Archaeal and eukaryotic MCM rings sequentially melt DNA for replication initiation

Domain: biology
Published: 2026-03-31
Editorial outcome: minor_revision

Abstract
DNA replication initiation requires local melting of fully base-paired DNA for a helicase to gain a foothold and initiate processive DNA unwinding. In eukaryotes and archaea, the helicase engine is the hexameric ring minichromosome maintenance (MCM) complex. In eukaryotes, a defined biochemical sequence assembles two Cdc45-MCM-GINS (CMG) complexes that provide limited DNA unwinding as the species that immediately precedes extensive unwinding. A prior structure revealed how MCM subunits interact with this form of DNA, but the atomic progression from undistorted DNA to this melted DNA species is unknown. Here, we present a sequential DNA melting mechanism determined by snapshots of an archaeal MCM ring with DNA in varying degrees of melting. In this mechanism, successive ATP-binding at MCM ATPase sites drives sequential discrete DNA melting steps mediated a specific MCM aromatic residue. Analysis of eukaryotic structures shows loaded MCM rings principally adopt only two molecular arrangements at the ATPase: one that does not melt DNA and one tuned to melt DNA with equivalent aromatic residues, indicating a universal sequential mechanism melts DNA in archaea and eukaryotes for replication initiation.

Introduction
DNA replication is a fundamental, intrinsic life process of duplicating genetic material to enable cell division. The act of DNA replication proceeds with a forked DNA structure where a helicase enzyme separates the constituent strands of the DNA double-helix, and polymerases use the exposed single strands as templates in the synthesis of new DNA1. The DNA replication process is tightly regulated by the cell cycle to preserve genomic integrity by ensuring that DNA is fully replicated once, and only once, each cell cycle2. An essential step towards regulated replication initiation is the establishment of a hexameric ring helicase to encircle one DNA strand while excluding the other3. Once established in this topology, the ring helicase propagates strand separation as it moves unidirectionally along the DNA. Establishment of the active helicase species where the helicase ring encircles only one DNA strand requires that the two DNA strands locally separate, termed melting, such that some base-pair interactions are lost. Bacteria first melt DNA with the initiator protein complex DnaA4-7, which allows subsequent loading of the hexameric DnaB ring helicase8,9 to encircle one of the exposed DNA strands6,7,10,11. In contrast, the sequence is reversed in eukaryotes and archaea with the minichromosome maintenance (MCM) complex loaded to fully double-stranded DNA12,13 followed by local DNA unwinding and helicase activation14 to ultimately yield a strand-excluded species that unwinds DNA and is competent for its replication.

In eukaryotes, the MCM complex is a heterohexamer of six similar subunits, Mcm2-715. All eukaryotic MCM proteins are highly similar in structure and sequence, especially in the ATPase domain16. The archaeal MCM complex is a homohexamer of six identical subunits17 that is highly similar in structure to eukaryotic Mcm2-7 with very strong sequence conservation in a 152-amino acid ATPase core that contains nearly all residues involved in both DNA-binding and ATP-binding16. MCM proteins belong to a large AAA+ family of ATPases18. These ATPases often function as motors that use energy derived from ATP hydrolysis to drive mechanical motion. The ATPase active sites are formed at subunit interfaces, analogous to the active sites of F1-ATPase19. One subunit contributes Walker-A and Walker-B residues, and the other contributes residues such as an arginine finger20,21. Based on analogy to F1-ATPase19,22, papillomavirus E1 helicase23, and SV40 large T-antigen24, tight subunit interfaces are expected to correlate with ATP-binding while loose interfaces are expected to correlate with the absence of nucleotide at the ATPase site. AAA+ helicases include MCM and the hexameric ring helicases of some viruses such as papillomavirus E123,25, SV40 large T-antigen24,26,27, and AAV Rep28. These helicases all form hexameric rings that project structural modules into the central channel to interact with DNA23,24,26,29-31. All of these AAA+ helicases use a pre-sensor-1 beta-hairpin (ps1\beta), named because its amino acids directly precede the AAA+ sensor-1 sequence motif32. The MCM helicases use an additional DNA-binding hairpin known as the helix-2-insert (h2i). This hairpin is a specialized insert in helix \alpha2 of the AAA+ topology18 that defines one AAA+ family clade33. We have previously shown that the h2i can adopt either a helix or horseshoe conformation in the \alpha2 region that is N-terminal to the insert hairpin31. The helix conformation of an h2i develops a parallel \beta-sheet interaction with the ps1\beta of an adjacent subunit that is not present in the horseshoe conformation31. As a result, subunits with the helix conformation have a tighter subunit interface than those in the horseshoe conformation.

The defined sequence for replication initiation in eukaryotes begins with recognition of a DNA replication origin by the origin recognition complex (ORC) 2,14,34 that recruits the MCM complex. Initial recruitment of MCM rings by ORC yields a salt-sensitive species where the association of MCM at the origin relies on its association with ORC35. This species is converted to a salt-stable species, termed loaded, where the MCM:DNA association is independent of ORC35,36. This provides an Mcm2-7 double-hexamer30 that encircles duplex DNA37 and can passively slide on DNA without unwinding it12,13. Loading of the Mcm2-7 ring involves opening the Mcm2/5 interface known as the gate 21. At the onset of S-phase, the Mcm2-7:DNA species is activated in a sequential series38 where it is converted from a stable double-hexamer that encircles both DNA strands to single hexamers that each encircle only one strand3. During this activation, Cdc45 is recruited to each MCM ring to seal the Mcm2/5 gate39. Cdc45 recruitment does not generate detectable DNA unwinding38. Next, the tetrameric GINS complex is recruited adjacent to Cdc45 to generate a larger CMG (Cdc45-MCM-GINS) complex40,41. GINS recruitment provides 0.6–0.7 turns of DNA unwinding at each CMG38 as the final detectable species that precedes the processive DNA unwinding of a replication fork.

Archaeal replication initiation is functionally similar to eukaryotic initiation. Archaeal ORC is homologous to eukaryotic ORC and binds at a DNA replication origin42,43, and two MCM hexamers assemble as a double hexamer17,44,45 that is highly similar in architecture to the eukaryotic Mcm2-7 double hexamer30. Archaea also possess GINS and Cdc45 homologs46,47 that stimulate the activities of MCM in biochemical DNA-unwinding assays48-50. Crystal structures of the archaeal proteins51-55 illustrate that they are indeed structural homologs of eukaryotic Cdc4556 and GINS57-59, suggesting fundamentally conserved mechanisms of action. For both archaea and eukaryotes, the MCM hexamer ring by itself is sufficient to unwind DNA in a biochemical strand displacement assay17,60, indicating general DNA unwinding is not the essential role of Cdc45 or GINS. Instead, eukaryotic Cdc45 and GINS are essential for the initial melting of DNA at a replication origin for initiation38 and are essential to establish origin-dependent DNA replication61. The precise roles of archaeal Cdc45 and GINS are not known, but presumably they function analogously to the eukaryotic proteins to enable specific activation of the MCM helicase at a replication origin.

Functionally, MCM has very strong similarities with the viral SuperFamily 3 (SF3) helicases62,63 papillomavirus E1 and SV40 large T-antigen (L-tag). Each assembles at a replication origin as a double-hexamer and unwinds duplex DNA bidirectionally once activated64,65. Early electron microscopy images showed that MCMs and SF3 helicases form highly similar dumbbell-shaped double-hexamers17,66,67. The underlying double-hexamer architecture is highly similar to that of MCM with the smaller N-terminal domains forming a head-to-head interaction at the middle and the larger ATPase domains located at each side17,67. All exhibit a 3’ -> 5’ polarity when unwinding DNA17,68,69. The ATPase domains share strong sequence homology and belong to the AAA+ family of ATPases18. All include a ps1\beta that interacts with the sugar-phosphate of the DNA backbone23,27,31. In the case of E1, the ps1\beta has two distinct functional residues: 1) a conserved lysine is essential for processive DNA-unwinding; and 2) an aromatic residue that is dispensable for DNA-unwinding is essential for DNA melting and DNA replication70.

The sequential generation of CMG requires prior exhaustion of ADP from the MCM ATPase sites followed by ATP-binding, but not ATP-hydrolysis38. Similarly, initial untwisting of DNA at each CMG relies upon ATP-binding, but not ATP-hydrolysis38. The cryo-EM structure of the species that immediately precedes processive unwinding reveals melted DNA and its interactions with the MCM subunits of CMG71,72. In particular, the ps1\beta and h2i of multiple adjacent ATPase domains interact with the melted DNA, principally with the strand that will subsequently be encircled to become the leading strand DNA template71. In this structure, the ATPase modules that interact with DNA belong to Mcm3, Mcm5, Mcm2, and Mcm671, which are adjacent subunits in the ring at the same side of the ring where Cdc45 and GINS are recruited71. The atomic progression from undistorted DNA to this melted DNA species is unknown because the MCM:DNA structures that precede this step, including the nucleotide-free species of the first step, are not known.

Here we show that DNA melting is mediated by MCM ATPase domains in a stepwise mechanism. We provide multiple high-resolution snapshots of DNA melting obtained by single particle cryo-EM analysis of an archaeal MCM encircling a segment of duplex DNA. The melting stages are driven by an aromatic residue on the h2i of three consecutive MCM subunits that collectively form an aromatic wedge. The subunits that participate in melting require a helix conformation in the h2i which in turn correlates with a tight subunit interface and an ATP mode of binding. The series of structures collectively suggest progressive DNA-melting by an MCM aromatic wedge driven by successive ATP-binding. The final structure in the series strongly resembles the structure of eukaryotic CMG with melted DNA71, suggesting a universal structure for the species that immediately precedes processive DNA unwinding by MCM. The arrangement of the aromatic residue with DNA is highly similar to that of E1 and L-tag, suggesting that an aromatic wedge mechanism to melt DNA for replication initiation is universal among archaea, eukaryotes, and DNA tumor viruses.

Results
With single particle cryo-EM (Supplementary Fig. 1-9, Supplementary Tables 1-6), we characterize multiple distinct MCM:DNA structures (Fig. 1) that are consistent with a sequential DNA melting mechanism mediated by a conserved aromatic residue of the h2i. The DNA used for the structure determinations are fully double-stranded, indicating that lost base-pairs correspond to bona fide DNA melting. The protein construct is a chimeric fusion of the Saccharolobus solfataricus MCM N-terminal domain and the Pyrococcus furiosus MCM AAA+ domain, Sso-PfMCM. We have previously determined the hexameric ring crystal structure of this construct and shown that it has a robust strand displacement activity29. This Sso-PfMCM chimera and a modified SsoMCM construct are the only two constructs that we have successfully crystallized in a hexameric form containing both the N-terminal and AAA+ domains. We previously reported cryo-EM structures of the modified SsoMCM construct encircling single-stranded DNA, which allowed us to identify additional states of DNA translocation16. For this study, we were motivated to elucidate how the archaeal MCM hexamer interacts with encircled double-stranded DNA, something we have never been able to capture by X-ray crystallography. The Pyrococcus furiosus genetic sequence has an intein in the AAA+ domain (PfMCM amino acids 362-72848) that was genetically removed from the construct29. In this report, residues are numbered with native amino acid numbers without intein removal such than residue 361 is directly linked to residue 729.

To verify that the MCM:DNA structures are general and not associated with a specific DNA sequence or DNA reagent, structures were determined for samples generated from two independent DNA sequences. The underlying structural classes of these two samples are highly similar, which enabled particle merging to generate higher resolution versions of each structural class. For each MCM:DNA species, the multiple structures were determined from a single cryo-EM grid as different 3-dimensional classes. These consist of single-hexamers encircling DNA and illustrate stepwise melting of DNA at the MCM ATPase tier and not the potential attributes of two hexamers together. All the structural classes adopt expected two-tiered ring-shaped architectures described previously12,17,30,37,41,73-76 with DNA located inside the central channel of the ring. One tier consists of the N-terminal domain (N-tier) in three subdomains defined previously44, consistent with many other MCM structures44,77,78. The other tier (C-tier) consists of the AAA+ family ATPase domain18,79,80. Nearly all structural classes adopt an expected hexameric architecture. A minor fraction of heptameric MCM rings encircling DNA are observed. Heptameric MCM rings have been observed previously45,74,81,82, but the heptameric MCM form is not expected to function biologically and will not be a focus of subsequent discussion.

Our major focus is the arrangement of the ATPase tier and its DNA-binding hairpins with DNA. Relative to this portion, the N-tier occupies two distinct rotational orientations when viewed down the central channel that define two Fundamental Classes. The distinct orientations will be referred to as eclipsed (Fundamental Class 1) and staggered (Fundamental Class 2) based on the alignment of one tier subunit:subunit interface with the corresponding subunit:subunit interface of the other tier. Inter-tier orientation differences have been described previously in comparing ORC/Cdc6/Cdt1/MCM2–7 to either Mcm2-7 double-hexamer or CMG83, and we will expand this comparison in a section below. The two forms exhibit three major differences (Fig. 1, Supplementary Fig. 10-11): 1) the central channel diameter of the ATPase tier is significantly wider in eclipsed Fundamental Class 1 versus staggered Fundamental Class 2; 2) duplex DNA is observed within the central channel of both tiers in eclipsed Fundamental Class 1 but is only observed at the ATPase tier of staggered Fundamental Class 2; and 3) the DNA is fully base-paired in the eclipsed hexamer, but a portion of the DNA in the staggered hexamer is single-stranded, indicating that DNA that was fully duplex during sample preparation had become melted.

Tight and Loose Subunit Interfaces
Each fundamental class encompasses multiple subclasses. Collectively, the subclasses provide a framework for sequential melting of DNA based on the ATPase tier. All the MCM ATPase sites of the structures indicate the presence of bound nucleotide with varying strengths. The Walker-A motif of an ATP-binding site binds anions, particularly sulfate or phosphate, or nucleotides if these are in sufficient concentration. Thus, the presence of nucleotide at these sites is anticipated at the high 5 mM Mg/ATP-\gammaS concentration used during sample preparation. We use two intersubunit criteria that are readily discerned in the cryo-EM maps to assign whether the interface is tight or loose and define the nucleotide status of each ATPase site. Interfaces that satisfy both criteria are assigned as tight. This differentiation method is conceptually similar to those applied in other helicase structural studies, such as bacteriophage T7 that differentiated ATPase site types by crystallographic occupancy84 and papillomavirus E1 that differentiated site types by the chemical architecture of the sites23. Both prior studies did not discern a formally different molecule bound in the site.

The first criteria we use to assign a tight subunit interface is the relative proximity of modules that comprise the bipartite ATPase site. Tight interfaces have a consistent and small appearance for a vacant volume (labeled T in Fig. 2) near a clearly visible \gamma-S-phosphate. The corresponding vacant volume of loose interfaces (labeled L in Fig. 2) is larger and variable in appearance. We adopt an intersubunit distance threshold of 6.8 Å for a P-loop proline C\alpha of one subunit to a C\alpha of the arginine finger motif (four residues prior to the conserved arginine) of the neighboring subunit (Fig. 2 and Table 1) for this tight interface criteria.

The second feature we use to define a tight subunit interface is the proximity of the h2i hairpin of one subunit to the ps1\beta of the neighboring subunit (Fig. 3). We have previously illustrated that the portion of helix-2 that faces the central channel (residues N-terminal to the insert) can adopt a traditional helix conformation or a horseshoe conformation 31. The helix conformation binds the DNA sugar-phosphate backbone with a fully conserved hydroxyl side chain, but the horseshoe conformation places this hydroxyl group too far from the DNA for direct interaction31. The horseshoe conformation also necessitates a loose subunit interface because the horseshoe structure would clash with the neighboring ps1\beta within the tight interfaces that are developed when adjacent DNA-binding hairpins interact with consecutive DNA nucleotides (Fig. 3). Analogous to the ATPase site distance, we have adopted a distance threshold of 8.0 Å for a specific pair of h2i-ps1\beta residues (a conserved h2i leucine and a conserved ps1\beta lysine, Fig. 3 and Table 1) for this tight interface criteria.

Based on these criteria (Fig. 3 and Table 1), all subunit interfaces of the Class 1 structures are loose. The Class 2a structure has 2 tight interfaces and 4 loose interfaces, and Class 2b has 3 tight interfaces and 3 loose interfaces (Fig. 2-3 and Table 1).

Comparative Analysis I: General Inter-tier Dihedrals Quantify Eclipsed Versus Staggered
The different inter-tier conformations of the eclipsed and staggered forms is qualitatively similar to the inter-domain orientation differences described previously in comparing ORC/Cdc6/Cdt1/MCM2–7 to either the Mcm2-7 double-hexamer or CMG83. In particular, prior analysis described in detail multiple MCM:Cdt1 interactions in driving different inter-domain orientations83. To quantitively compare the inter-tier conformations of our structures to eukaryotic complexes, we calculated a dihedral angle at each subunit of a complex85,86 along with the mean dihedral angle. The NTD subunit position selected for the dihedral (large sphere in Fig. 4a inset) is near a subunit interface on a conserved loop87,88 at the bottom of the NTD. The CTD subunit position selected for the dihedral (second large sphere in Fig. 4a inset) is near the same subunit interface at the top of the CTD. With these definitions, a 0º dihedral (perfectly eclipsed) approximately generates a continuous vertical interface seam across the N-tier and C-tier. Class 1a has a mean dihedral of +3 that approximately eclipses the interface seams of the two tiers. In contrast, Class2a and Class2b have mean dihedral angles of -32º and -31º that dramatically offset two interface seams in a staggered conformation. The different seam alignments for the Fundamental Classes can be viewed at the sides of the green subunits in the side views of Fig. 1 approximately along the horizontal midline of the molecule.

This algorithm was extended to all eukaryotic MCM complex structures of the PDB that contain six AAA+ domains and six OB-folds (72 total eligible) 85. This analysis (Fig. 4) illustrates that all eukaryotic MCM structures fall into two obviously different categories (category 1 mean dihedral range: -6 to -15; category 2 mean dihedral range: -29 to -38). These values are similar to those of Class 1a and Class 2, and so we will group them in the same eclipsed or staggered categories, with 11 in an eclipsed conformation and the majority (61) in a staggered conformation. Consistent with the prior description of the molecular basis for Cdt1 to drive specific inter-tier orientations83, 100% of the structures that contain Cdt1 (9GM589, 9GJP89, 9BCX90, 9GJW89, 6WGI91, 5V8F92, 6WGG91, 5XF893) adopt an eclipsed conformation in the dihedral analysis. Only three structures that lack Cdt1 adopt an eclipsed conformation (8W0G94, 8W0I94, 6WGF91), and these structures all possess a contiguous opening at the Mcm2/5 interface across both tiers. Considered with the role of Cdt1 in generating an open Mcm2-7 ring93, all eclipsed structures are either functionally prone to open across both tiers due to the presence of Cdt1, or the ring shows a bona fide continuous opening across both tiers. In contrast, 100% of CMG structures in the analysis are in a staggered conformation and closed ring. The overall analysis indicates that Cdt1 drives an eclipsed conformation while Cdc45 and GINS drive a staggered conformation. The Class 1a structure is unique within the eclipsed category because it consists solely of MCM proteins in a closed ring. Class 1a also has dihedral values that are significantly more positive than all other structures analyzed (Fig.4a). The dihedral values of the Class 2 structures are very similar to those of eukaryotic staggered complexes (Fig 4). The gross similarities of Class 1a to several eukaryotic eclipsed structures (5XF893, 6WGG91, 5V8F92, 9GJW89, and 9BCX90) and Class 2b to a eukaryotic staggered structure (PDB 7Z1371) is illustrated with a molecular morph in Supplementary Movie 1. Our subsequent analysis will focus on the ATPase C-tier and ignore the N-tier.

Comparative Analysis II: AAA+ Hexamer Core RMSD Values Define two Principal Forms of Staggered Eukaryotic Mcm2-7
We also quantitatively compared MCM hexamer structures based on the collective architecture of all six ATPase domains. Analogous to the highly specific inter-subunit arrangement for catalytically competent F1-ATPase22, a highly specific MCM inter-subunit molecular architecture is required to generate a tight MCM ATPase site poised for hydrolysis. We have previously shown that MCM proteins have an ATPase core that is nearly invariantly 152-amino acids in length and highly conserved in sequence and structure16. We have used both properties to identify the eukaryotic Mcm2-7 structures that most similarly resemble our structures for overall distribution of tight and loose ATPase sites. To identify the most appropriate comparative eukaryotic MCM structures for the collective arrangement of the six MCM ATPase cores as a unit, RMSDs of the hexameric ATPase core of all eukaryotic Mcm2-7 structures with six AAA+ domains of the PDB (77 eligible structures, including all 72 of the dihedral analysis) were calculated86,95 against all six permutations of the Class 1a structure and against all six permutations of the Class 2b structure (462 comparisons each, see Supplementary Fig. 12). Each of the six permutations will be referred to as a specific setting of the complex. The top 30 RMSD alignments (Supplementary Tables 7-8) were visually inspected. The Class1 structures did not have significant matches (Supplementary Table 7), suggesting that no eukaryotic Mcm2-7 structures with six loose interfaces are represented in the PDB. In contrast, the Class 2 structures provided several strong matches (Supplementary Table 8).

Within the RMSD analysis, two principal forms of staggered Mcm2-7 structures are obvious. The 72 Mcm2-7 structures eligible for both the dihedral and RMSD analysis (above) were binned to eclipsed (11 structures) or staggered (61 structures) categories, sub-binned by the setting of Class 2b that provided the lowest RMSD, and each bin was reverse-sorted by the RMSD value of that setting (Fig. 4). The staggered conformation has two obvious forms with the RMSD values of all settings in unison. These two forms comprise nearly all staggered Mcm2-7 complexes (56 of 61 structures) and will be referred to as Staggered Form I (32 structures) and Staggered Form II (24 structures) with Form I best fitting Class 2b in setting 4 and Form II best fitting Class 2b in setting 1. The dihedral analysis similarly identifies two principal forms that include the same respective structures (Fig. 4a). Form I systematically has Mcm4 and Mcm6 dihedrals above the mean; Mcm3 and Mcm7 dihedrals near the mean; and Mcm2 and Mcm5 dihedrals below the mean. Form II has Mcm3 and Mcm5 dihedrals above the mean; Mcm2 and Mcm7 dihedrals near the mean; and Mcm4 and Mcm6 dihedrals below the mean. Notably, settings 1 and 4 that define these two principal forms are offset by 180° (Fig. 4c) and thus appear approximately opposite one another, suggesting they could fulfill opposing functions.

We associate Form I with stable association with fully duplex DNA and Form II with DNA melting. Form I structures exist within multiple species, including single-hexamers, double-hexamers, and CMG. Some Form I structures encircle DNA, either dsDNA or ssDNA, while others do not contain DNA. Importantly, all Form I structures that encircle dsDNA have fully base-paired DNA at the ATPase tier. All Form II structures (24 total) are CMG structures. Of these, 3 have melted DNA bound by the ATPase tier hairpins (PDB 7PMK96, 7Z1371, and 7QHS71, see below), and 19 have ssDNA bound by the hairpins. Two structures are low-resolution structures of complexes that included ssDNA during sample preparation but did not include DNA in the structural models (PDB 6PTN97: 5.80 Å, PDB 6PTO97: 7.00 Å). These two structures may have ssDNA bound at the ATPase tier hairpins that was not readily discerned at the reported resolution. If so, then all 24 Form II structures consist of CMG molecules with ssDNA regions bound by the ATPase hairpins.

Based on inspection of the top-30 RMSD fitting structures of Class 2b (Supplementary Table 8), we selected PDB 7PMN96, 7PMK96, 7Z1371, 9E2X98, and 9GJW89 for more thorough comparison and analysis below. None of these comparative structures belong to staggered Form I. With no strong RMSD fits to Class 1a, we selected two staggered Form I Eukaryotic MCM double-hexamer structures encircling DNA that is fully duplexed at the ATPase tier (PDB 5BK437 and PDB 7W1Y99) as comparative cases for the Class 1 structures. The tight versus loose criteria outlined in the section above was applied to these selected structures (Table 1). The structures and their comparative equivalents are analyzed in finer detail below.

Fundamental Class 1: Eclipsed Hexamer; Fully Base-paired DNA at Both Tiers
The first fundamental class places the two tiers of the MCM hexameric ring in an eclipsed conformation with both tiers encircling a contiguous dsDNA molecule that appears undistorted and fully base-paired. The central channel is more dilated than in the other fundamental hexameric class (see below), and the DNA-binding hairpins of the ATPase domain only marginally interact with the encircled DNA (Fig. 5, Supplementary Fig. 13-14). For each structure of Fundamental Class 1, all h2i are in the horseshoe conformation (Fig. 3, Supplementary Fig. 15-17), likely contributing to the limited direct binding between the h2i hairpin and the DNA. All Class 1 structures therefore have loose interfaces, consistent with nucleotide-free states, as required as a first step towards replication initiation38. Within Fundamental Class 1, three subclasses are identified with different numbers of well-ordered AAA+ ATPase subunits.

Class 1a has six well-defined AAA+ domains at the C-tier and a well-defined B-form DNA duplex (Fig. 1a, Supplementary Fig. 10a, 11a). Ignoring the encircled DNA, the protein complex appears 6-fold symmetric such that six permutations are indistinguishable. As such, the origin of the DNA helical axis defines the specific MCM:DNA permutation of each cryo-EM particle. The hairpins of the ATPase domain do not closely contact the DNA (Fig. 5a, Supplementary Fig. 13a, 14a). All six h2i are in the horseshoe conformation with corresponding loose subunit interfaces (Supplementary Fig. 15a, 16a, 17a).

Class 1b has five well-defined AAA+ domains at the C-tier and a B-form DNA duplex (Fig. 1b, Supplementary Fig. 10b, 11b). This protein complex is not C6-symmetric due to the poor order of one of the AAA+ domains. The arrangement of five ordered ATPase domains defines a specific permutation of MCM hexamer particles. Two different DNA modes are observed for this asymmetric species. In both cases, the h2i of the orange subunit located clockwise to the disordered ATPase subunit (in the Top view from the N-terminal side of Fig. 1) makes the closest approach to a DNA strand. The polarity of the contacted strand differs with the cyan strand in closest proximity to the orange subunit h2i in this subclass. Although this h2i is the closest approach between the ATPase tier and the DNA, it does not appear to form specific interactions (Fig. 5b, Supplementary Fig. 13b, 14b). All five ordered ATPase domains are in the horseshoe conformation with corresponding loose subunit interfaces (Supplementary Fig. 15b, 16b).

Class 1c also has five well-defined AAA+ domains at the C-tier and a B-form DNA duplex (Fig. 1c, Supplementary Fig. 10c, 11c). This protein complex is also not C6-symmetric due to the poor order of one of the AAA+ domains. The arrangement of five ordered ATPase domains defines the specific permutation of MCM hexamer particles and does not appear distinguishable from Class 1b based on the protein. In this subclass, the orange subunit located clockwise to the disordered ATPase subunit (in the Top view from the N-terminal side of Fig. 1) makes the closest approach to the magenta DNA strand, the opposite strand polarity as Class 1b. Although this h2i is the closest approach between the ATPase tier and the DNA, it does not appear to form specific interactions (Fig. 5c, SupplementaryFig. 13c, 14c). All five ordered ATPase domains are in the horseshoe conformation with corresponding loose subunit interfaces (Supplementary Fig. 15c, 16c).

The Class 1 structures are similar to those of the eukaryotic pre-melting complexes within the yeast Mcm2-7 double hexamer (PDB 5BK437, Fig. 5d) and the human Mcm2-7 double hexamer (PDB 7W1Y99, Fig. 5e) because the DNA is fully duplex at the C-tier, and the aromatic groups of the h2i hairpin (yellow and highlighted with dashed outline) are distant from the DNA. While all Class 1 structure subunits are in the horseshoe conformation with corresponding loose interfaces, the structure of PDB 5BK437 has three horseshoe (Mcm3, 5, and 2) and three helix conformations (Mcm6, 4, and 7; Supplementary Fig. 17e). The structure of PDB 7W1Y99 only differs from the PDB 5BK437 helix/horseshoe arrangement at Mcm3, which appears to be horseshoe in PDB 5BK437 and helix in of PDB 7W1Y99. The transformation of an MCM ATPase tier structure within the Mcm2-7 double hexamer (PDB 5BK437 or PDB 7W1Y99) from a species with some tight interfaces to 0-tight/6-loose interfaces in Class 1a provides a representation of the initial exhaust of ADP from the MCM double-hexamer that is an essential replication initiation early step38.

Fundamental Class 2: Staggered Hexamer; DNA Partially Opened and Exclusively at C-tier
The second fundamental class approximately staggers the domains that comprise each tier (Fig. 1, Supplementary Fig. 10-11). This inter-tier rotation is similar to that observed in the crystal structure of an archaeal MCM hexamer in complex with single-stranded DNA31. It is also similar to the orientations of eukaryotic CMG41,71,72,96-98,100-110 and for each MCM hexamer of the eukaryotic Mcm2-7 double-hexamer30,37,111-114 (see Fig. 4). In contrast to Fundamental Class 1, Fundamental Class 2 has extensive interactions between the C-tier DNA-binding hairpins and one DNA strand in a binding mode that is highly similar to that with single-stranded DNA31. Different numbers of h2i helix/horseshoe conformations (Fig. 3, Supplementary Fig. 15-17) with concurrent differences in the numbers of bound nucleotides define two subclasses. These differences suggest successive steps in a melting mechanism that will be elaborated in a subsequent section.

In Class 2a, the h2i and ps1\beta hairpins of three adjacent subunits form a spiral staircase that binds 5 consecutive nucleotides of one DNA strand (cyan strand, Fig. 1d, Supplementary Fig. 10d, 11d). Two nucleotides of the opposing strand (magenta) are disordered. These nucleotides would complement the first two cyan strand nucleotides that are tightly bound by the MCM hairpins. The loss of these base-pair interactions (Fig. 6a, Supplementary Fig. 13d, 14d) indicates that this region of DNA has melted and that this melting is driven by interaction of the cyan strand with the MCM hairpins. The binding mode with the cyan strand is analogous to the 6-subunit spiral staircase that the MCM ring uses to bind 10 consecutive nucleotides of ssDNA16,31. To bind consecutive nucleotides in this mode, participating subunits must adopt the helix h2i conformation for two reasons: 1) to position the conserved hydroxyl group where it can bind a DNA phosphate group; and 2) to develop a tight subunit interface such that adjacent subunits are positioned to bind DNA in the same mode. The configuration of Class 2a with two consecutive helix conformations (Supplementary Fig. 15d, 16d, 17b) enables three consecutive subunits to engage in binding the cyan strand (Fig. 6a, Supplementary Fig. 13d, 14d). This structural class has two tight subunit interfaces and four loose subunit interfaces that are defined by the two helix conformations and four horseshoe conformations of the h2i. This structure is highly similar to a eukaryotic CMG structure that also melts 2 DNA base-pairs (PDB 7PMN96, Fig. 6b). The two nucleotides of unpaired DNA in the structure of PDB 7PMN96 represent bona fide DNA melting because the DNA substrate does not possess an unpaired 5’-tail. As with the Class 2a structure, the two nucleotides of the complementary strand are disordered. Notably, both structures stack an h2i aromatic side-chain on the sugars and bases of DNA nucleotides that are melted (yellow and highlighted in dashed outline in Fig. 6).

Class 2b has four subunits engaged in binding DNA (Fig. 1e, Supplementary Fig. 10e, 11e). This class is analogous to Class 2a with three consecutive subunits in the helix conformation (Supplementary Fig. 15e, 16e, 17c), enabling four consecutive subunits to bind the 7 nucleotides of the cyan strand (Fig. 5c, Supplementary Fig. 13e, 14e). Four nucleotides of the opposing strand (magenta) are disordered. These nucleotides complement the first four cyan strand nucleotides that are tightly bound by the hairpins. The loss of these base-pair interactions (Fig. 6c, Supplementary Fig. 13e, 14e) indicates that this region of DNA has melted and increased DNA melting by two nucleotides relative to Class 2a. Class 2b has three tight subunit interfaces and three loose subunit interfaces (Fig. 2) with three helix conformations and three horseshoe conformations of the h2i (Fig. 3). Class 2b is highly similar to two different eukaryotic CMG structures with melted DNA (PDB 7PMK96 and PDB 7Z1371), suggesting a universal role for this structural form. Specifically, these structures adopt 3-helix/3-horseshoe conformations and similarly bind the DNA strand depicted in cyan with analogous hairpin-DNA interactions (Fig. 6d, e, Supplementary Fig. 17d). The four nucleotides of unpaired DNA in the structure of PDB 7PMK96 represent bona fide DNA melting because the DNA substrate does not possess an unpaired 5’-tail. As with the Class 2b structure, the four nucleotides of the complementary strand are disordered. The structures consistently stack an h2i aromatic side-chain on the sugars and bases of the bound DNA strand that is melted (yellow and highlighted in dashed outline in Fig. 6). The helix/horseshoe distribution of PDB 7Z1371 is opposite that of PDB 5BK437 (Supplementary Fig. 17) and reflects the opposite configurations of eukaryotic staggered Form I and eukaryotic staggered Form II (Fig. 4).

Fundamental Class 3: Staggered Heptamer; Fully Base-paired DNA at Both Tiers.
A third fundamental class consists of a heptameric MCM ring encircling fully duplexed DNA. (Supplementary Fig. 18). Ignoring the encircled DNA, the protein complex appears 7-fold symmetric such that seven permutations are indistinguishable. As such, the origin of the DNA helical axis sets the specific MCM:DNA permutation of each cryo-EM particle. The two tiers are in a staggered conformation and not eclipsed. This is a minor class and is not predicted to function biologically. It is presented here out of completeness and not discussed further.

DNA-interactions
Both fundamental hexameric ring structural classes encircle DNA, but they interact with DNA differently. While Fundamental Class 1 has limited direct interaction with the DNA, Fundamental Class 2 strongly interacts with the DNA sugar-phosphate backbone, principally with one strand (depicted in cyan in Fig. 1, 6). For a given subunit, four total polar interactions of the two hairpins with the cyan strand match those observed for ssDNA-binding by the corresponding residues of a related archaeal MCM hexamer (PDB 6MII, Fig. 7a). These interactions are between phosphate groups of the DNA backbone and atoms that are fully conserved in all MCM proteins and are therefore feasible for all eukaryotic and archaeal MCM subunits. These interacting atoms reside on the h2i and ps1\beta DNA-binding hairpins of the ATPase domain. The h2i is visually more prominent than the ps1\beta because it extends further into the central channel. This hairpin forms two polar interactions with the cyan strand DNA backbone. For DNA-interacting h2i, the conserved hydroxyl side-chain of S357 and main-chain amide of A732 interact with the phosphates of consecutive nucleotides of DNA. Notably, S357 is within the residues that comprise the helix/horseshoe conformations, and the helix conformation uniquely places it in position to interact with the DNA backbone. For DNA-interacting ps1\beta, the conserved positively charged side-chain of K785 and the main-chain amide of A786 interact with adjacent DNA phosphate groups. The polar interactions of the two hairpins are shown in magenta (Fig. 7b). Mutation of the ps1\beta lysine completely disables the strand displacement activity of MCM31,115. Mutation of the h2i hydroxyl group in SsoMCM (T369A) greatly reduces the cooperativity of unwinding31. SsoMCM T369A mutants were not well-behaved, and the only construct successfully isolated had a specialized internal loop deletion31. The reduced unwinding cooperativity and construct behavior are consistent with a role for this residue to interact with a neighboring subunit to develop a tight interface.

The W741 aromatic group stacks on the sugar of the DNA sugar-phosphate backbone (yellow, Fig. 7b). This stacking interaction is conserved in the ssDNA bound structure of SsoMCM (Y386, Fig. 7a). This stacking interaction is not needed to unwind DNA in a strand displacement assay based on alanine mutagenesis with SsoMCM31, and we have previously suggested that the stacking interaction could be important for DNA melting. Notably, the SsoMCM Y386A mutation eliminates the aromatic group from all 6 subunits of the SsoMCM homo-hexamer.

Aromatic Wedge is Conserved in Archaeal MCM; Eukaryotic Mcm5, 2, 6; SV40 Large T-antigen and Papillomavirus E1
As illustrated above, the DNA-interacting residues of the MCM ATPase hairpins have two categories: 1) polar residues (K785 and S357) that interact with the DNA backbone and are essential for DNA unwinding; and 2) aromatic residues (W741) that stack on DNA sugars and bases and are dispensable for DNA-unwinding. The separate functions of these residues strongly resemble the separate functions of two residues of the papillomavirus E1 ps1\beta where the analogous lysine (K506) is essential for unwinding DNA while the aromatic histidine (H507) is dispensable for unwinding DNA70. Instead, H507 has a critical role in DNA replication, DNA melting, and the functional progression from a double-trimer to a double-hexamer70. Of the 8 H507 mutants tested (A, V, L, R, M, N, F, and Y), only H507F supported in vivo replication in a transient DNA replication assay70, and only the aromatic mutants (H507F and H507Y) supported in vitro replication in a cell-free replication system70. Based on permanganate reactivity, only the H507F mutant melted DNA in the T-rich region that is anticipated to be located at the E1 ATPase tier70, and only the H507F mutant provided unwinding in an origin unwinding assay70.

We have previously noted that the SsoMCM h2i aromatic residue Y386 stacks on DNA sugars and bases very similar to the E1 ps1\beta aromatic H507 residue and suggested these residues may serve similar functions for melting and replication initiation31. We now strengthen this analogy based on a recent structure of SV40 L-tag, a close relative of E1, bound to melted DNA (PDB 9KAE27, Fig. 7c). This structure shows five unpaired nucleotides of DNA that represent bona fide DNA melting because the substrate did not have an exposed single-stranded region. In this structure, the aromatic histidine side-chain H513 stacks on the sugars and bases of the melted DNA with strong similarity to the aromatic residue of archaeal MCM (our Class 2 structures) and eukaryotic Mcm5, 2, and 6 (PDB 7PMN96, PDB 7PMK96, PDB 7Z1371, and PDB 7QHS71). The nucleotides not bound by L-Tag that have lost base-pairing interactions are disordered, analogous to the behavior of the melted DNA of our Class 2 structures and the eukaryotic CMG structures of PDB 7PMN96 and 7PMK96 (Fig. 6). Interestingly, although the central channel hairpins bind one DNA strand with identical polarity, the intact double-stranded region is observed at the N-tier for L-tag (PDB 9KAE27) and at the C-tier for the MCM structures (Class 2a, Class 2b, 7PMN96, 7PMK96, 7Z1371, and 7QHS71). Similar to MCM and E1, residues of the L-tag ps1\beta have different functions. Lysine K512 is essential for unwinding DNA in a strand-displacement assay116, but DNA unwinding in a strand displacement assay is less sensitive to H513 mutation (6 of 10 H513 mutants retain activity) 116. Based on these factors, we will refer to these aromatic residues as constituting an aromatic wedge to melt DNA for each complex.

Aromatic wedge residues are inherently present at all six subunits of archaeal homohexamers, but they follow a striking conservation pattern in eukaryotic Mcm2-7 that matches the arrangements of eukaryotic staggered Form I and staggered Form II (Fig. 4). Three consecutive subunits in the ring, Mcm5, Mcm2, and Mcm6, conserve an aromatic residue on the h2i at the same residue position as archaeal MCM (Fig. 7d). These aromatic groups stack on DNA sugars and bases in complexes with melted DNA (Fig. 6) but are distant from DNA in complexes with unmelted dsDNA (Fig. 5). In contrast, the other three subunits, Mcm4, Mcm7, and Mcm3, are nearly always not aromatic (Fig 7d). These two categories of h2i groups (aromatic: Mcm5, 2, 6 vs. non-aromatic Mcm4, 7, 3) correlate with the arginine finger side of tight interfaces for the two eukaryotic staggered forms (Fig. 4). Specifically, Mcm4, 7, and 3 are the subunits at the arginine finger side of three tight interfaces of Form I (Setting 4 of Fig. 4), and Mcm5, 2, and 6 are the subunits at the arginine finger side of three tight interfaces of Form II (Setting 1 of Fig. 4). Thus, the presence or absence of an aromatic wedge provides the underlying basis for Form I to adopt a stable structure that specifically does not melt DNA, and Form II to adopt an alternate stable structure that melts DNA.

Analogous to the conserved ps1\beta of E1 and L-tag, all archaeal and eukaryotic Mcm2-7 conserve a lysine on the ps1\beta29,31,115 (magenta, Supplementary Fig. 19) and conserve a hydroxyl group of the h2i31 (magenta, Fig. 7d). These residues very likely are functionally equivalent to the conserved lysines of L-tag and E1 (Fig. 7e-g, Supplementary Fig. 20). Interaction of these residues with DNA is sufficient to unwind DNA in a strand displacement assay, but DNA melting requires the functionally distinct aromatic residues of the hairpins.

Mechanism for Sequential Melting at a Single Hexamer
The distinct, related, Class 2 structures and their similarity to eukaryotic CMG (Fig. 6) suggest a sequential DNA melting mechanism based on successive conversion of subunit interfaces from loose to tight. The process begins with 6 loose interfaces and all subunits in the horseshoe conformation, represented by the C-tier of Fundamental Class 1. This form is consistent with a nucleotide-free MCM:DNA species that would exist after the necessary exhaust of ADP from the Mcm2-7 double-hexamer38. Successive conversion of adjacent interfaces to a tight conformation with associated h2i helix conformations drives sequential binding of one DNA strand by the ps1\beta and h2i hairpins to sequentially push the aromatic wedge into the DNA minor groove and increase DNA melting. The interfaces are converted from loose to tight by ATP-binding, as shown to occur during eukaryotic CMG activation38. Our structures show the disposition of MCM:DNA with 0, 2, and 3 tight interfaces. We have not detected the presence of a species with one tight interface among the cryo-EM particles, but we presume that such a species must exist so that multiple ATP-binding events are not precisely concerted. Although we have not identified a structure with a single tight interface, the comparative RMSD analysis revealed a candidate structure (PDB 9E2X98) of CMG encircling one strand of DNA with a G-quadraplex and a 3’-ssDNA extension (Fig. 8a). This structure had one tight interface according to the distance criteria (Table 1) and adopts the anticipated setting for a sequential mechanism (see below). Two of the h2i aromatic side-chains, Mcm2 and 6, stack on nucleotides of the ssDNA, but the aromatic side-chain of Mcm5 sits below the DNA. This structure does not allow assessment of the degree of melting because only one strand is present. The structure of loading intermediate 9GJW89 has a low RMSD in the same setting as 9E2X98 (Supplementary Table 8) and shows a similar degree of engagement of the h2i aromatic residues with dsDNA that is not melted (Fig. 8b). Two of the h2i aromatic side-chains, Mcm2 and 6, are projected to the minor groove, but the aromatic side-chain of Mcm5 is more distant from the DNA.

The first mechanistic step is to tighten one interface of a C-tier with 6 loose interfaces (represented by Class 1a) to a structure that resembles that of PDB 9E2X98 (Fig. 9a). Tightening a second, adjacent, interface yields the Class 2a species with two tight interfaces and two DNA base-pairs melted (Fig. 9a). This MCM:DNA species is also represented by the 7PMN96 structure. Conversion of another adjacent interface to tight yields the Class 2b structure with three tight interfaces and 4 bases of DNA melted (Fig. 9a). This structure strongly matches the eukaryotic melted DNA species that follows GINS recruitment (PDB 7Z1371) and matches the stable eukaryotic staggered Form II structure (Fig. 4).

For this sequential mechanism, the transition from Class 2a to Class 2b involves tightening one interface that is adjacent to two consecutive tight interfaces. The tightened interface could be the first empty interface clockwise or counterclockwise. Considered at the ATP site level, the ATP site that is tightened could involve the Walker-A site of one of the subunits participating in tight binding or the arginine finger of one of these subunits. These scenarios are not distinguishable for archaeal homo-hexameric structures because the subunits are identical in sequence. The comparative structures of PDB 7PMN96 and 7PMK96 definitively show the directional order because they derive from heterohexameric structures with distinguishable subunits. These structures illustrate that the tightened interface is at the counterclockwise side (3/5 interface tightened rather than 6/4; see Table 1 and Fig. 6). Notably, the directional order of ATP-binding during sequential melting is identical to the directional order of ATP-binding in subsequent DNA translocation31.

Double-hexamer Consideration
Single CMG hexamers are sufficient to provide limited DNA unwinding117 that is consistent with the structural species described above. Extended origin unwinding, which is an essential component of replication initiation, requires formation of a competent double-hexamer117. The structure of the human Mcm2-7 double-hexamer encircling dsDNA (PDB 7W1Y99) includes a melted DNA bubble at the center of the complex at the double-hexamer interface. The DNA properties that make it susceptible to forming a bubble may be an important part of origin identification that precedes the DNA melting at the ATPase tiers. The central DNA bubble of the human structure (PDB 7W1Y99) could potentially migrate to the C-tier based on activities of the MCM ring, but we feel that the DNA replication initiation mechanism will generally tolerate re-annealing of this central DNA bubble. The central bubble (PDB 7W1Y99) seems to at least partly derive from the double-hexamer architecture because the corresponding single-hexamer structures (PDB 8S0A114 and PDB 8W0E94) are very similar in underlying hexamer:DNA arrangement, but they but do not exhibit melted DNA at the N-tier. The central N-tier bubble of PDB 7W1Y99 thus appears to derive from simultaneous interaction of each constituent hexamer C-tier with DNA in conjunction with a double-hexamer interaction that enforces a greater distance between the C-tiers than undistorted B-DNA allows. If any of these three interaction modes are lost, the additional degrees of freedom would likely allow the DNA to re-anneal. The exhaustion of nucleotides from the MCM complex at the onset of DNA replication is expected to release the grip of both hexamers on DNA. As illustrated in the Class 2 and comparative structures above, only MCM subunits that participate in a tight interface appropriately position DNA-binding modules of adjacent subunits for binding DNA. Exhaustion of all nucleotides from the MCM ATPase sites is expected to convert all interfaces to loose with concurrent loss of interactions with DNA (analogous to the Class 1a structure). Although the hexamers would continue to encircle DNA, the lost grip would likely allow the central bubble to re-anneal.

To demonstrate our preferred path for the sequential melting mechanism of Fig. 9a in the context of a double-hexamer, a molecular morph movie was generated using the rigimol routine of PyMOL86. The process begins with one hexamer losing its grip on DNA and sliding away from the other hexamer, breaking the double-hexamer interface. The sliding hexamer’s lost grip on DNA allows the central DNA bubble to re-anneal as described above. The constituent hexamers are then converted to the melting form (eukaryotic staggered Form II) by the sequential melting mechanism of Fig. 9a. We illustrate the transitions at each hexamer independently to emphasize that the transformations among the ATPase sites are likely not precisely concerted. Following conversion of both hexamers, the final melting structure of PDB 7Z1371 is established (Supplementary Movie 2). The morph movie is intended to provide general qualitative visualization of our preferred path for sequential melting and not to convey specific molecular dynamics.

Proposed Mechanism for a Fixed MCM Aromatic Wedge to Extend Origin Unwinding
The specific placement of aromatic wedge residues at Mcm5, 2, and 6 and absent from Mcm4, 7, 3 profoundly implies that the MCM ring does not extend initial DNA melting via the rotary translocation mechanism31 that the mature helicase uses at the replication fork to pull one encircled DNA strand through its central pore. For translocation and melting activities, MCM subunits (predominantly) bind one strand of DNA in a spiral staircase (see Fig. 7a-b). In the rotary DNA translocation activity, the collective subunit staircase moves down, pulling the bound DNA through the central pore. Next, the subunit at the bottom of the staircase releases its grip on DNA and moves to the top to bind the next incoming nucleotide of ssDNA. The binding and release events proceed around the ring, with each subunit sequentially taking its turn at the top of the staircase. This activity is possible for the eukaryotic heterohexamer in CMG because all Mcm2-7 subunits conserve the polar ssDNA-contacting residues of the ps1\beta and h2i. In contrast, only Mcm5, 2, and 6 have aromatic wedge residues, and hence the other subunits (Mcm4, 7, and 3) cannot take a turn to serve as an aromatic wedge. A sequential rotary action in the melting complex (such as PDB 7Z1371) will convert it to a form where aromatic wedge subunits no longer participate in tight interfaces or interact with DNA and thus will not be able to melt DNA.

We therefore suggest that the overall protein complex of the melting conformation (Class 2b, PDB 7PMK96, PDB 7Z1371, PDB 7QHS71) is static with an aromatic wedge persistently poised to melt DNA. In this form, melting can be extended if an external force pulls the DNA relative to the protein (Fig. 9b, Supplementary Movie 3). An alternative description is that melting can be extended by an external force that pushes the aromatic wedge, like a plow, into undistorted DNA and sequentially melts one base-pair at a time. In this mechanism, the DNA nucleotides all move one sequential position in their arrangement with the MCM DNA-binding hairpins, which contrasts the continuous association of each DNA nucleotide with a specific hairpin that occurs during rotary translocation. If the DNA is viewed as a chain, and the MCM DNA-binding hairpins as teeth of a sprocket, the rotary translocation mechanism follows a standard chain drive where the teeth maintain continuous association with specific chain links, and rotation of the sprocket drives the entire chain to move. In the above mechanism for extension of DNA melting, the sprocket remains fixed while an external force pulls the chain such that it slips over the sprocket in one increment. Based on this analogy, we refer to this as a gear slip (Fig. 9b, Supplementary Movie 3). Notably, this melting extension is not based on ATP hydrolysis by MCM because the MCM ring is static, and the MCM:DNA movement is caused by an external factor.

A likely candidate to provide an external force is Mcm10. First, Mcm10 has been shown to function as a wedge that pries the constituent hexamers of the double hexamer apart72. Second, Mcm10 is the decisive factor to transition from initial melting to extensive origin unwinding38. With both hexamers in a static melting conformation, as illustrated by PDB 7Z1371, the action of prying the two hexamers apart from each other will directly push the aromatic wedge into the undistorted DNA beyond the C-tier of the hexamer (Fig. 9c, Supplementary Movie 4). Notably, this movement of the MCM ring with respect to the DNA is opposite in direction to that used during rotary ssDNA translocation31. In the absence of contrary information, we presume that archaeal MCM will melt DNA with a mechanism that is highly similar to that of eukaryotes with an external force pushing a static aromatic wedge. Although an archaeal counterpart of Mcm10 has not been identified118, we predict that an unidentified factor pushes the MCM ring in the static melting conformation towards undistorted DNA (or pulls the DNA over the wedge) to extend DNA melting for replication initiation.

Lagging Strand Exit from the Ring
We suggest that final exit of the lagging strand from the ring occurs stepwise at the two tiers because all CMG structures adopt a staggered conformation (Fig. 4). The staggered conformation cannot readily develop a contiguous opening across both tiers to enable a strand to exit both tiers in a single step. All staggered conformation structures have a closed ring at the N-tier. In contrast, the staggered conformation is able to generate a wide gap at the C-tier as illustrated by our earlier ssDNA translocation structure where one ATPase domain is disordered (PDB 8EAM16). This gap is sufficiently large to enable the lagging strand to pass through and exit the C-tier ring, and the gap is naturally re-sealed by the rotary DNA translocation activity of the ATPase tier16. We suggest that such lagging strand exit from the C-tier is preceded by its exit from the N-tier, and such N-tier exit may occur when Mcm10 wedges the two hexamers apart72 with Mcm10 remodeling the MCM ring119,120 at the N-tier to enable strand exit. Mcm10 may also bind to exposed ssDNA121 to assist strand exit.

Biological Context
The single-hexamer structures reported here show progressive melting at the end of DNA. Biological DNA replication begins with an MCM double-hexamer at interior regions of DNA and requires multiple firing factors and additional protein recruitment38,61,71. The exclusive MCM:DNA composition of the samples reported here assures that the DNA melting is mediated by the MCM proteins, but this composition does not show DNA at the N-terminal tier for a melted DNA complex, which would be present when melting occurs an interior region of DNA71. The structures of PDB 7PMN96 and PDB 7PMK96 similarly show CMG with DNA melted at an end. Within these structures, the MCM ring likely sits at the end of the DNA because it is more energetically favorable to melt DNA at the end than in the middle. In contrast, the structure of PDB 7Z1371 was generated on a piece of DNA flanked at both ends by nucleosomes to enforce an interior DNA positioning, and this structure uniquely shows CMG with DNA melted by the C-tier at an interior DNA region.

The homo-hexameric construction of the archaeal MCM hexamer positions conserved aromatic wedge residues equivalently in all settings. As such the distinct, stable staggered forms of eukaryotic Mcm2-7 (Fig. 4 Staggered Form I and Staggered Form II) may not be possible for archaeal MCM. The additional form in eukaryotes is that of the stable and idle MCM:DNA double-hexamer and suggests that a stable staggered form that does not melt DNA does not exist in archaea. Development of the capacity to generate these two fundamental, stable states is likely part of the basis for the evolution of the MCM helicase from homohexamers in archaea to heterohexamers in eukaryotes.

Methods
Cloning, Expression, and Purification
The previously described protein construct (pJM001.329) is a chimeric fusion of the Saccharolobus solfataricus MCM N-terminal domain and the Pyrococcus furiosus MCM AAA+ domain (Sso-PfMCM) with an N-terminal His6-SUMO tag. The original SUMO vector was the generous gift of Dr. Christopher D. Lima122. The Pyrococcus furiosus genetic sequence has an intein in the AAA+ domain (PfMCM amino acids 362-72848) that was genetically removed from the construct29. The protein construct expresses SsoMCM residues 1-269, followed by PfMCM residues 257-361, and then PfMCM residues 729-96629. Freshly transformed BL21(DE3)-RIPSL chemically competent cells were grown overnight in a 100 mL starter culture of LBE media with 30 mg/L kanamycin and 2% glucose. The starter culture was distributed to 6 L of LB media containing 0.4% glucose and 30 mg/L kanamycin. The cultures were grown at 37°C to O.D. 0.3 and subsequently grown at 18°C. IPTG was added to 0.4 mM at O.D. 0.7 followed by overnight growth at 18°C. Cells were harvested by centrifugation, resuspended in 50 mM Tris pH 8.3, 250 mM NaCl, 10% glycerol, 5 mM \beta-ME, and lysed with a microfluidizer. The soluble fraction was isolated by centrifugation. Nucleic acids were removed by adding 5 M NaCl to 1 M final concentration followed by 10% polyethylenimine-HCl to 0.3% final concentration. The soluble fraction was isolated by centrifugation, and the protein was precipitated by adding 43.6 g of ammonium sulfate per 100 mL. The precipitated protein was isolated by centrifugation and resuspended in 50 mM Tris pH 8.3, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 5 mM \beta-ME followed by batchwise binding to Ni-NTA agarose (Qiagen) with five subsequent washes with the same buffer. The protein was eluted with 50 mM Tris pH 8.3, 500 mM NaCl, 10% glycerol, 250 mM imidazole, 5 mM \beta-ME and subjected to size-exclusion chromatography in 20 mM HEPES pH 7.6, 200 mM NaCl, 5 mM \beta-ME, eluting at a volume consistent with a hexamer. Ulp1 protease was added, and the sample was dialyzed overnight against 20 mM Tris pH 8.3, 100 mM NaCl, 5 mM \beta-ME to cleave the SUMO tag. The Ulp1 protease plasmid was the generous gift of Dr. Christopher D. Lima122. The cleaved MCM protein was purified by anion exchange with a 0.1 – 1 M NaCl linear gradient in 20 mM Tris pH 8.3, 5 mM \beta-ME. Selected fractions were purified by gel-filtration chromatography in 20 mM HEPES pH 7.6, 200 mM NaCl, 5 mM \beta-ME; dialyzed against 25 mM HEPES pH 7.6, 10 mM NaCl, 5 mM magnesium acetate; and concentrated to 20 mg/mL for storage.

Cryo-EM Sample Preparation, Data Collection, and Processing
Cryo-EM samples were prepared to consist of 2 mg/mL Sso-PfMCM, 5 mM ATP-\gammaS, 0.25 mM 60-mer palindromic DNA substrate (see below for specific sequences) in 25 mM HEPES pH 7.6, 10 mM NaCl, 5 mM magnesium acetate. Samples were incubated for 2 hours at room temperature and then passed through a 0.22 \mu m spin filter. Filtered samples were plunge frozen on a Vitrobot using C-flat grids. Cryo-EM data were collected using a Titan Krios (Thermo Fisher) transmission electron microscope, equipped with a K3 direct electron detector and post column GIF (energy filter). K3 dark and gain reference were collected just before data collection. Data collection was performed in SerialEM software123 with image shift protocol (9 images were collected with one defocus measurement). Movies were recorded at defocus values from -0.8 to -1.8 \mu m at a magnification of 81kx, which corresponds to the pixel size of 1.08Å at the specimen level (super resolution pixel size is 0.54). Motion correction was performed on raw super resolution movies stacks and binned by 2 using MotionCor2 software124. For each structure, an initial evaluation of a subset of the micrographs was performed with Relion125, CTFFind126, cisTEM127, Cryosparc128, pyem129, and Chimera130.

For the Sso-PfMCM:Mg:ATP-\gammaS:DNA1 complex, the protein solution and was combined with a 60-mer palindromic oligonucleotide that contained a repetitive sequence, ACTGACTGACTGACTGACTGACTGACTGACGTCAGTCAGTCAGTCAGTCAGTCAGTCAGT. This DNA sequence was inspired by the deposited oligonucleotide sequence of the cryo-EM structure of eukaryotic Mcm2-7 double-hexamer encircling duplex DNA (PDB 5BK437). The sample was frozen to grids with a vitrobot, and 5,668 micrographs were collected with the following parameters: Magnification: 81.000; Pixel size: 1.08 (Counting)/0.54 (Super Resolution); Exposure time: 3sec / 0.05 sec per frame/ 60 frames; Dose: 29.74 e-/physical pixel/second or 25.5 e-/Å2/second; Total dose: 76.5e-.

The motion-corrected micrographs were imported to Cryosparc128, and its Patch CTF routine was applied to each micrograph. 45,565 particles were picked by the Cryosparc128 Blob picker routine from 50 micrographs. Particle images were extracted and used in 2D classification to generate templates that were used in the Cryosparc128 Template Picker for the full dataset. 15,146,308 particles were identified, and their positions were converted for use in Relion125 by pyem129. In Relion, the micrographs were imported, and CTF parameters were determined using CTFFind126. The imported particle positions were used to extract particle images at 8.64 Å per pixel. These particle images were imported to Cryosparc128, subjected to 2D Classification, and 4,851,305 particles were selected. These particle positions were converted for use in Relion125 by pyem129 and were extracted at 4.32 Å per pixel by Relion. The particle images were imported to Cryosparc128, subjected to 2D Classification, and 3,369,871 particles were selected. These were extracted at full 1.08 Å per pixel resolution by Cryosparc128 to yield 3,304,031 particles. These particles were subjected to two rounds of 2D classification followed by duplicate removal in Cryosparc128 to yield 2,388,484 particles for use in subsequent refinement. These particles were subjected to Heterogeneous Refinement in Cryosparc128 with 6 classes. Two of the classes had hexameric ring structures with obvious DNA in the central channel: Class 1 (DNA passing through both tiers, 353,074 particles) and Class 2 (DNA at C-tier, 579,241 particles).

Class 1 was subjected to homogeneous refinement followed by 3D-classification with 10 classes in Cryosparc128. Ten volumes were used to seed 10 classes of Heterogeneous Refinement in Cryosparc128. One of these subclasses (74,910 particles) had six well-ordered ATPase domains and appeared C6-symmetric with cylindrical, featureless DNA. This subclass ultimately generated the Class1a structure by symmetry expansion and 3D classification (40,953 particles; described further in section below on Merged particles). The final refinement was based on Local Refinement in Cryosparc128. Two additional subclasses had only five well-ordered ATPase domains and thus intrinsically asymmetric. An initial homogeneous refinement in Cryosparc128 (95,316 particles) showed DNA strand features, but the DNA structure was noisy and suggested multiple conformations. These refined particles were subjected to 3D classification in Cryosparc128 in 6 classes using a cylindrical focus mask that encompassed the central channel. This classification revealed conceptually similar structures where the h2i of a subunit of an ATPase domain located adjacent to the disordered ATPase domain significantly approached one of the DNA strands, but the polarity of this interaction strand differs. These two subclasses were refined and partitioned as described under Merged particles. The refined particles of Merged particle refinement were separated by dataset for final set-specific refinements. Class1b was based on Cryosparc128 Local Refinement of 32,139 particles, and Class1c was based on Cryosparc128 Local Refinement of 15,877 particles.

Class 2 was subjected to homogeneous refinement followed by 3D-classification with 10 classes in Cryosparc128. The particles from 9 of 10 classes (517,028 particles) were pooled and used in Heterogeneous Refinement in Cryosparc128 seeded with 4 of the volumes from the prior 3D-classification job. This refinement distinguished two forms within the C-tier, Class2a with 4 horseshoe helix-2-inserts (148,429 particles) and Class2b with 3 horseshoe (196,176 particles). The final refinements for each were obtained by Homogeneous Refinement in Cryosparc128.

Class 3 (heptamer) was obtained by Cryosparc128 Local Refinement (25,549 particles) using the structure derived from the Merged Particles heptamer (see below) after separating the particles by dataset.

The Sso-PfMCM:Mg:ATP-\gammaS:DNA2 sample was prepared by combining the protein solution with a 1 mM 60-mer oligonucleotide that contained a random palindromic sequence, TCCGTCTCACAGTAACAACCCTCCGCCTGATCAGGCGGAGGGTTGTTACTGTGAGACGGA. The sample was frozen to grids with a vitrobot, and 6633 micrographs were collected with the following parameters: Magnification: 81.000; Pixel size: 1.08 (Counting)/0.54 (Super Resolution); Exposure time: 4.9sec / 0.07 sec per frame/ 70 frames; Dose: 18.829 e-/physical pixel/second or 15.963 e-/Å2/second; Total dose: 78.22e-.

The motion-corrected micrographs were imported to Cryosparc125, and its Patch CTF routine was applied to each micrograph. 79,822 particles were identified by Blob Picker from 40 micrographs and extracted. These were subjected to two rounds of 2D classification to generate templates for Cryosparc’s Template Picker128, which identified 14,853,988 particles. These particles were exported to Relion by pyem129 and extracted in Relion125 at 4.32 Å per pixel. The particles were subjected to 3 rounds of Cryosparc128 2D classification to yield 4,211,984 particles. The particles were then extracted at 1.08 Å per pixel in Cryosparc128 to yield 4,174,824 particles followed by duplicate removal to retain 4,104,256 particles. Following a final 2D classification, 3,793,296 particles remained in the set.

These particles were subjected to Heterogeneous Refinement in Cryosparc128 with 6 classes. Two of these classes had hexameric ring structures with obvious DNA in the central channel: Class 1 (DNA passing through both tiers, 527,777 particles) and Class 2 (DNA at C-tier, 879,551 particles).

Class 1 was subjected to homogeneous refinement followed by 3D-classification with 10 classes in Cryosparc128. The 10 volumes were used to seed 10 classes of Heterogeneous Refinement in Cryosparc128. One of these subclasses (110,906 particles) had six well-ordered ATPase domains and appeared C6-symmetric with cylindrical, featureless DNA. This subclass ultimately generated the Class1a structure by symmetry expansion and 3D classification (60,755 particles; described further in section below on Merged particles). The final refinement was based on Local Refinement in Cryosparc128. Two additional subclasses had only five well-ordered ATPase domains and thus intrinsically asymmetric. An initial homogeneous refinement in Cryosparc128 (85,099 particles) showed DNA strand features, but the DNA structure was noisy and suggested multiple conformations. The refined particles were subjected to 3D classification in Cryosparc128 in 6 classes using a cylindrical focus mask that encompassed the MCM central channel. This classification revealed conceptually similar structures where the h2i of a subunit of an ATPase domain located adjacent to the disordered ATPase domain significantly interacts with one of the DNA strands, but the polarity of this interaction strand differs. These two subclasses were refined and partitioned as described under Merged particles. The refined particles of Merged particle refinement were separated by dataset for final set-specific refinements. Class1b was based on Cryosparc128 Local Refinement of 28,511 particles, and Class1c was based on Cryosparc128 Local Refinement of 14,925 particles.

Class 2 was subjected to Heterogeneous Refinement in Cryosparc128 with 3 classes seeded with volumes corresponding to class2a, class2b, and DNA-free models (see below for seed volume generation description). One class was selected (291,379 particles), subjected to Homogeneous Refinement, followed by 3D classification to generate 10 seed volumes that were used in Heterogeneous Refinement of the 10 classes. Seven classes were selected, pooled, and subjected to a final Homogeneous Refinement (Class 2a, 235,648 particles). A second class from the initial Class 2 Heterogeneous Refinement job was selected (328,400 particles), subjected to Homogeneous Refinement followed by 3D classification to generate 10 seed volumes that were used in Heterogeneous Refinement of the 10 classes. Six classes were selected, pooled, and subjected to a final Homogeneous Refinement (Class 2b, 275,045 particles).

The seed volumes for the first Heterogeneous Refinement of Class2 were obtained by first subjecting the 879,551 particles of class 2 to 3D classification in 10 classes. One of these classes appeared DNA-free and formed one seed volume after Homogeneous Refinement. Eight of the classes had obvious DNA at the central channel of the ATPase domain, and these were pooled and subjected to Heterogeneous Refinement seeded by four classes of the prior 3D Classification job. One of these was used as a seed volume following its Homogeneous Refinement (Class 2a), and two appeared similar and were pooled and subjected to Homogeneous refinement, generating the third seed volume (Class 2b).

Class 3 (heptamer) was obtained by Local Refinement (25,290 particles) using the structure derived from the Merged Particles heptamer (see below) after separating the particles by dataset.

Merged Particle Structures
For the two datasets, the particles comprising Class 2a and Class 2b were pooled respectively and subjected to Homogeneous Refinement in Cryosparc128 to generate refined structures based on the merged particles. The other two classes of structure were very nearly symmetric for the protein component and required special classification treatment to elucidate the features of the central DNA that was enhanced by maximizing the total particles. As a result, these computations were performed on the merged particles of the two datasets, and final structures for the individual datasets were then obtained by separating the particles based on the respective datasets.

Class1a is nearly C6-symmetric at the protein level, and classification jobs tended to be dominated by versions of the complex that illustrated varying disorder of the peripheral helical bundle subdomains of the NTD. The following procedure provided classification based on the central DNA features. First, the Class1a particles of the two datasets (185,816) were pooled and subjected to Homogeneous Refinement in C6 symmetry. The resulting particles were 6-fold expanded about the C6-axis (1,114,896 particles) and subjected to 3D Classification in 10 classes using a central channel focus mask. The focus mask was created with 5-pixel soft-padding from a cylinder shape (9 Å radius, 100 Å height) created in Chimera130. The best-looking class was selected and subjected to Local Refinement (113,110 particles) using the full solvent mask of the previous classification job. Duplicate particles were removed (101,708 particles retained), and the final Class 1a structure was obtained by final Cryosparc128 Local Refinement. The particles were separated into their respective datasets and generated final structures by Local Refinement (see above).

The particles of the structures of 5 well-ordered ATPase domains for both datasets were combined and subjected to 3D classification in Cryosparc128 using the cylindrical focus mask (above). Two classes were pooled to generate Class1b (60,650 particles) following Cryosparc128 Local Refinement. One class of 30,802 particles generated Class1c following Cryosparc128 Local Refinement. The particles were separated into their respective datasets and generated final structures by Local Refinement (see above).

Class 3 is nearly C7-symmetric at the protein level, analogous to the near C6-symmetry of Class 1a. The smaller number of particles of the heptameric form greatly benefited by merging the two dataset particles in a procedure similar to that of Class 1a. 2D classification indicated a minor presence of heptameric rings in views parallel to the ring channel. All other views were not obviously in a specific oligomeric form, and thus the particles comprising these class averages might contain hexamer, heptamer, or other forms. An initial map for the heptamer was obtained from a subset of particles in Cryosparc128 by calculating 6 ab initio classes and selecting a class that was recognizable when looking down the channel axis at one side with decreasing order when moving away from that view. This structural class appeared to derive from superimposed particles of hexamer and heptamer. The particles were subjected to homogeneous refinement followed by 3D classification in 10 classes, which improved the density across the ring. The most well-defined class that appeared to encircle DNA was selected and subjected to homogeneous refinement followed by 3D classification in 3 classes. The best class was selected and subjected to homogeneous refinement in C1-symmetry to generate a reference map that was used in a subsequent refinement in C7-symmetry, which aligned the structure to the symmetry axis. The particles were then symmetry expanded about the C7-axis and then subjected to 3D classification in 7 classes using the same cylindrical focus mask used for Class 1a above. The best-looking structure was selected and subjected to Local Refinement using the full solvent mask of the preceding 3D classification job. This heptamer volume provided one class for a large-scale classification job of all the particles of the two datasets.

The full set of initially selected particles for the two datasets were used for Cryosparc128 ab initio reconstructions in 6 classes. The six generated volumes and the heptameric volume (see above) were used in a 7-class Heterogeneous Refinement job with the full set of particles. The heptameric class (379,160 particles) was subjected to homogeneous refinement followed by 3D classification in 10 classes. The 10 volume classes were used as the initial volumes in a Cryosparc128 Heterogeneous Refinement job. The heptameric class was selected (89,337 particles), and the particles were used to generate 3 ab initio classes in Cryosparc128, and the resulting volumes were used as initial volumes in a 3-class Cryosparc128 Heterogeneous Refinement. The best class (71,238 particles) was selected and subjected to homogeneous refinement in C1 symmetry followed by refinement in C7 symmetry. The particles were expanded about the C7-axis and subjected to 3D classification in 7 classes using a cylindrical focus mask (20 Å radius, 100 Å height). The best class was selected and subjected to Local Refinement (61,530 particles) using the full solvent mask of the previous 3D classification. Duplicate particles were then removed (50,889 particles retained), and an additional Local Refinement job was performed. The Cryosparc128 Particle Sets tool was then used to generate the final particle set (50,839 particles) by removing the particles contributing to Classes 1 and 2. A final Cryosparc128 Local Refinement job with this particle set generated the structure of heptamer for the merged particle set. The structures for the individual datasets were then obtained by Local Refinement using the particles of the respective datasets.

Model-building and Refinement
Initial models were constructed from the crystal structure of Sso-PfMCM, PDB 4R7Y29, by placing its N- and C-terminal domains in the respective density. The structures were refined with phenix.real_space_refine131,132 and rebuilt in Coot133-135. The final refined coordinate files contain either ATP-\gammaS or ADP at each site according to the strength of the \gamma-phosphate in the EM map. The inclusion of ADP molecules in the model is to model the apparent density and does not indicate that hydrolysis of the \gammaS-phosphate has occurred. The refinements included bond distance and octahedral angle restraints for the associated magnesium ion. For ATP-\gammaS, the magnesium was bonded to a Walker-A S335 side-chain, an ATP-\gammaS \beta-phosphate oxygen, an ATP-\gammaS \gamma-phosphate oxygen, and three water molecules. For ADP, the magnesium was bonded to a Walker-A S335 side-chain, an ADP \beta-phosphate oxygen, and four water molecules. The polypeptide construct consists of SsoMCM residues 1-269, followed by PfMCM residues 257-361, and then PfMCM residues 729-96629. In the deposited molecular coordinate files, these residue spans are numbered 1-269, 1257-1361, and 1729-1966.

Figure Preparation
Figures were prepared with the programs PyMol86, Chimera130, ChimeraX136, and Adobe Illustrator. Residues are numbered with native amino acid numbers without intein removal. Cartoon representations depict MCM hexamers as six-color rings with tight and loose interfaces and DNA as beads on a string, similar to the style we have used previously16,31.

RMSD Comparisons
A report from the PDB with all structures meeting the search term “mcm” and a 200 kDa molecular weight cutoff was downloaded. For each, a hexameric MCM structure consisting of six 152-amino acid ATPase core16 structures was constructed by automated alignment in PyMOL86 of the 152-amino acid SATPase core of chain Class 2b chain F to each chain 2-7 (residues 319:361 / 729:837 renumbered to a contiguous span, 332-483). An ATPase core hexamer for a candidate alignment model (such as Class 2b) was similarly constructed by automated alignment in PyMOL86 of the same 152-amino acid ATPase core of Class 2b chain F to each chain A-F, and six different settings were constructed by modifying the chain identifiers in all six permutations of the directional order of eukaryotic subunits around the ring (such as 2, 6, 4, 7, 3, 5— see Supplementary Fig. 12 for illustration of the process including the six settings of Class 2b for candidate alignments). The RMSD of each setting against each eukaryotic Mcm2-7 structure of the downloaded list was calculated and sorted to compare the collective disposition of the six MCM ATPase cores as a unit. Altogether, 77 structures were compared in 6 settings for a total of 462 comparisons. The top 30 RMSD alignments for Class 2b (Supplementary Table 8) were visually inspected to identify the comparative structures used. The RMSD analysis of Class 1a (Supplementary Table 7) did not identify strong candidates for comparison.

Dihedral Analysis
For each of the 77 structures identified above, an MCM N-tier model was generated by automated alignment in PyMOL86 of the Class 2b chain F OB-fold (residues 105:130 / 185:265) to each subunit of the candidate Mcm2-7 structure. An MCM C-tier model was generated by automated alignment in PyMOL86 of the Class 2b chain F 152-amino acid AAA+ core16 (residues 319:361 / 729:837 renumbered to a contiguous span, 332-483) to each subunit of the candidate Mcm2-7 structure. The chains for each were renamed to correspond to the Mcm2-7 structure, and centroid positions for each tier were calculated by PyMOL86. For each chain of the constructed alignments, a dihedral was calculated by PyMOL86 from the following four sequential positions (with original Class 2b residue numbering): SsoMCM residue P201 C-alpha, OB-fold centroid, AAA+ centroid, followed by PfMCM residue Y755 C-alpha. Five structures did not contain all 6 OB-folds and were excluded from the analysis.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Rasouli, Myasnikov and Enemark report a sheer number (18) structures of a duplex DNA bound protein chimera with a fusion of the Sulfolobus MCM N-terminal oligomerisation domain and the Pyrococcus MCM ATPase motor domain. Enemark has shown previously that this protein is catalytically active. Its use in structural biology studies already provided insights that proved precious for the field over time. The current work constitutes an important addition to this body of work. Here the authors report various mode of duplex DNA engagement for a single MCM ring. While the authors propose a mechanism whereby the structural transitions in this single hexamer could lead to DNA melting, they choose not to acknowledge some recent work on the mechanism of loading of a single MCM ring, and the structures of a fully loaded single hexamer around duplex DNA, from both human and yeast MCM (though, admittedly, the latter is only a bioRxiv uploaded manuscript). The recent identification of a single duplex DNA loaded MCM in humans was a big deal (with two high profile papers published simultaneously). The discovery that this is a stable intermediate also in archaea is remarkable and makes these data worth publishing in Nature Communications. That said, a rework of the introduction and discussion is recommended, which will provide a proper framework for the discoveries reported. Specific points are detailed below.\nMajor issues\n1. Is it correct to define Class 2 as a DNA melting intermediate, considering the discovery from Steve Bell’s lab (Champasa et al 2019) that a yeast MCM variant that cannot homodimerise can form CMG but not open DNA? Would it not be best to compare this structure to the human MCM single hexamer (Weissmann et al 2024 and Yang et al 2024)?\n2. The authors observe Class 2 where the leading but not the lagging strand template is visible. They interpret this as a melting intermediate. This could also reflect the fact that the leading strand is visible because it is stabilised by pore loop contacts. This is indeed the case for a helicase loading intermediate (single loaded yeast MCM hexamer) described in a bioRxiv manuscript by Miller, Diffley and colleagues. The PDB coordinates (8RIG) can be downloaded from the PDB already. This is an intermediate on the path to double hexamer formation. In the final product (the DNA is not melted).\n3. Along the same lines, I feel the authors should do a better job at drawing a parallel between the mechanism of MCM loading onto duplex DNA and the mechanism of DNA melting by the CMG. In this respect, I note that no reference was made to recent work published in Nature Communications by Butryn et al (2025), which describes a single-loaded yeast hexamer in different duplex DNA binding states, captured by using inactivating ATPase mutants. These structures show a DNA engagement state similar to that of the DNA-melting (or single-stranded DNA translocating) CMG, concluding that the mechanism of MCM interaction and translocation along DNA is always the same, and implying that melting occurs when two CMGs are facing one another. I feel this is another example of a missed opportunity to show how the structure of an archaeal single loaded hexamer on duplex DNA is relevant to mechanistic studies in eukaryotes (both for DNA melting, as stated in this manuscript, but also for helicase loading).\nSmaller issues\n1. The authors claim that no contacts exist between MCM and DNA in the class 1 structure. They should clarify how the DNA register is established such that duplex DNA can be averaged in the structures. If not contacted by the ATPase domain, duplex DNA must surely be engaged by elements of the N-terminal oligomerisation domain. Otherwise, DNA would be lost from the central channel or only result in a featureless sausage through single-particle averaging. You could refer to studies on the FANCI-FANCD2 DNA encircling ring by Alcon et al Nature 2024, Fig. 4a and Fig. 4b for comparison.\n2. Heptameric MCMs had been previously observed in studies by Jose’ Maria Carazo, Silvia Onesti and Laura Spagnolo. Their work should be cited.\n3. The resolution of the structures reported is high enough for discriminating nucleotides in the ATPase pockets but the authors showed not to show them. I think this should be rectified.\n4. Line 37 page 2 “Similar, but different subunits” sounds odd. To me, similar implies that they are different, otherwise they would be identical.\n5. Figure 1 is very similar to Extended Data Figure 2 and 3. Figure 2 appears very similar to Extended Data Figure 4 and 5. Could the authors check whether any panels have been mistakenly mispaced?\n6. Page 3 line 72 “GINS recruitment provides 1.5–2 turns of DNA unwinding at each CMG” is incorrect. Douglas 2018, which the authors cite in this passage reads “each MCM hexamer untwists around 0.6–0.7 turns of DNA”.", "reviewer_2": "A large number of biochemical and cryo-EM studies in the past few years have focussed on the molecular characterisation of the initiation stages of DNA replication. Progress in this area has been significantly advanced by the ability to reconstitute the process in vitro using purified yeast proteins, and solve structures of various intermediate states. Although much is now known about this process, a complete molecular explanation of the very first stages of DNA melting is still lacking. This manuscript focusses on this key question, using the archaeal homo-hexameric MCM replicative helicase as a model system. The authors present multiple cryo-EM structures of an archaeal MCM single hexamer bound around DNA. Their sample preparation method facilitates the capture of multiple different forms of the MCM-DNA complex, either containing B-form double stranded DNA, or in various states that the authors propose mimic the first stages of replication origin melting. By integrating these structures with previously published structures of archaeal and yeast MCM complexes, the authors propose a compelling model for DNA melting, involving sequential conversion of inter-subunit interfaces from loose to tight states. The manuscript is well structured and written, and clearly explains some quite complex structural conclusions and hypotheses. Further interrogation of the structural data using functional experiments (e.g. with specific mutants in vitro or in vivo) is completely lacking. Overall, however, given the high quality of the structural data and the general interest in this area of the replication field, I would suggest that the structures alone are sufficiently interesting to warrant publication without functional validation, particularly given the difficulty of such experiments in the archaeal system.\nMajor points:\n• The author’s models are heavily dependent on nucleotide-dependent transitions between loose and tight states at inter-subunit interfaces in the MCM ring. To this reviewer, it was unclear whether the authors actually had high enough resolution in their structures to model nucleotide in any of the MCM active sites? If not, this should at least be stated.\n• The model in Fig. 5 is dependent on sequential transitions in inter-subunit interfaces around the MCM ring. One tight interface appears to favour the conversion of the neighbouring interface from loose to tight. Can the authors explain how this would work? Is there a structural explanation for why ATP binding at one interface would, for example, favour ATP binding at the adjacent interface?\n• Recent structures have demonstrated that a small section of DNA, positioned at the inter-hexamer interface, is already distorted in the human MCM double hexamer (e.g. PDB:7W1Y). Thus, unlike in yeast, melting (or at least distortion of the duplex DNA) has already been initiated before CMG assembly. On the one hand, the authors make almost no mention of the human structures. The authors repeatedly refer to ‘eukaryotic’ structures, when in reality they are only referring to yeast. Given that a major selling point of this manuscript is the derivation of a generalised model of MCM-driven melting, the authors need to properly consider the implications of the human MCM structures for their models. For example, how do the authors envisage that the initial bubble of distorted DNA trapped near the N-tier of MCM (in the human structures) is converted to melted DNA in the MCM C-tier of each single hexamer, as has been demonstrated previously by the Diffey-Costa groups, as well as in this manuscript?\n• In the yeast system, the ejection of ADP from MCM and the initiation of origin melting is dependent on the conversion of MCM double hexamers into pairs of CMG helicases, driven by multiple so-called firing factors (Douglas et al., Nature, 2018). Given this, it is quite surprising that the authors can observe DNA melting by simply loading an MCM single hexamer onto double-stranded DNA in ATP S. Do the authors think this reflects a biologically meaningful difference between the ways in which melting is initiated in archaea and eukaryotes?\n• The authors prepare their samples in the slowly hydrolysable ATP analogue ATP S. Presumably the origin melting they observe should be dependent on ATP hydrolysis? What do the authors observe if they prepare their complexes in non-hydrolysable nucleotide (e.g. AMP-PNP)?\n• Although the authors are primarily using the archaeal system as a model to understand general principles of MCM function, the lack of discussion regarding the biological mechanisms of DNA replication initiation in archaea seemed an oversight. The authors should describe what is known about the various steps in MCM loading and activation (e.g. as part of the introduction). This is also relevant to point 4 (above).\nMinor points:\n• Lines 312-315: the authors note that the archaeal structure with 4 tight interfaces also a disordered subunit, potentially providing an exit route for strand exclusion. Is the same true for eukaryotic CMGE (PDB: 7QHS)? If not, does this reflect mechanistic differences between the archaeal and eukaryotic systems?\n• Why do the authors use a chimeric MCM fusion, with different domains from different archaeal species?\n• More detail should be given on the conditions (concentrations, buffers, times, temperatures) used for preparing the samples before grid preparation\n• The different coloured circles that the authors use to explain the different subunit conformations was barely visible on my printed version", "reviewer_3": "Rasouli et al have investigated the DNA unwinding ability of a hybrid archaeal MCM helicase. The authors employed double-stranded DNA for their cryo-EM analysis of the protein-DNA complexes. ATPgS was employed to slow down ATP-hydrolysis. The sample yielded multiple complex variants. Two classes were observed – one, where dsDNA is observed in the NTD and CTD, the MCM CTD and NTD are twisted , and ATO occupancy is low. The second class, with partially unwound DNA in the CTD, the MCM CTD and NTD are not twisted and nucleotide is observed in several subunits. Within each class, several variants are observed. Class 1 variants detail CTD flexibility, while class 2 variants have different numbers of nucleotides bound and unwound nucleotides. The work is exciting, as it shows the stepwise activation of an MCM hexamer. One could complain that the MCM complex is a hybrid between two different species, but I would argue that the construct is well suited to decipher a mechanism. Nevertheless, some justification should be given.\nThe structure is well described, but figures must be improved to highlight key structural changes, especially for a general audience. Also, the processing workflow is missing and the identified changes could benefit from some verification. The comparison to eukaryotic MCM is well done.\nMajor comments:\nCould the authors test their DNA unwinding model biochemically e.g. by mutating S357 or similar.\nThe authors need to provide a detailed EM data processing workflow.\nThe description of the main structural changes e.g. eclipsed vs staggered need to be illustrated by a cartoon. The vertically oriented a-helices are mentioned but not clearly shown in any figure. The structural changes need to be conceptualised and shown in the form of a cartoon.\nMinor comments:\nIn the context of the staggered and eclipsed conformation, the eukaryotic should be discussed more, here, MCM2-7 NTD/CTD undergo structural changes during the MCM2-7 hexamer/OCCM/DH/CMG transition. One review mentioned this (Riera et al 2017), but more data have been published since then.\nExtended data 7: Please show one specific example of the horseshoe/helix conformation in isolation to clarify what structural change happens. This isn't easy to see in the figure, as several elements overlap."}, "author_response": "We thank the reviewers for their careful consideration of our manuscript and suggestions for its improvement. Beyond their helpful suggestions, the reviewers raised profoundly important questions for us to consider, and our responses to these have led us to reframe the manuscript to emphasize an aromatic wedge mechanism for melting DNA that is conserved in archaea, eukaryotes, and DNA tumor viruses. We are grateful to the reviewers for pushing us to consider these questions. Key insights for this revision were obtained by calculation of RMSD of all 6 permutations of our structures (particularly Class 2b) against all eukaryotic MCM structures of the PDB. This analysis identified additional comparative structures and a consistent arrangement of the MCM ring when melting DNA that involves subunit-specific, conserved aromatic residues that we now describe as an “aromatic wedge”. Further details on the RMSD comparison are provided in specific responses below. The computer scripts and output for this analysis are provided as a supplemental archive file for interested reviewers to evaluate.\nThe reviewers had significant agreement on several points for improvement, including figure clarity, ATP site density figures, and the suggestion to incorporate human MCM among the structural comparisons. We have revised the manuscript based on these comments as detailed in our specific responses to each review below. Since the original submission, we have reversed the structural classes that we describe as “staggered” and “eclipsed.” This originally referred to the positions of vertically oriented helices of each tier, but we did not meaningfully discuss either helix. We now use “staggered” and “eclipsed” based on the positions of the underlying domains, which is more intuitively meaningful. We quantify the extent of such alignment with defined dihedral angles between the tiers and compare these to all eukaryotic MCM structures of the PDB. The computer scripts and output for this analysis are provided as a supplemental archive file for interested reviewers to evaluate.\nWe have also improved the structure of the 3 heptameric complexes by using a larger diameter focus mask during 3D classification. Although the heptameric structures are not a major focus of the manuscript, the modified 3D classification improved the DNA features of the map. The heptameric complexes have a larger diameter than the hexameric complexes, but our earlier treatment applied the same cylindrical focus mask that was used for the hexameric complexes. The EM maps for the heptameric complexes showed improved DNA density with the application of a larger diameter focus mask. The DNA appears to reside against one side of the channel rather than in its center, and this was more fully enclosed by the larger diameter focus mask. New PDB validation reports are uploaded. All other structures (each hexameric MCM and not heptameric) are unchanged from the original submission. The improved DNA density for the heptameric structure for the merged particle structure is shown below:\nFrom original submission, Fig. S8: Revised, now Fig. S15:\nResponses to the comments of each reviewer are provided below.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nRasouli, Myasnikov and Enemark report a sheer number (18) structures of a duplex DNA bound protein chimera with a fusion of the Sulfolobus MCM N-terminal oligomerisation domain and the Pyrococcus MCM ATPase motor domain. Enemark has shown previously that this protein is catalytically active. Its use in structural biology studies already provided insights that proved precious for the field over time. The current work constitutes an important addition to this body of work. Here the authors report various mode of duplex DNA engagement for a single MCM ring. While the authors propose a mechanism whereby the structural transitions in this single hexamer could lead to DNA melting, they choose not to acknowledge some recent work on the mechanism of loading of a single MCM ring, and the structures of a fully loaded single hexamer around duplex DNA, from both human and yeast MCM (though, admittedly, the latter is only a bioRxiv uploaded manuscript). The recent identification of a single duplex DNA loaded MCM in humans was a big deal (with two high profile papers published simultaneously). The discovery that this is a stable intermediate also in archaea is remarkable and makes these data worth publishing in Nature Communications. That said, a rework of the introduction and discussion is recommended, which will provide a proper framework for the discoveries reported. Specific points are detailed below.\nWe thank the reviewer for encouraging comments and enthusiasm for the consistency between the archaeal structures and eukaryotic ones. The manuscript now cites recent loading structures and eukaryotic single-hexamer:DNA structues. The central question posed by the reviewer is which structures are most appropriate for comparison. To address this objectively, we now compute an RMSD of each permutation of our structure against each eukaryotic MCM ring in the PDB. Based on this analysis, we identified two strong yeast CMG comparative structures (PDB 7PMN and 7PMK; now Fig. 6b and Fig. 6d), and we are now very confident to describe each of these structures as generating DNA melting. The RMSD analysis is detailed further in our response to point 1.\nA logical extension of this question is why encircled double-stranded DNA appears intact in some complexes with MCM and melted in others. We show structurally that melting occurs based on subunit-specific, conserved aromatic residues of MCM that we term an “aromatic wedge.” This aromatic wedge and its role in DNA melting is conserved in our archaeal MCM structures, the comparative eukaryotic CMG structures, and in a recent structure of the more distantly related SV40 Large T-antigen (PDB 9KAE, shown in Fig. 7c), which also contains AAA+ ATPase domains and forms a double-hexamer to initiate unwinding DNA for replication. We have revised the focus of the manuscript and given it a new title in providing answers to the reviewer’s incisive questions. Further details are provided in response to point 3.\nMajor issues\n1. Is it correct to define Class 2 as a DNA melting intermediate, considering the discovery from Steve Bell’s lab (Champasa et al 2019) that a yeast MCM variant that cannot homodimerise can form CMG but not open DNA? Would it not be best to compare this structure to the human MCM single hexamer (Weissmann et al 2024 and Yang et al 2024)?\nWe are grateful for these two questions. First, we are grateful for the opportunity to add the Champasa citation and improve the clarity of our manuscript. As noted by the reviewer, the Champasa et al 2019 article describes a yeast MCM variant that does not robustly form a double-hexamer of two Mcm2-7 rings but does form CMG. On the specific point on whether this variant can “open DNA”, the article shows that the variant can generate initial DNA melting, but it does not produce extensive origin unwinding. We feel that the confined DNA melting that we report for both Class 2 structures is consistent with the initial melting described in Champasa et al 2019. We have added this reference and spelled out that we feel our structures correspond to initial melting and not extensive origin unwinding. We also feel that it is correct in literal definition to describe the lost base-pair interactions of the Class 2 structures as “melting”. As described further below, disorder of nucleotides following loss of base-pair interactions is a common theme in DNA-melting structures and is observed in multiple newly added comparative structures (PDB 7PMN of Fig. 6b, PDB 7PMK of Fig. 6d, and PDB 9KAE of Fig. 7c).\nRegarding the second question on structural comparison choice, the density of PDB 7Z13 is very clear among the features that were the focus of our initial submission, and we were intrigued by its strong visual similarity with Class 2b. The reviewer’s question of whether a different structure might provide a more suitable comparison for the Class 2 structures led us to objectively verify that we were not comparing to PDB 7Z13 solely out of admiration. For this verification, we downloaded a report from the PDB with all structures meeting the search term “mcm” and a 200 kDa molecular weight cutoff. For each, we generated a hexameric MCM structure consisting of six 152-amino acid ATPase core structures (see Meagher et al IJMS 2022. doi: 10.3390/ijms232314678). The RMSD of this structure with each of the six permutations of the corresponding 6 X 152-amino acid hexameric ATPase core of Class 2b was calculated and sorted to compare the collective disposition of the six MCM ATPase domains as a unit (see Supplementary Fig. 10 for illustration of the process including the six settings of Class 2b for candidate alignments). Altogether, 77 structures were compared in 6 settings for a total of 462 comparisons. The top 30 RMSD alignments (Supplementary Table 1) were visually inspected. The alignment of PDB 7Z13 with Class 2b in the setting used for comparison in the initial submission (with chain E corresponding to Mcm3) was among the top 30, and we would like to retain this comparison in Fig. 6e.\nThis analysis also revealed two structures with striking similarity to the Class 2 structures of our manuscript that we now include among the comparisons of Fig. 6. PDB 7PMN (Fig. 6b) is similar to Class 2a with two tight interfaces (ANP-bound) and two unpaired DNA bases. PDB 7PMK (Fig. 6d) is similar to Class 2b with three tight interfaces (ANP-bound) and four unpaired DNA bases (structures from doi: 10.1038/s41586-021-04145-3). These structures align in the same setting as PDB 7Z13 with Class 2 chain E matched to Mcm3. Importantly, the structures of PDB 7PMN and PDB 7PMK were determined with a substrate that does not have an exposed 5’-end, and hence the DNA in these structures represents bona-fide melted DNA. As with the Class 2 structures that we describe, DNA melting occurred at an end of the DNA molecule, and nucleotides not bound by MCM that have lost base-pair interactions are disordered.\nNotably, 28 of the top 30 RMSD alignments are CMG structures. One loading complex that does not contain Cdc45 or GINS (PDB 9GJW) was among the top 30 and is now compared to Class 2b in Fig. 8b. This structure/alignment was similar to a structure of CMG bound to a G-quadruplex/ssDNA hybrid substrate, and this is now included in Fig. 8a.\nThe specific subunit arrangements (Table 1) of our presented structures and the comparative structures now form the basis for a sequential melting mechanism illustrated as a cartoon in Fig. 9a to correspond to the initial melting described in the Champasa et al 2019 article referred to above.\n2. The authors observe Class 2 where the leading but not the lagging strand template is visible. They interpret this as a melting intermediate. This could also reflect the fact that the leading strand is visible because it is stabilised by pore loop contacts. This is indeed the case for a helicase loading intermediate (single loaded yeast MCM hexamer) described in a bioRxiv manuscript by Miller, Diffley and colleagues. The PDB coordinates (8RIG) can be downloaded from the PDB already. This is an intermediate on the path to double hexamer formation. In the final product (the DNA is not melted).\nWe believe that interactions between the pore loops and this strand directly assist its melting from the complement. PDB 8RIG is among the structures used in the above RMSD analysis. The lowest RMSD for PDB 8RIG with Class 2b was ranked 46 (in the setting where Class 2b chain E is matched to Mcm6). As this was not among the top-30, we do not consider it as strong a match for comparison as the ones that we have selected.\n3. Along the same lines, I feel the authors should do a better job at drawing a parallel between the mechanism of MCM loading onto duplex DNA and the mechanism of DNA melting by the CMG. In this respect, I note that no reference was made to recent work published in Nature Communications by Butryn et al (2025), which describes a single-loaded yeast hexamer in different duplex DNA binding states, captured by using inactivating ATPase mutants. These structures show a DNA engagement state similar to that of the DNA-melting (or single-stranded DNA translocating) CMG, concluding that the mechanism of MCM interaction and translocation along DNA is always the same, and implying that melting occurs when two CMGs are facing one another. I feel this is another example of a missed opportunity to show how the structure of an archaeal single loaded hexamer on duplex DNA is relevant to mechanistic studies in eukaryotes (both for DNA melting, as stated in this manuscript, but also for helicase loading).\nThe central question that the reviewer raises for consideration is why MCM-encircled double-stranded DNA is intact for some complexes (such as the noted loading complexes) and melted in others (such as PDB 7Z13 or Class2b). We now discuss that we feel these differences arise from the aromatic wedge (see below) and its precise placement on DNA (its specific “setting”). We specifically favor the setting illustrated when Class 2b chain E aligns to Mcm3 (as in PDB 7Z13, and 7PMK) as a melting conformation. We note that this structural “melting conformation” appears to be especially stable because 21 of the top-30 alignments are in “setting 1”, which aligns Class 2b chain E with Mcm3. Another illustration of the stability of this form is its abundance as “staggered Form II” in the new Fig. 4.\nThis setting develops tight ATPase interfaces among the Mcm5, 2 and 6 subunits, which are precisely the subunits that stack an aromatic residue on the bases and sugars of the melted DNA. Further, these subununits specifically conserve the aromatic residue (“aromatic wedge”) while the other subunits are nearly always not aromatic (Fig. 7d; also see Supplementary Figure 3 of Meagher et al 2019). As a result, only eukaryotic Mcm2-7:DNA:ATP arrangements of Setting 1 (that match the setting of PDB 7Z13) appropriately position the aromatic residues of the 3 adjacent subunits to assist in DNA melting. Other settings do not optimally position each aromatic group for melting and therefore are not expected to melt DNA as robustly. Due to the homo-hexameric construction of the archaeal MCM hexamer, all settings would position the conserved aromatic group equivalently.\nWe previously showed that the corresponding aromatic residue in a homo-hexameric archaeal MCM (SsoMCM Y386, Meagher et al 2019, doi: 10.1038/s41467-019-11074-3) stacks on the bases and sugars of single-stranded DNA and showed that an aromatic residue is not needed in this position to unwind DNA in a strand displacement assay based on mutation to alanine (Supplementary Figure 4 of Meagher et al 2019). Notably, this mutation eliminates the aromatic group from all 6 subunits of the homo-hexamer. We further noted that the position of the aromatic group is similar to that of a conserved histidine of the related hexameric helicase E1 from papillomavirus (Compare h2i Y386 to ps1b H507 in Supplementary Figure 2 of Meagher et al 2019, doi: 10.1038/s41467-019-11074-3). This was especially intriguing to us because Arne Stenlund had previously mutated E1 H507 to 8 possible residues (A, V, L, R, M, N, F, and Y) did not prevent unwinding DNA in a strand displacement assay, but several melting and replication activities critically relied on an aromatic residue in this position (doi: 10.1016/j.molcel.2007.02.009). Of the mutants tested, only H507F supported in vivo replication in transient DNA replication assay, and only the aromatic mutants (H507F and H507Y) supported in vitro replication in a cell-free replication system. Based on permanganate reactivity, only H507F melted DNA in the T-rich region that is anticipated to be located at the E1 ATPase tier, and only H507F provided unwinding in an origin unwinding assay (doi: 10.1016/j.molcel.2007.02.009). Accordingly, we postulated (Meagher et al 2019) that the MCM h2i aromatic residue and the E1 ps1b histidine have similar roles in melting DNA but are dispensable for strand displacement during processive unwinding.\nWe now strengthen this analogy based on a recent structure of SV40 T-antigen, a close relative of E1, bound to melted DNA (PDB 9KAE, Fig. 7c). In this structure, the aromatic histidine side-chain stacks on the bases of the melted DNA similar to the aromatic residue of archaeal MCM (our Class 2 structures) and eukaryotic Mcm5, 2, and 6 (PDB 7PMN, PDB 7PMK, and PDB 7Z13). Further, the nucleotides not bound by T-antigen that have lost base-pairing interactions are disordered, analogous to the behavior of the melted DNA of our Class 2 structures and the eukaryotic CMG structures of PDB 7PMN and 7PMK.\nBased on this collective comparative analysis, we have adjusted the focus of the article and the article’s title to explicitly describe a role for an aromatic wedge in DNA melting in archaea (Class 2 structures of this manuscript), eukaryotes (PDB 7PMN, 7PMK, 7Z13), DNA tumor viruses (E1 and SV40 T-antigen, PDB 9KAE).\nSmaller issues\n1. The authors claim that no contacts exist between MCM and DNA in the class 1 structure. They should clarify how the DNA register is established such that duplex DNA can be averaged in the structures. If not contacted by the ATPase domain, duplex DNA must surely be engaged by elements of the N-terminal oligomerisation domain. Otherwise, DNA would be lost from the central channel or only result in a featureless sausage through single-particle averaging. You could refer to studies on the FANCI-FANCD2 DNA encircling ring by Alcon et al Nature 2024, Fig. 4a and Fig. 4b for comparison.\nThe DNA does not appear bound in the Class 1 structures with specific interactions. When inspecting the maps, the most prominent approach of the protein to DNA appears at one of the helix-2-insert hairpins. However, this appears too distant for a direct interaction such as a lysine-phosphate interaction. It strikes us more like a dipole-dipole interaction that might contribute to a weakly preferred orientation. During refinement and classification of the structures, the DNA did appear as a featureless sausage. We now show this in Supplementary Fig. 6 as part of the cryo-EM workflow and illustrate below:\nPrior to 3D classificaition with focus map Prior to 3D classificaition with focus map\nTo become Class 1a To become Class 1b and Class 1c\nThe DNA was subsequently subjected to 3D classification with a focus mask that encompasses the central channel. For the Class 1a structure that is essentially C6-symmetric at the protein, this classification would be expected to identify six different permutations of DNA structure. These structures would each span a 60° rotation range about the DNA helical axis if the DNA is a regular helix and its orientation is fully random. For illustrative purposes, hypothetical versions of such DNA maps were constructed by rotating a B-DNA molecule through a 360° range about its helical axis in 1° increments, and composites of the structures were used to generate maps in chimera at 6 Å resolution (for example: molmap #0-359 6). The resulting maps are depicted below and equally weighted by setting the threshold level according to the number of contributing models. Rotation of a full 360° about the B-DNA helical axis generates the featureless sausage described by the reviewer. Individual 60° classes resemble the final model, indicating that the DNA of Class 1a is likely close to undistorted B-form and that this DNA probably does not strongly interact with the MCM ring because such interaction would likely provide a more featured DNA appearance than the modest resolution of the 60° wedge examples.\nPredicted map for\nB-DNA in all helical 6 3D classes (predicted) from all orientations of B-DNA about helical axis\naxis orientations\n0-359 6 0-59 6 60-119 6 120-179 6 180-239 6 240-299 6 300-359 6\nlevel 91.139 level 15.19 level 15.19 level 15.19 level 15.19 level 15.19 level 15.19 Fig. 1a (Class 1a)\n100% 16.67% 16.67% 16.67% 16.67% 16.67% 16.67%\nIn the case of the Class 1b and 1c structures where the protein complex is asymmetric due to a disordered ATPase domain, the initial map has minor double-helix features (see above from Supplementary Fig. 6), indicating a slight orientational preference. As with Class 1a, the most prominent approach between the protein and DNA appears at one of the helix-2-insert hairpins—specifically the hairpin of a subunit adjacent to the disordered ATPase domain. However, this hairpin also appears too distant from the DNA for a direct interaction (such as a lysine-phosphate interaction). As with Class 1a, this strikes us more like a dipole-dipole interaction that might contribute to a weakly preferred orientation.\nFor these structure classes, we selected two classes from 3 using the cylindrical focus map (see Supplementary Fig. 6). For this case, a random DNA orientation can be emulated by rotating a DNA molecule through larger ranges as illustrated below where two 101-degree ranges provide hypothetical maps with discernable major and minor grooves and approximately align one DNA strand in opposite polarity as in the structures of Class 1b and Class1c. The remaining hypothetical class has a double-helix appearance without assignable major and minor grooves. Thus, the overall appearance of our structures suggest that the DNA orientations have orientation preferences that are likely very small but slightly better than random.\nPredicted map for 3 3D classes (predicted) from\nB-DNA in all helical all orientations of B-DNA about helical axis\naxis orientations\n3’ -> 5’ 5’ -> 3’\n3’ -> 5’ 5’ -> 3’\n143-243 6 101-142,244-359 6 0-100 6\n0 le - v 3 e 5 l 9 9 6 1.139 Fig. 1b (Class 1b) l 2 e 8 v . e 1 l % 25.57 l 4 e 3 v . e 9 l % 40 l 2 e 8 v . e 1 l % 25.57 Fig. 1c (Class 1c)\n100% Major/Minor double-helix Major/Minor\ngrooves No distinguishablegrooves\ndiscernable; major/minor grooves 5’-> 3’ strand\n3’->5’ strand at center of\nat center of view\nview\n2. Heptameric MCMs had been previously observed in studies by Jose’ Maria Carazo, Silvia Onesti and Laura Spagnolo. Their work should be cited.\nYes- this should be cited. Thank you for pointing this out. References #47, 74, 81, and 82 of the revised submission now refer to heptameric structures. One suggested article (Cannone et al, 2017; DOI: 10.1038/srep42019) describes an octamer rather than a heptamer.\n3. The resolution of the structures reported is high enough for discriminating nucleotides in the ATPase pockets but the authors showed not to show them. I think this should be rectified.\nWe have prepared a figure to illustrate the triphosphate regions of the maps (Fig. 2) that also demonstrates one of the intersubunit distances that we use to assign a “tight interface”. In the presented maps, the triphosphate is generally clearly visible for the tight interfaces while triphosphate density is less clear for the loose interfaces. We have modeled weaker cases as ADP molecules, but we note that we are not able to differentiate disordered g-phosphate from hydrolyzed g -phosphate, and so we emphasize that the molecule assigned in the PDB model does not indicate that hydrolysis must have occurred.\n4. Line 37 page 2 “Similar, but different subunits” sounds odd. To me, similar implies that they are different, otherwise they would be identical.\nWe have deleted “but different”\n5. Figure 1 is very similar to Extended Data Figure 2 and 3. Figure 2 appears very similar to Extended Data Figure 4 and 5. Could the authors check whether any panels have been mistakenly mispaced?\nWe have inspected all placed panels of:\nFig. 1/Supplementary Fig. 8/Supplementary Fig.9\nFig. 5/Supplementary Fig. 11/Suplementary Fig. 12\nFig. 6/Supplementary Fig. 11/Suplementary Fig. 12\nThe correct panels are placed (figure numbers above are the new figure numbers). Each are viewed from the same perspective, and the structures are highly similar. In particular, the merged particles structures are derived from the particles that generated the individual DNA species structures— and so similarity is expected. One area where differences can be perceived is in the cutaway views that illustrate encircled DNA (Rightmost panels of Fig.1/Supplementary Fig. 8/Supplementary Fig.9). The DNA1 structures have DNA order that persists further below the ring than those of DNA2. The corresponding merged particle structures show an intermediate length of order. On the other hand, the DNA2 structures are higher resolution than the corresponding structures of DNA1. The merged particle structures appeared to provide a good compromise between structure resolution and extent of DNA visualization.\n6. Page 3 line 72 “GINS recruitment provides 1.5–2 turns of DNA unwinding at each CMG” is incorrect. Douglas 2018, which the authors cite in this passage reads “each MCM hexamer untwists around 0.6–0.7 turns of DNA”.\nThank you for pointing this out. We misinterpreted a different sentence from the Douglas et al manuscript as the unwinding number (“Thus, each of the two activated CMG helicases constrains approximately 1.5-2 helical turns of unwound DNA.”). We have changed the cited number.\nReviewer #2 (Remarks to the Author):\nA large number of biochemical and cryo-EM studies in the past few years have focussed on the molecular characterisation of the initiation stages of DNA replication. Progress in this area has been significantly advanced by the ability to reconstitute the process in vitro using purified yeast proteins, and solve structures of various intermediate states. Although much is now known about this process, a complete molecular explanation of the very first stages of DNA melting is still lacking. This manuscript focusses on this key question, using the archaeal homo-hexameric MCM replicative helicase as a model system.\nThe authors present multiple cryo-EM structures of an archaeal MCM single hexamer bound around DNA. Their sample preparation method facilitates the capture of multiple different forms of the MCM-DNA complex, either containing B-form double stranded DNA, or in various states that the authors propose mimic the first stages of replication origin melting. By integrating these structures with previously published structures of archaeal and yeast MCM complexes, the authors propose a compelling model for DNA melting, involving sequential conversion of inter-subunit interfaces from loose to tight states. The manuscript is well structured and written, and clearly explains some quite complex structural conclusions and hypotheses. Further interrogation of the structural data using functional experiments (e.g. with specific mutants in vitro or in vivo) is completely lacking. Overall, however, given the high quality of the structural data and the general interest in this area of the replication field, I would suggest that the structures alone are sufficiently interesting to warrant publication without functional validation, particularly given the difficulty of such experiments in the archaeal system.\nWe thank the reviewer for supportive comments that the model is compelling and that the manuscript has achieved clarity in describing structural concepts that are potentially complex. We strived to make the writing accessible to both experts and non-experts, and so these comments are especially gratifying.\nMajor points:\n• The author’s models are heavily dependent on nucleotide-dependent transitions between loose and tight states at inter-subunit interfaces in the MCM ring. To this reviewer, it was unclear whether the authors actually had high enough resolution in their structures to model nucleotide in any of the MCM active sites? If not, this should at least be stated.\nWe have prepared a figure to illustrate the triphosphate regions of the maps (Fig. 2) that also demonstrates one of the intersubunit distances that we use to assign a “tight interface”. In the presented maps, the triphosphate is generally clearly visible for the tight interfaces while triphosphate density is less clear for the loose interfaces. We have modeled weaker cases as ADP molecules, but we note that we are not able to differentiate disordered g-phosphate from hydrolyzed g -phosphate, and so we emphasize that the molecule assigned in the PDB model does not indicate that hydrolysis must have occurred.\n• The model in Fig. 5 is dependent on sequential transitions in inter-subunit interfaces around the MCM ring. One tight interface appears to favour the conversion of the neighbouring interface from loose to tight. Can the authors explain how this would work? Is there a structural explanation for why ATP binding at one interface would, for example, favour ATP binding at the adjacent interface?\nThe directional order illustrated in Fig. 5 (now numbered Fig. 9) was originally based on the presumed directional order that the complex uses in a rotary DNA translocation mechanism (Meagher et al 2019, doi: 10.1038/s41467-019-11074-3). The DNA-binding and ATPase site differences between Class 2a and Class 2b are readily distinguished, and a structural transition from Class 2a to Class 2b would involve tightening one subunit interface that is adjacent to two consecutive tight interfaces (See Fig. 6 or Fig. 9). The tightened interface could occur at the first empty interface clockwise or counterclockwise. Considered at the ATP site level, the ATP site that is tightened could involve the Walker-A site of one of the subunits participating in tight binding or the arginine finger of one of these subunits. These scenarios are not distinguishable for archaeal homo-hexameric structures because the subunits are identical in sequence. In the absence of contrary information, we presumed that the directional order for tightening ATPase sites would be consistent with that used for rotary DNA translocation and thus occur counterclockwise in the view of Fig. 9a. The newly added comparative structures of PDB 7PMN and 7PMK definitively resolve the directional order because they derive from heterohexameric structures with distinguishable subunits. These structures illustrate that the tightened interface is at the counterclockwise side (3/5 interface tightened rather than 6/2), and hence the directional order of ATP-binding during melting is indeed identical to the directional order used for DNA translocation.\n• Recent structures have demonstrated that a small section of DNA, positioned at the inter-hexamer interface, is already distorted in the human MCM double hexamer (e.g. PDB:7W1Y). Thus, unlike in yeast, melting (or at least distortion of the duplex DNA) has already been initiated before CMG assembly. On the one hand, the authors make almost no mention of the human structures. The authors repeatedly refer to ‘eukaryotic’ structures, when in reality they are only referring to yeast. Given that a major selling point of this manuscript is the derivation of a generalised model of MCM-driven melting, the authors need to properly consider the implications of the human MCM structures for their models. For example, how do the authors envisage that the initial bubble of distorted DNA trapped near the N-tier of MCM (in the human structures) is converted to melted DNA in the MCM C-tier of each single hexamer, as has been demonstrated previously by the Diffey-Costa groups, as well as in this manuscript?\nThe reviewer raises a good point to enhance the generality of eukaryotic comparison, and we have added PDB 7W1Y to what is now Fig. 5.\nThe 7W1Y to 7Z13 transition has been very intriguing to ponder, and we are grateful to the reviewer for providing the motivation to do this. PDB 7W1Y is a highly informative structure that beautifully illustrates the structure of a human double-hexamer encircling DNA in a conformation poised to proceed to replication initiation and provides exquisite resolution for the constituent components. We do not intend our response below to the reviewer’s question about the N-tier bubble to diminish the remarkable significance or technical achievement of the PDB 7W1Y structure.\nThe bubble at the N-tier of PDB 7W1Y seems to at least partly derive from the double-hexamer architecture because the corresponding single-hexamer structures (PDB 8S0A and PDB 8W0E) are quite similar in underlying hexamer:DNA arrangement, but they but do not exhibit melted DNA at the N-tier. The N-tier bubble in PDB 7W1Y appears to derive from simultaneous interaction of each constituent hexamer C-tier with DNA in conjunction with a double-hexamer interaction that enforces a greater distance between the C-tiers than undistorted B-DNA allows. If any of these three interactions are lost, the additional degrees of freedom potentially will allow the DNA to re-anneal. For multiple arguments below, we feel that at least one of the hexamers of PDB 7W1Y must release its grip on DNA (while continuing to encircle DNA) and that such release would generally permit the N-tier DNA bubble to re-anneal.\nFirst, one of the initial steps towards replication initiation is exhaustion of nucleotides from the MCM ATPase sites. Based on several MCM:DNA structures and arguments presented in this manuscript, we fundamentally associate DNA-interaction with the presence of nucleotides at the corresponding ATPase sites to create a “tight” interface to appropriately position DNA-binding modules of adjacent subunits for binding DNA (now illustrated in Fig. 3 that demonstrates helix versus horseshoe). Exhaustion of all nucleotides from the MCM ATPase sites is expected to convert all interfaces to “loose” with concurrent loss of interactions with DNA (analogous to the Class 1a structure that we present, see Fig. 5). Hence, exhaustion of nucleotides is predicted to release the grip of both hexamers on DNA. Although the hexamers would continue to encircle DNA, the lost grip would likely allow the N-tier bubble to re-anneal.\nSecond, a fundamental global difference between the 7W1Y and 7Z13 structures strongly suggests that at least one hexamer dissociates its grip on DNA in order to transition between the two arrangements. Each structure is effectively 2-fold symmetric with a hexamer:hexamer dyad co-aligned with a DNA dyad, but in opposite directions. The 7W1Y dyads place the DNA major groove at the Mcm3:Mcm3’ side while the 7Z13 dyad has the DNA minor groove at the Mcm3:Mcm3’ side (see below). Interconversion of these forms very likely involves at least one hexamer losing its grip on DNA. The forms can interconvert by either: 1) rotating each hexamer 180º about the DNA helical axis (both rotating the same direction or each rotating in opposite directions); 2) by sliding the hexamers one-half helical turn along the helical axis; or 3) by a combination of rotation and slide of the hexamers with respect to the DNA helical axis. In each scenario, at least one hexamer would need to disengage is binding grip on DNA (detailed below), which in turn would likely allow the N-tier bubble to re-anneal.\nA more detailed explanation that at least one MCM hexamer would need to release its grip on DNA can be viewed with the DNA-binding register of the two complexes (see illustration below). For this comparison, the author-provided nucleotide numbers are provided along with the number of nucleotides from the DNA dyad, which provides a consistent reference for comparison. As illustrated, nucleotides at dyad+16 and dyad+17 are bound at Mcm7 in PDB 7W1Y, but dyad+16 and dyad+17 are bound at Mcm3 and Mcm5 and PDB 7Z13. Two scenarios are possible with this difference: 1) the DNA disengages binding to Mcm7 and re-engages binding at Mcm5 and 3; or 2) the DNA dyad that aligns to the double-hexamer dyad changes during the transformation (e.g. the midpoint of the two MCM hexamers translates with respect to DNA). The protein disengagement of Scenario 1 implicitly provides a period where hexamers lose their grip on DNA to likely allow a capacity for the N-tier bubble to re-anneal.\nFor the second scenario, we feel it could be possible for the MCM double-hexamer to translate along the DNA axis based on the translocation activity of one hexamer. In this action, an active translocating hexamer could maintain its grip on DNA, but the second hexamer would need to release its grip on DNA and passively slide (perhaps viewed as being pushed by the first hexamer). This scenario therefore also involves at least one hexamer losing its grip on DNA and thus provides the capacity for the N-tier bubble to re-anneal.\n3’ 5’ 3’ 5’ PDB 7W1Y\n2 register\n5 6\nMcm3 Mcm3\nS/-9\n(dyad+16)\n(dyad+17)\nMajor Dyad = Minor Dyad = 7 4\nGroove Groove\nS/-25 - O/25 A/27 - B/27\nside side\nPDB 7Z13 register 2\nA/46\n(dyad+19) 6\nA/45 5\nMcm3’ Mcm3’ (dyad+18)\nA/44\n(dyad+17)\nA/43\n3’ 5’ 3’ 5’ (dyad+16)\nPDB 7W1Y Mcm3:Mcm3’ at major groove side PDB 7Z13 Mcm3:Mcm3’ at minor groove side A/42 3 4\n(dyad+15) 7\nWith a likely loss of at least one hexamer:DNA contact, it strikes us as too demanding for the replication initiation mechanism to require that the N-tier bubble must not re-anneal in order for replication initiation to be successful. Once the two hexamers have separated, we feel that it would not be possible to regenerate this N-tier bubble if these nucleotides were to re-anneal unless the overall double-hexamer is regenerated. In contrast, if melting is based on an aromatic wedge within single hexamers (as in our structures and in PDB 7PMN and PDB 7PMK), melting and re-annealing can potentially occur repeatedly prior to successful advancement to extended origin unwinding and replication initiation.\n• In the yeast system, the ejection of ADP from MCM and the initiation of origin melting is dependent on the conversion of MCM double hexamers into pairs of CMG helicases, driven by multiple so-called firing factors (Douglas et al., Nature, 2018).\nGiven this, it is quite surprising that the authors can observe DNA melting by simply loading an MCM single hexamer onto double-stranded DNA in ATPgS. Do the authors think this reflects a biologically meaningful difference between the ways in which melting is initiated in archaea and eukaryotes?\nWe believe that melting at the MCM ATPase domain occurs fundamentally the same way in archaea and eukaryotes and is derived from an aromatic wedge that is now a central focus of the manuscript. All eukaryotic structures of MCM with DNA melted at the MCM ATPase domain are CMG structures. The aspect that the reviewer likely finds surprising is that the Class 2 structures reported in our manuscript do not have Cdc45 or GINS but nevertheless exhibit melting. Our newly added RMSD-based structure comparisons indicate that the Class 2 structures with melted DNA most closely resemble eukaryotic CMG structures. Of the top-30 scoring RMSD eukaryotic comparative structures, 28 are CMG structures. Thus, it appears to us that although the Class 2 structures do not possess Cdc45 or GINS, they somehow structurally mimic a CMG arrangement for the collective ATPase tier.\n• The authors prepare their samples in the slowly hydrolysable ATP analogue ATPgS. Presumably the origin melting they observe should be dependent on ATP hydrolysis? What do the authors observe if they prepare their complexes in non-hydrolysable nucleotide (e.g. AMP-PNP)?\nWe ascribe the different conformations observed to whether/how ATPgS is bound at the respective ATP sites. We believe that the limited melting in our Class 2 structures (and in PDB 7PMN and PDB 7PMK, which are both with AMP-PNP) is based on ATP-binding and that hydrolysis is not necessary. This is not processive DNA unwinding that would require ATP hydrolysis in a rotary cycle.\n• Although the authors are primarily using the archaeal system as a model to understand general principles of MCM function, the lack of discussion regarding the biological mechanisms of DNA replication initiation in archaea seemed an oversight. The authors should describe what is known about the various steps in MCM loading and activation (e.g. as part of the introduction).\nWe have added additional details to the introduction about replication initiation in archaea.\nMinor points:\n• Lines 312-315: the authors note that the archaeal structure with 4 tight interfaces also a disordered subunit, potentially providing an exit route for strand exclusion. Is the same true for eukaryotic CMGE (PDB: 7QHS)? If not, does this reflect mechanistic differences between the archaeal and eukaryotic systems?\nIn order to focus on an aromatic wedge melting mechanism, we have removed the description of an archaeal MCM ring encircling ssDNA where one ATPase domain is disordered. However, to answer the reviewer’s question, we believe that the mechanism that the lagging strand exits the ring is not well-understood in any system, and we think this represents a highly intriguing question. Without contrary information, we presume that archaea and eukaryotes use similar mechanisms for such strand exit. The open interface of the 4-tight interface structural complex is an intriguing potential candidate for part of how a strand might exit. An analogous eukaryotic structure with a mobile ATPase domain has not been described to our knowledge, but may exist. We now only briefly refer to the 4-tight interface structure in a short lagging strand exit section.\n• Why do the authors use a chimeric MCM fusion, with different domains from different archaeal species?\nWe first generated this chimeric fusion to investigate its ability to unwind DNA in a strand displacement assay. We empirically found it to be extremely robust as a hexamer and active for DNA-unwinding in a strand displacement assay. The construct also proved very useful for structural studies because we were able to determine a crystal structure of the MCM hexamer (Miller et al 2014, doi: 10.7554/eLife.03433). This structure was cited as the template model in the first high-resolution cryo-EM structure of eukaryotic Mcm2-7 (Li et al 2015, doi: 10.1038/nature14685). In over 10 years of work with several archaeal MCM constructs for structural studies by X-ray crystallography, we have only successfully crystallized two constructs in a hexameric form that contains both the N-terminal domain and the AAA+ domain: this Sso-PfMCM chimera and a modified SsoMCM construct (Meagher et al 2019, doi: 10.1038/s41467-019-11074-3). We have since used cryo-EM to identify and analyze additional MCM:DNA forms for both constructs, and these allow direct comparison with the original crystal structures. We previously reported cryo-EM structures of the SsoMCM construct encircling single-stranded DNA, which allowed us to identify additional states of DNA translocation (Meagher et al IJMS 2022. doi: 10.3390/ijms232314678). In the current manuscript, we were motivated to elucidate how the archaeal MCM hexamer interacts with encircled double-stranded DNA, something we have never been able to capture by X-ray crystallography.\n• More detail should be given on the conditions (concentrations, buffers, times, temperatures) used for preparing the samples before grid preparation\nWe have added experimental details for protein purification and for generation of the sample of cryo-EM analysis. A comprehensive cryo-EM workflow is now provided as Supplementary Fig. 1-7.\n• The different coloured circles that the authors use to explain the different subunit conformations was barely visible on my printed version\nWe have removed the colored circles from the main figures and have added dashed outlines to highlight structural features.\nReviewer #3 (Remarks to the Author):\nRasouli et al have investigated the DNA unwinding ability of a hybrid archaeal MCM helicase. The authors employed double-stranded DNA for their cryo-EM analysis of the protein-DNA complexes. ATPgS was employed to slow down ATP-hydrolysis. The sample yielded multiple complex variants. Two classes were observed – one, where dsDNA is observed in the NTD and CTD, the MCM CTD and NTD are twisted , and ATO occupancy is low. The second class, with partially unwound DNA in the CTD, the MCM CTD and NTD are not twisted and nucleotide is observed in several subunits. Within each class, several variants are observed. Class 1 variants detail CTD flexibility, while class 2 variants have different numbers of nucleotides bound and unwound nucleotides. The work is exciting, as it shows the stepwise activation of an MCM hexamer. One could complain that the MCM complex is a hybrid between two different species, but I would argue that the construct is well suited to decipher a mechanism. Nevertheless, some justification should be given.\nThe structure is well described, but figures must be improved to highlight key structural changes, especially for a general audience. Also, the processing workflow is missing and the identified changes could benefit from some verification. The comparison to eukaryotic MCM is well done.\nWe thank the reviewer for supportive comments. In particular, the reviewer’s comment that the comparison to eukaryotic MCM is well-done is gratifying because we strived to create a cogent comparison to eukaryotic MCM. We feel the revised manuscript has strengthened this comparison.\nWe first generated this chimeric fusion to investigate its ability to unwind DNA in a strand displacement assay. We empirically found it to be extremely robust as a hexamer and active for DNA-unwinding in a strand displacement assay. The construct also proved very useful for structural studies because we were able to determine a crystal structure of the MCM hexamer (Miller et all 2014, doi: 10.7554/eLife.03433). In over 10 years of work with several archaeal MCM constructs for structural studies by X-ray crystallography, we have only successfully crystallized two constructs in a hexameric form that contains both the N-terminal domain and the AAA+ domain: this Sso-PfMCM chimera and a modified SsoMCM construct (Meagher et al 2019, doi: 10.1038/s41467-019-11074-3). We have since used cryo-EM to identify and analyze additional MCM:DNA forms for both constructs, and these allow direct comparison with the original crystal structures. We previously reported cryo-EM structures of the SsoMCM construct encircling single-stranded DNA, which allowed us to identify additional states of DNA translocation (Meagher et al IJMS 2022. doi: 10.3390/ijms232314678). In the current manuscript, we were motivated to elucidate how the archaeal MCM hexamer interacts with encircled double-stranded DNA, something we have never been able to capture by X-ray crystallography.\nMajor comments:\nCould the authors test their DNA unwinding model biochemically e.g. by mutating S357 or similar.\nThe reviewer raises an interesting point about S357 because this residue appears to play a prominent role in two aspects of the sequential melting that our manuscript describes—both in the development of tight interfaces and in DNA-binding among the subunits that adopt a tight interface. We have previously tested unwinding activities for mutants of the related archaeal SsoMCM (see Supplementary Figure 4 and Table 2 of Meagher et al 2019, doi: 10.1038/s41467-019-11074-3) as part of a structure-function report on DNA translocation and unwinding. The structure from the earlier report is now illustrated in Fig. 7a for comparison. Although the present manuscript is on the topic of melting rather than processive DNA unwinding, SsoMCM T369A mutants of the helix-2-insert hydroxyl group (T369 corresponds to S357 of the present structure, see Fig. 7a-b) are consistent with a role for this residue to function with neighboring subunits. In our 2019 manuscript, mutational analysis was performed in two contexts- 1) wild-type SsoMCM; and 2) a construct that lacks the C-terminal HTH domain (that is dispensable for unwinding) and incorporates a shortened linker between the N-terminal domain and the C-terminal domain (SsoMCM-GGSGGS-ΔC). In the absence of mutations, the modified construct showed much stronger cooperativity for unwinding than wild-type (h=6.610 ± 2.030 versus h=1.160 ± 0.2619). Among mutants tested, the T369A mutant was unique in that it could not be expressed and purified in the full-length protein context. The T369A mutant could be isolated in the more cooperative SsoMCM-GGSGGS-ΔC context, but with a vastly reduced cooperativity (h=0.7792 ± 0.2188). Collectively, the results indicate a role for T369 in the cooperative function of MCM ATPase subunits and that added cooperativity of the SsoMCM-GGSGGS-ΔC context may partly compensate for the lost cooperativity upon mutation of residue 369 to alanine. We have added a description of the reduced cooperativity of SsoMCM-GGSGGS-ΔC T369A to the current manuscript.\nThe authors need to provide a detailed EM data processing workflow.\nWe have added a detailed workflow with intermediate images as Supplementary Fig. 1-7.\nThe description of the main structural changes e.g. eclipsed vs staggered need to be illustrated by a cartoon. The vertically oriented a-helices are mentioned but not clearly shown in any figure. The structural changes need to be conceptualised and shown in the form of a cartoon.\nWe attempted to mark the vertically oriented helices with partially transparent circles, and these clearly did not work well. Reviewer 2 also noted that these are barely visible. We now provide a staggered/eclipsed cartoon in Fig. 4. We have changed the fundamental description of eclipsed versus staggered to not focus on the vertical helices. The vertical helices were prominently different to us during structure determination, and we used them to distinguish the forms. The revised description focuses on overall domain positions, which are more mechanistically meaningful. The domain positions are also amenable to the dihedral calculations of Fig. 4. We feel that the quantified dihedrals are more meaningful and interpretable and allow comparison with heterohexameric eukaryotic Mcm2-7 complexes.\nMinor comments:\nIn the context of the staggered and eclipsed conformation, the eukaryotic should be discussed more, here, MCM2-7 NTD/CTD undergo structural changes during the MCM2-7 hexamer/OCCM/DH/CMG transition. One review mentioned this (Riera et al 2017), but more data have been published since then.\nThis reviewer comment greatly strengthened the manuscript because it prompted the dihedral analysis of Fig. 4 to explore the scope of inter-tier conformations among eukaryotic Mcm2-7 structures. The reviewer is correct about the suggested analogy—thank you for pointing this out. We examined the inter-tier rotation of all 77 eukaryotic Mcm2-7 structures of the PDB used for the above RMSD calculations of the AAA+ hexamer. The inter-tier rotation was quantified by dihedral angles defined by consistent positions of the N-tier OB fold, the C-tier AAA+ fold, and two positions of the ring central channel. Five of the eukaryotic models were then excluded (8RWV, 6SKO, 8q6P, 8s0e, 8s0f) because they do not have a full set of 6 OB-folds and 6 AAA+ domains in the model. The 72 models remaining have two obviously distinct categories that we now classify as eclipsed (mean dihedral: -6 to -15) and staggered (mean dihedral: -29 to -38). The review article mentioned by the reviewer (Riera et al 2017) ascribes a major role for Cdt1 in generating a different conformation for OCCM than in DH or CMG. The dihedral analysis is fully consistent with such a role because 100% of the structures with Cdt1 are classified as eclipsed based on the dihedrals. We have added a plot of the complete dihedral analysis for each subunit of the 72 analyzed structures in Fig. 4. A movie illustrating the general theme of eclipsed and staggered among the presented structures and some eukaryotic structures is presented as Supplementary Movie 1.\nExtended data 7: Please show one specific example of the horseshoe/helix conformation in isolation to clarify what structural change happens. This isn't easy to see in the figure, as several elements overlap.\nWe now illustrate the helix-horseshoe conformations for three of our structures in Fig. 3, highlighting the specific regions with dashed outline. These figures also illustrate an intersubunit distance that correlates the helix conformation. Along with the ATPase site distance of Fig 2, these two distances define whether we assign subunit interfaces as “tight” (Table 1)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed all of my comments satisfactorily and I recommend that this manuscript is published in Nature Communications.", "reviewer_2": "I am satisfied that the authors have addressed my concerns. I recommend publication.", "reviewer_3": "The authors have done an excellent job, and I am happy with the revised manuscript."}, "author_response": ""}]

Decision letter