RecJ domain of Cdc45 is oriented to favor the capture of DNA segments that might accidently escape the Mcm2-7 pore. Comparison of the new structure with a previously-determined apo CMG model (Costa et al., 2011) shows that the Mcm2-7 ATPase domains of the complex transition from a planar, open ring into a closed, right-handed spiral in the presence of both DNA and nucleotide. Analysis of this state alongside other ring-ATPases shows that the MCM spiral is most similar to that adopted by the bacterial DnaB helicase upon engaging single-stranded DNA (Itsathitphaisarn et al., 2012), and that the GINS•Cdc45 complex bridges the junction between the ends of the spiral in a manner similar to that by which the Rpn1 accessory subunit spans a spiral Rpt1-6 ATPase assembly in the eukaryotic proteasome (Lander et al., 2012). Interestingly, examination of a subpopulation of CMG dimers present in our EM data shows how two Mcm2-7 complexes associate within a double hexamer and suggests that this dimerized state persists during CMG formation, prior to separation during fork progression (Ilves et al., 2010; Yardimci et al., 2010). Collectively, our observations establish that Mcm2-7 unwinds DNA using an approach distinct from that of superfamily III helicases and highlight several new Mcm2-7 ring configurations and assembly states accessed by the motor during the initiation of DNA replication. Results and discussion Determination of a higher-resolution CMG model In a previous study, we determined the medium-resolution (28 Å) structures of the Drosophila melanogaster CMG helicase in both an apo state and bound to a non-hydrolyzable ATP analog (Costa et al., 2011). Though sufficient for mapping individual subunits within the CMG, both models revealed a planar structure for Mcm2-7, with GINS and Cdc45 spanning a gap that appeared between Mcm2 and Mcm5 when nucleotide was omitted. Since insights into where DNA might bind to the CMG or how binding might potentially alter the structure of complex were unclear, we set out to trap and image a prospective translocation intermediate of the CMG using 3D single-particle electron microscopy. A purified solution of the CMG was first mixed with a 20 bp duplex DNA substrate bearing a single-stranded 3′-dT(40) tail and passed over a sizing column in the presence of the non-hydrolyzable ATP analog, ATPγS, to form a ternary complex. The complex did not behave well during cryo-preservation attempts using holey-carbon EM grids, so samples were instead deposited onto continuous carbon grids and exposed to uranyl formate for negative staining. A total of 29,913 particles were selected from EM micrographs acquired with JADAS automated data collection software (JEOL, Zhang et al., 2009) on a JEM2100 electron microscope. Following particle picking and 2D averaging, a 3D model was generated by projection matching using a low-pass filtered (60 Å), free-hand test-validated, nucleotide-bound structure of the CMG as a starting model (‘Materials and methods’; Rosenthal and Henderson, 2003; Lyubimov et al., 2012). CMG particles imaged with DNA and ATPγS turned out to be quite uniform, permitting structure determination to a higher resolution than that obtained previously (18 Å vs 28 Å resolution, Figure 1—figure supplement 1). The resultant model (Figure 1A) in turn allowed for a more accurate fitting of the Mcm2-7 and GINS subunits (Figure 1B,C), revealing several new features. For instance, the location of Psf1 C-terminus, which was previously not visible, was now clearly evident, and could be readily fit to a recently-published full-length structure of Psf1 from an archaeal ortholog (Oyama et al., 2011; Figure 1C). Flexing within the Mcm2-7 ring was also apparent with the C-terminal lobes of different MCM subunits displaying markedly distinct degrees of movement with respect to their associated N-terminal regions (Figure 1D,E). Asymmetric positioning between the two tiers of an MCM ring has not been reported previously, demonstrating that these elements are conformationally independent of each other to some extent in the presence of DNA substrates. Figure 1 with 1 supplement see all Download asset Open asset 18 Å resolution of a CMG–DNA–ATPγS complex. (A) Top-down view (N-terminal MCM face) of the CMG highlighting subunit positions. (B) Docking of homology models into the assembly. (C) Docked structures into segmented density for: top—a near-full-length, archaeal MCM monomer Mcm4 (PDB ID 3F9V); middle—the GINS complex (PDB ID 2Q9Q and 3ANW, ‘Materials and methods’); bottom—the archaeal Mcm N-terminal domain hexamer (PDB ID 1LTL and 2VL6, ‘Materials and methods’). (D) The N- and C-terminal domains of Mcm2-7 (colors) differentially flex around the helicase ring, with GINS–Cdc45 (white) wedging open Mcm5 in particular. (E) The N-terminal domains of Mcm2-7 are relatively planar, and are fit best by a hexameric, DNA-free structure of the archaeal MCM NTDs, indicating the observed intra-subunit flexing derives from ATPase domain movement. https://doi.org/10.7554/eLife.03273.003 The CMG advances on duplex DNA from the C-terminal, ATPase side of Mcm2-7 The increase in resolution obtained for the CMG in the presence of DNA provided initial, support evidence for nucleic acid binding to the complex. More concrete evidence for DNA association was apparent in electron density maps generated for the CMG, which showed a rod-shaped feature jutting away from the C-terminal face of Mcm subunits 2 and 5 (Figure 2A)—this feature is absent in DNA-free 3D reconstructions of the CMG (Costa et al., 2011). Because negative-stains are non-ideal for visualizing nucleic acids (Grob et al., 2012), we further assessed DNA binding by biotin-labeling the duplex end of the oligonucleotide, mixing the CMG–DNA samples with streptavidin, and collecting new single-particle EM data. Inspection of the resultant 2D class averages from this approach revealed clear additional density compared to the unlabeled CMG–DNA particles (Figure 2B), demonstrating that the tailed substrate indeed associates with the complex particles. Given the electron density features seen for the DNA and the distance the streptavidin ‘pointer’ resides from the complex, the EM data show that the CMG binds to the single-stranded end of the 3′-tailed DNA substrate, corroborating biochemical data indicating that the complex preferentially associates with and translocates along single-stranded DNA over duplex substrates (Ilves et al., 2010; Fu et al., 2011). Figure 2 Download asset Open asset Polarity of DNA binding by the CMG. (A) Observed experimental density seen at low contours reveals a rod-shaped extension (green) of comparable length to that expected for a 20mer DNA duplex that extends from the Mcm C-terminal motor domain. This feature is absent in DNA-free CMG reconstructions (Costa et al., 2011). A schematic of the relative single- and double-stranded DNA regions of the substrate used for the present studies is shown at left. (B) Comparison of DNA–CMG class averages with and without streptavidin-labeling clearly marks the duplex end of the 3′-tailed duplex substrate. Note how the streptavidin density sits at a distance from the body of the CMG, indicating that the majority of the duplex region of the substrate is not bound by the CMG. https://doi.org/10.7554/eLife.03273.005 The ability to visualize not only DNA binding to the CMG but also the position of the duplex end with respect to the particle, resolves a key question concerning the polarity by which MCM helicases engage a presumptive translocation strand. MCMs and viral SF3 helicases, such as SV40 LTag and the papilloma virus E1 protein, are both AAA+ ATPases (Neuwald et al., 1999). This relationship, coupled with shared ability of MCMs and SF3 enzymes to translocate along DNA in a 3′→5′ direction (Kelman et al., 1999; Chong et al., 2000; Bochman and Schwacha, 2008; Moyer et al., 2006), has suggested that members of two helicase families might operate by a common translocation mechanism. However, studies of E1 and archaeal MCMs bound to DNA substrates have yielded conflicting data concerning the direction by which DNA threads through the helicase pore. In E1, the 3′ end of DNA has been observed by X-ray crystallography to lie proximal to the C-terminal motor domains of the helicase (Enemark and Joshua-Tor, 2006). By contrast, based on FRET measurements between a dye-labeled DNA/MCM pair, the converse has been reported for Sulfolobus solfataricus MCM (McGeoch et al., 2005; Rothenberg et al., 2007). In the new CMG structure, the streptavidin appended to the duplex DNA end can be clearly seen to localize next to the C-terminal, AAA+ domain face of the particle (Figure 2B). This finding not only demonstrates that a DNA segment bound by an MCM runs from the N-terminal collar to the ATPase motor region in a 3′ to 5′ direction, but also indicates that MCM and SF3 helicases bind substrates with opposing polarities. The RecJ domain of Cdc45 is poised to assist in the capture of DNA that might escape the Mcm2/5 gate When the structure of the CMG was first reported, the fold of the associated Cdc45 subunit was unknown. As a consequence, although the general location of Cdc45 could be identified in both apo and ATP-bound forms of the CMG, the orientation and role of this subunit was left unresolved (Costa et al., 2011). Recently, however, the N-terminus of Cdc45 was shown to belong to the RecJ family of ssDNA exonucleases (Sanchez-Pulido and Ponting, 2011). Interestingly, within this grouping, Cdc45 belongs to an offshoot branch that can still bind DNA, but that also possesses natural amino acid substitutions which would appear to inactivate any native hydrolase functions (Krastanova et al., 2012). To understand how the RecJ fold of Cdc45 interfaces with Mcm2-7 and GINS, we built a homology model for DmCdc45 based on Thermus thermophilus RecJ and docked it into the higher-resolution, DNA-bound CMG reconstruction. The catalytic core and DNA tracking domain of the homology model fit unambiguously into only one region of the Cdc45 density (Figure 3A), leaving only a single, unaccounted for region (most likely corresponding to the C-terminal segment of Cdc45 outside the defunct exonuclease core, or possibly to the N-terminal extension present in Mcm2) that interdigitates between the N-terminal ‘A-domains’ of Mcm5 and Mcm2 (Figure 3B, Figure 3—figure supplement 1). Notably, in placing the Cdc45 RecJ domain, we found that this element appeared to contact the now-apparent C-terminal ‘B-domain’ of Psf1 (Figure 3A). To test whether this interaction might be real or fortuitous, we subjected the Psf1 B-domain surface to site-directed mutagenesis and tested the ability of the mutant subunits to support binding to both Mcm2-7 and Cdc45 (‘Materials and methods’). Ablation of either the Psf1 B-domain region (residues 185–202) or Cdc45 N-terminal region (residues 1–99) prevented CMG formation under the conditions used to purify the intact assembly (Figure 3—figure supplement 2). Likewise, while point mutations were unable to disrupt CMG formation as judged by co-immunoprecipitation, a quadruple Psf1 mutant (E190A/L192A/V193A/R194A) proved unable to interact with Cdc45. Together, these data indicate that the Cdc45–Psf1 interaction evident from the EM data plays a critical role in CMG formation and/or stability. Figure 3 with 2 supplements see all Download asset Open asset Cdc45 is positioned to permit trapping of single-stranded DNA. (A) Top—Segmented electron density corresponding to Cdc45. A prominent horseshoe-shaped region fits well to the catalytic core of the homologous RecJ exonuclease (PDB ID 1IR6). Bottom—Docked models of RecJ and full-length GINS (generated using an archaeal Psf1 homolog, PDB IDs 2Q9Q and 3ANW) into DNA-bound CMG reconstructions highlight a previously unobserved interaction between the B-domain of Psf1 and the exonuclease-like domain of Cdc45. (B) An extension of the Cdc45 RecJ-like region contacts and interdigitates between the A-domains of the Mcm2 and Mcm5 N-terminal regions. (C) The Mcm2-7 central channel (black line) and the Cdc45 DNA tracking groove (red arrow) are offset by ∼90°. (D) Schematic showing how the single-stranded DNA-binding groove of the Cdc45 RecJ-like domain could facilitate the capture of a leading strand segment if the Mcm5-2 DNA gate were to transiently open. https://doi.org/10.7554/eLife.03273.006 In previous apo and ATP-bound models of the CMG, the particle was seen to transition from a conformation in which the Mcm2/5 interface was open to one in which it was closed (Costa et al., 2011). This transition in turn pinched off the large single channel that ran through the particle into two smaller channels, sealing the interior of Mcm2-7 away from the inner surface of GINS–Cdc45. In the DNA-bound model, the CMG still exhibits two channels; however, docking of the Cdc45 RecJ domain shows that its exonuclease/DNA-tracking groove is offset by 90° with respect to the central axis of the Mcm2-7 pore (Figure 3C). This orientation indicates that, were Cdc45 to bind DNA in a manner similar to RecJ, it would be poised to capture the leading DNA strand that might escape from Mcm5-2 gate (Figure 3D). Consistent with this idea, cross-linking data in work to be published elsewhere (Petojevic et al.) show both that Cdc45 engages the leading strand of a fork substrate only in the absence of nucleotide and that this interaction is ablated by the mutation of residues suggested by the model to be important for DNA binding. DNA and nucleotide remodel the Mcm2-7 AAA+ ATPase subunits into a right-handed spiral How ATP-dependent physical movements within hexameric helicases are coupled to DNA binding and unwinding has long been a central question in the field (Singleton et al., 2007; Enemark and Joshua-Tor, 2008; Lyubimov et al., 2011). Notably, when comparing the new DNA-bound CMG model to the prior substrate-free state, we found that the AAA+ ATPase ring is no longer flat, but instead adopts a clear right-handed spiral (Figure 4A,B). This change in conformation does not propagate into the N-terminal domains, which maintain a roughly planar character (Figure 1E), but is instead offset by the variable flexing seen for the C-terminal domains in different positions around the ring (Figure 1D). The observed asymmetry between the two MCM tiers indicates that the N-terminal domains form a relatively stable collar that likely helps to coordinate and restrain movements of the associated C-terminal ATPase regions. Figure 4 Download asset Open asset Global comparison of the DNA-bound Mcm2-7 region of the CMG with other hexameric helicases and ATPases. (A) Cut-away view (removing Mcm5) of the Mcm2-7 central channel highlights a spiral organization for the Mcm2-6-4-7-3 AAA+ ATPase regions. Colored spheres demarcate the approximate center of mass for AAA+ pore loops as derived from the docking of MCM AAA+ domain as shown in Figure 1B. (B) Top-down view (from the N-terminal face) of MCM AAA+ domains docked into the DNA-bound CMG reconstruction showing the existence of a right-handed spiral. The CMG density has been removed for clarity. (C) In the presence of a single-stranded DNA, bacterial DnaB can adopt a right-handed spiral with a moderately-wide pore (PDB ID 4ESV, Itsathitphaisarn et al., 2012). (D) The E1 helicase assembles into a right-handed spiral with a relatively narrow pore (PDB ID 2GXA, Enemark and Joshua-Tor, 2006). (E) Comparison of the AAA+ ring of the eukaryotic proteasome with Mcm2-7 region of the DNA- and ATPγS-bound CMG. The non-ATPase subunit Rpn1 binds to the side of the Rpt1-6 hetero-hexamer, wedging itself between the N-terminal and C-terminal tiers of the ATPase ring and helping to promote the formation of a right-handed ATPase domain spiral. Similar architectural features are apparent within the DNA–ATPγS–CMG complex, where GINS–Cdc45 occupy an analogous position. https://doi.org/10.7554/eLife.03273.009 Closer analysis of the AAA+ spiral reveals several features that have important implications for the action of MCM subunits during DNA unwinding. First, the largest inter-subunit shifts within the Mcm2-7 ring, which occur between Mcm subunits 2 and 5, also correspond to the point where the GINS–Cdc45 complex docks against the helicase. Inspection of the DNA-bound model reveals that GINS–Cdc45 does not simply straddle the Mcm2/5 interface, but that portions of the accessory subunits actually wedge themselves between the N- and C-terminal tiers of the Mcm2-7 ring (Figure 1D). This action widens the exterior groove between the MCM N- and C-terminal domains at their points-of-contact with GINS–Cdc45, and is offset by a concomitant narrowing of the groove on the exterior MCM face opposite the GINS–Cdc45 binding site (i.e., Mcm4, Figure 1D). The structural consequences resulting from GINS–Cdc45 binding suggests that these accessory subunits not only play a role in blocking access through the Mcm2/5 gate, as has been seen previously (Costa et al., 2011), but that they also help stabilize a spiral configuration of ATPase centers when DNA is present. Since the mutation of active site residues at the Mcm2/5 interface ablates helicase activity (Bochman et al., 2008; Ilves et al., 2010), it is likely that the spiral state observed here, which positionally offsets the ATP-binding site of Mcm5 from the arginine-finger residue of Mcm2, inter-converts with another conformation in which the Mcm2/5 interface is remodeled to form a catalytically functional ATPase center during the translocation cycle. Hence, a need for GINS–Cdc45 in preventing DNA from escaping Mcm2-7 would likely be infrequent and limited to instances when the Mcm2/5 gate accidentally opens for an extended period of time, such as at a roadblock created by other nucleoprotein complexes or DNA damage. A second unexpected feature of the DNA-bound CMG complex is that the spiral is more pronounced than that seen in SF3 helicases, and instead more closely approximating the spiral evinced by a RecA-family helicase, DnaB, in the presence of DNA (Figure 4B–D). The width of the Mcm2-7 central channel (as measured from homology models of the motor domains docked into the EM density) is likewise significantly larger (∼30–35 Å) compared to E1 (∼14 Å), and more closely approaches that of DnaB (∼22 Å). Interestingly, in E1 and DnaB the difference in channel diameter and subunit rise between the two proteins sculpts the DNA substrate bound by each helicase into a single-stranded helix whose relative pitches differ significantly; these geometric differences allow each subunit of DnaB to engage two nucleotides of DNA (Itsathitphaisarn et al., 2012), whereas E1 binds only a single nucleotide per protomer (Enemark and Joshua-Tor, 2006). The similarity of the Mcm2-7 spiral to DnaB raises the interesting possibility that the helicase might translocate with a step-size greater than one nucleotide per ATP consumed; consistent with this notion, a recent study has shown that the MCM N-terminal DNA-binding collar of Pyrococcus furiosus binds four nucleotides per subunit (Froelich et al., 2014). A third notable attribute of the ternary DNA–CMG–ATPγS model is that several structural features of the complex turn out to be most similar not to replicative helicases, but to a completely orthogonal system, namely, the regulatory subcomplex of the eukaryotic proteasome. The proteasome consists of several discrete subcomplexes including a heterohexameric unfoldase region, termed the ‘base’, which (like the CMG) contains six homologous AAA+ ATPase subunits (Rpt1-6) (Forster et al., 2013). Recent cryo-EM studies have imaged the complete 26S yeast proteasome bound to ATP at ∼9 Å resolution showing that the AAA+ subunits of the base also form a right-handed spiral (Lander et al., 2012). Comparison of proteasome spiral with that seen here for the CMG shows that these regions of the two systems exhibit a surprisingly similar global architecture (Figure 4E). Moreover, the proteasome also contains an accessory subunit (Rpn1) that—as observed here for GINS–Cdc45 in the context of the CMG—wedges itself between a subset of ATPase and OB-fold domains present in Rpt1-6 (Lander et al., 2012; Figure 4E). The structural congruencies exhibited between the CMG and proteasome ATPase subcomplexes suggest that, even though the substrates for the two systems differ greatly, both motors may share certain commonalities in how ATP turnover is coupled to movements that promote translocation. Such a similarity could underlie both the pronounced asymmetry of the CMG and proteasome ATPase rings, and the relatively high degree of tolerance shown by both systems toward active-site mutations within certain subunits (Moreau et al., 2007; Ilves et al., 2010; Beckwith et al., 2013). Identification of a dimeric CMG configuration Although the CMG has been observed to operate as a discrete single complex during replication (Yardimci et al., 2010), the loading of the Mcm2-7 hexamer onto DNA by ORC, Cdc6, and Cdt1 during initiation results in the transient formation of a catalytically inactive, head-to-head double hexamer intermediate (Evrin et al., 2009; Remus et al., 2009). The MCM N-terminal domains have been shown to comprise the dimer interface of the double hexamer (Fletcher et al., 2003; Costa et al., 2006; Remus et al., 2009), and create" @default.
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W4240271709 title "Decision letter: DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome" @default.
W4240271709 doi "https://doi.org/10.7554/elife.03273.012" @default.
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