CryoEM structures of human CMG - ATP

S - DNA and CMG - AND-1 complexes

Neil J Rzechorzek*, Steven W Hardwick, Vincentius A Jatikusumo, Dimitri Y Chirgadze & Luca

Pellegrini*

Department of Biochemistry, Tennis Court Road, Cambridge CB2 1GA, UK

* To whom correspondence should be addressed. Email: njr48@cam.ac.uk, lp212@cam.ac.uk

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DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS

(CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of

DNA unwinding and replisome interactions remain poorly understood. Here we report

the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue

ATP

S. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of

MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the

5′-end to MCM5 at the 3′-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full

set of interactions. DNA binding correlates with nucleotide occupancy: five MCM

subunits are bound to either ATP

S or ADP, whereas the apo MCM2-5 interface remains

open. We further report the cryoEM structure of human CMG bound to the replisome

hub AND-1 (CMGA). The AND-1 trimer uses one

-propeller domain of its trimerisation

region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the

resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA

while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to

recruit partner proteins.

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Accurate and faithful duplication of our chromosomal DNA in preparation for mitosis is

essential for cellular life (Michael O'Donnell, Langston, & Stillman, 2013). DNA synthesis in

S-phase is a highly complex biochemical process carried out by the replisome, a large and

dynamic multi-protein assembly of about thirty core components (Bell & Labib, 2016). The

replisome contains all necessary enzymatic activities for copying the genetic information

encoded in the parental DNA, as well as non-enzymatic factors that guarantee efficient DNA

synthesis under normal conditions and during replicative stress.

Central to the replication process is the physical separation of the parental strands of DNA, to

allow the templated polymerisation of new leading and lagging strands, according to the semi-

discontinuous model of DNA replication. Replicative DNA helicases form hexameric rings

that thread single-stranded DNA through their ring channel and achieve unwinding of double-

stranded (ds) DNA by a process of strand exclusion (Lyubimov, Strycharska, & Berger, 2011;

Michael E O'Donnell & Li, 2018). Each helicase subunit consists of an N-terminal domain and

a C-terminal ATPase domain, which form a double stack of N-tier and C-tier rings. ATP

binding takes place at the subunit interface and its hydrolysis requires residues from both

subunits. Processive strand separation results from the allosteric coupling of ATP hydrolysis

to concerted movements of the DNA-binding elements that line the ring pore in each subunit,

as first shown for the replicative viral E1 DNA helicase (Enemark & Joshua-Tor, 2006) and

the hexameric Rho RNA helicase (Thomsen & Berger, 2009).

Unwinding of parental DNA in eukaryotic cells is performed by the 11-subunit Cdc45-MCM-

GINS assembly or CMG (Moyer, Lewis, & Botchan, 2006). The six MCM proteins, MCM2-

7, belong to the AAA+ family of ATPases and form a hetero-hexameric ring that translocates

on single-strand DNA (Abid Ali & Costa, 2016). In the absence of DNA substrate, MCM2-7

adopts predominantly an open spiral conformation; the co-factors Cdc45 and GINS — a hetero-

tetramer of Psf1-3 and Sld5 — bind to the MCM5-2 interface and lock MCM2-7 into the

closed-ring conformation required for robust DNA unwinding (Costa et al., 2011). In vivo, a

multi-step process operates to assemble and activate the eukaryotic CMG DNA helicase at the

start of S-phase. In the model system budding yeast, CMG activation involves loading of the

MCM2-7 proteins at origin DNA as an inactive double hexamer, which is then activated by

phosphorylation-dependent recruitment of the Cdc45 and GINS cofactors, intervention of

MCM10, and ATP hydrolysis (Douglas, Ali, Costa, & Diffley, 2018). Activation yields two

CMG assemblies that segregate on opposite template DNA strands and move past each other

to establish two independent replication forks (Georgescu et al., 2017). The translocating CMG

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tracks along the leading-strand template in the 3′-to-5′ direction (Fu et al., 2011), with the N-

tier ring of MCM2-7 at the leading edge of the advancing helicase (Georgescu et al., 2017).

Strand separation is proposed to be achieved by a modified version of steric exclusion, whereby

the lagging strand penetrates the N-tier of the CMG before separation (Langston & O'Donnell,

2017).

The mechanism of translocation by which the CMG couples ATP hydrolysis to processive

DNA unwinding is the current focus of intense research efforts. Based on structural analysis of

bacteriophage, viral and bacterial systems (Enemark & Joshua-Tor, 2006; Gao et al., 2019;

Itsathitphaisarn, Wing, Eliason, Wang, & Steitz, 2012; Singleton, Sawaya, Ellenberger, &

Wigley, 2000) a consensus has emerged for a sequential rotary mechanism of DNA unwinding

by replicative DNA helicases. In this mechanism, ATP is sequentially hydrolysed by

successive ring subunits so that each ring position cycles through ATP, ADP and apo states. In

turn, the ATP state determines allosterically the position of the DNA-binding loops, that adopt

a staircase arrangement matching the DNA spiral bound within the ring pore. The sequential

hydrolysis of ATP around the ring causes the coordinated motion of the DNA-binding loops,

resulting in translocation of the DNA substrate through the ring.

A complicating feature when trying to analyse CMG translocation is that, unlike the homo-

hexameric helicases of simpler organisms, the MCM2-7 motor of the CMG is a hetero-hexamer

of six related but distinct subunits (Bochman, Bell, & Schwacha, 2008). Indeed, biochemical

measures of DNA unwinding by purified fly CMG showed that ATP binding and hydrolysis

are not equally important at all MCM ring interfaces (Eickhoff et al., 2019; Ilves, Petojevic,

Pesavento, & Botchan, 2010). Furthermore, biological evidence in yeast shows that the

importance of DNA binding is different among MCM subunits (Lam et al., 2013; Ramey &

Sclafani, 2014). Recent cryoEM analyses of yeast CMG have led to the proposal of alternative

translocation mechanisms, based on ‘pumpjack’ or ‘inchworm’ movements of the N- and C-

tier of the MCM ring (Abid Ali et al., 2016; Yuan et al., 2016). A recent structural study of the

fly CMG in conditions of DNA-fork unwinding (Eickhoff et al., 2019) imaged four distinct

states of the helicase; the states formed the basis for an asymmetric model of DNA unwinding

that accounted for the different roles of the MCM2-7 subunits in translocation.

The critical insights provided by these initial landmark studies have not been sufficient to settle

the important issue of the mechanism of DNA translocation by the CMG, and therefore further

structural investigations are needed. It is especially important to obtain high-resolution cryoEM

maps that will allow the determination of accurate atomic models of the helicase bound to fork

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DNA substrates, to elucidate unambiguously key aspects of the mechanism of translocation on

DNA such as the protein-DNA interface and the geometry of the ATP-binding sites. Equally

important is to obtain high-resolution information on the interactions of the CMG with other

core replisome components. Furthermore, published structural analyses focused on CMGs

from simpler model systems such as yeast or Drosophila, and no structural evidence is currently

available for vertebrate CMG.

Here we report the cryoEM structure at 3.3 Å of human CMG bound to a fork DNA substrate

in the presence of ATPgS. We also present the cryoEM structure of human CMG bound to

AND-1, a core replisome component that acts as a platform for recruitment of replisome

components to the replication fork. Unique features captured in our structures provide insights

into DNA translocation and formation of larger replisome assemblies by the human CMG

helicase.

RESULTS

Expression and purification of human CMG-ATP

S-DNA

To maximise our chances of producing correctly-assembled human CMG, we used transient

transfection of suspension-free HEK293 cells with a plasmid system encoding all 11 subunits

of the CMG assembly. After co-expression of MCM2-7, Cdc45 and GINS, human CMG was

purified by Ni

- and Streptactin-affinity chromatography (Supplementary figure 1A). A

large endogenous protein that co-purified at sub-stoichiometric levels with the CMG over the

two-step purification was identified as AND-1, a known replisome component. AND-1 co-

purification indicates a tight constitutive association with the CMG in the human replisome, in

agreement with the known association of AND-1’s orthologue Ctf4 with the yeast CMG

(Gambus et al., 2006).

To capture a high-resolution snapshot of human CMG poised to translocate on a fork DNA

substrate, we decided to use the ATP analogue ATPgS. Streptactin-bound CMG was incubated

with buffer containing a fork DNA substrate and ATPgS before elution with desthiobiotin

(Supplementary figure 1B). The DNA consisted of a 40 bp duplex region with 30 nt tails and

resembled closely a fork DNA that had been designed to measure CMG’s helicase activity,

with a 3¢ polydT tail for helicase loading in the correct orientation for fork unwinding and a 5¢

GC-rich tail that inhibits helicase binding (Petojevic et al., 2015).

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CryoEM data were collected on a Titan Krios operating at 300 keV using a K2 Summit detector

and processed with Relion-3 (Scheres, 2012). After 2D and 3D classification and refinement,

we obtained a 3.29 Å map of CMG-DNA-ATPgS from a set of 213,527 particles, and a 3.41 Å

map of the CMG C-tier, comprising the ring of AAA+ ATPase domains, after masking of the

N-tier (Supplementary figures 2 and 3). Both maps were used to build a molecular model of

CMG--ATPgS-DNA. The excellent quality and high resolution of the map allowed an accurate

description at atomic level of the protein-DNA interface and ATP-binding sites of the human

CMG (Figure 1A).

Overall structure

The 11-subunit assembly of the human CMG shows the familiar architecture first demonstrated

for the yeast and drosophila CMG (Costa et al., 2011; Georgescu et al., 2017): a two-tiered ring

of MCM2-7 proteins, with the Cdc45 and GINS coactivators bound together to the N-tier

portion of MCM2, MCM5 and MCM3, so that Cdc45 faces the MCM2-5 interface of ATPase

domains (Figure 1B). Each AAA+ ATPase domain contains a nucleotide-binding site at the

subunit interface in the C-tier ring, and interacts with DNA via two b-hairpin loops named pre-

sensor-1 (PS1) and helix-2 insert (H2I) (Iyer, Leipe, Koonin, & Aravind, 2004) that line the

pore of the C-tier ring (Supplementary figure 4A). Flexible anchorage between MCM

subunits is provided by a domain-swapped helix in each ATPase domain, which tethers each

MCM to its neighbour subunit (Supplementary figure 4B).

A continuous chain of eleven thymidine nucleotides is bound in a right-handed B-form spiral

within the C-tier channel of ATPase domains. The single-stranded (ss) DNA contacts all 6

MCM subunits, from MCM2 with its 5′-end to MCM5 with the 3′-end, and thus traverses all

MCM interfaces except MCM2-5 (Figure 2A, B). No clear density is visible within the N-tier

of the CMG for either single-stranded DNA or the double-strand portion of our fork DNA

substrate. The likely explanation for this observation is that the slowly-hydrolysable ATPgS

nucleotide has permitted the engagement of the CMG helicase with the leading-strand portion

of the fork DNA substrate but prevented its translocation to the ss-dsDNA nexus.

Three of the six MCM interfaces in the ring: MCM6-4, MCM4-7 and MCM7-3, are bound to

ATPgS, whereas the MCM3-5 and MCM2-6 interfaces contain ADP as product of ATPgS

hydrolysis, while the MCM2-5 interface is empty (Figure 2B). In accordance with the apo

status of MCM5, the MCM2-5 gate remains ajar and the MCM2-7 C-tier ring adopts a shallow

right-handed spiral conformation (Supplementary Figure 5).

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In addition to their N- and C-tier domains, each MCM subunit contains a smaller C-terminal

winged helix (WH) domain. The WH domains of MCM2 and MCM6 were well resolved in

the focused C-tier map and could be therefore be modelled in the density (Figure 1B). Density

for the WH domain of MCM5 could be identified in the lumen of the C-tier pore, but was not

of sufficient quality to allow modelling. The similar MCM2 and 6 WH domains sit on the rim

of the C-tier and interact with each other with approximate two-fold symmetry. The first 14

proline-rich amino acids of the MCM3 isoform used in our study bind at the interface between

the MCM3 N-tier and the GINS Psf3, likely extending and stabilising the GINS-MCM ring

interface (Supplementary figure 6).

DNA binding

In the structure, the ssDNA is embedded within the pore of the C-tier ring (Figure 2A). Nine

of the eleven nucleotides from the 3′-end of the ssDNA adopt a right-handed spiral

conformation that follows closely that of B-form DNA. Four MCM subunits, MCM6, 4, 7 and

3 make an identical set of contacts with DNA, involving both PS1 and H2I loops. MCM6, 4, 7

and 3 interact with four nucleotides each, with a two-nucleotide offset between contiguous

subunits (Figure 2B). The DNA-binding loops in are arranged in a staircase matching the DNA

spiral, from MCM2 at the top of the staircase (5′-end of the DNA) to MCM5 at the bottom

(3′-end) (Figure 2C). DNA binding correlates with nucleotide occupancy, as nucleotide-bound

MCM6, 4, 7 and 3 make a full set of interactions with DNA.

Within each four-nucleotide footprint, an invariant serine at the start of loop H2I (MCM6 S425,

MCM4 S539, MCM7 S410, MCM3 S419) is hydrogen bonded to the 5′-terminal phosphate,

whilst an invariant lysine in loop PS1 (MCM6 K486, MCM4 K600, MCM7 K471, MCM3

K480) is ion paired to the subsequent phosphate (Figure 3 and Supplementary figures 7, 8A).

The role of the invariant PS1 lysine is remarkably similar to that of K506 of the E1

papillomavirus replicative DNA helicase (Enemark & Joshua-Tor, 2006). The ssDNA is kept

in close contact with each MCM subunit by two hydrogen bonds between phosphates of the

second and third nucleotide in each binding site and main-chain nitrogens of the PS1 residue

after the invariant lysine and of a first-strand residue in the H2I hairpin (Figure 3).

Besides these polar contacts, the protein-DNA interface has substantial hydrophobic character:

small aliphatic side chains of valine and alanine in both H2I and PS1 loops pack against the

ribose-phosphate backbone of the DNA, creating a continuous hydrophobic surface in the C-

tier pore that matches the spiral of the DNA (Figure 3). In addition to making extensive

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contacts with the DNA backbone, the MCM subunits use the H2I loop to interact with the

bases: a pair of conserved H2I residues, consisting of a basic and an aromatic/hydrophobic

amino acid six residues apart ([+]x

[W/Y] motif; +, basic; W, aromatic; Y, large aliphatic)

contact the third and fourth thymidine in each binding site (Figure 3 and Supplementary

figure 7).

The B-form spiral of the DNA is interrupted at the MCM6-DNA interface. MCM6 F442 —

the aromatic residue in the [+]x

[W/Y] motif — unstacks the second and third nucleotide from

the 5′-end of the DNA by inserting its side chain between the thymine bases (Figure 3). The

disruption of B-form DNA is reinforced by the equivalent residue in MCM2, W569, that

engages in a similar interaction, by stacking against the base of the 5′-end thymidine (Figure

3). All MCMs, with the exception of MCM3, have either an aromatic or a bulky hydrophobic

residue at this position, suggesting that they can all in principle engage in a similar interaction

(Supplementary figure 7). The position of MCM6 F442 at the MCM-DNA interface is

reminiscent of that of H507 in the DNA-binding loop of the viral E1 helicase (Enemark &

Joshua-Tor, 2006). As proposed for E1 H507 (Liu, Schuck, & Stenlund, 2007), the

aromatic/hydrophobic residue of the [+]x

[W/Y] motif might have an additional or alternative

role at an earlier stage in replication, by helping drive melting of origin DNA.

Correct positioning of the invariant serine at the start of the H2I loop for interaction with the

phosphate backbone of the DNA requires adoption of a helical conformation by the five

residues succeeding the serine (H2I a

; Figure 3 and Supplementary figure 7). This local

helical folding is driven by anti-parallel b-strand pairing of the two residues preceding the

serine with the second b-strand of the PS1 loop in the preceding MCM subunit. This inter-

subunit interaction helps merge the H2I and PS1 loops of individual MCMs into a continuous

DNA-binding staircase that extends around the pore in the C-tier, as noted recently for the

archaeal homo-hexameric MCM ring (Meagher, Epling, & Enemark, 2019). As expected for

the MCM subunit at the bottom of the staircase, H2I a

is disordered in MCM5.

ATP binding and hydrolysis

In the structure, five of the six ATP-binding sites in the MCM2-7 ring are occupied by a

nucleotide (Figure 4 and Supplementary figure 8B, C). The three contiguous ATP-binding

sites of MCM6, MCM4 and MCM7 contain ATPgS, with a Mg

ion coordinated between b

and g phosphates. Interestingly, the cryoEM map shows clearly that MCM2 and MCM3 have

hydrolysed their ATPgS to ADP. Furthermore, the MCM5 nucleotide-binding site is empty. It

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is possible that MCM5 had hydrolysed ATPgS and released ADP, or alternatively that ATPgS

was never bound: both possibilities are compatible with the observed open state of the MCM5-

2 interface.

All residues previously identified as involved in ATP binding and hydrolysis engage as

expected with the ATPgS moieties bound at the three MCM7-3, MCM4-7 and MCM6-4

interfaces, including Walker A and B residues and sensor-1 asparagine of the P-loop subunit,

and arginine finger, sensor-2 arginine and sensor-3 histidine residues in the contiguous ‘sensor’

subunit (Figure 4 and Supplementary figure 9). The two ADP-bound interfaces between

MCM3-5 and MCM2-6 show a very similar set of contacts, except that the arginine fingers in

MCM5, R513, and MCM6, R529, are partially disordered, likely as a consequence of the

absence of the g phosphate. A noteworthy feature of ATP binding by MCM2-7 is the extensive

range of hydrophobic interactions that shield the aromatic base of the nucleotide from solvent

(Figure 4). These interactions include a ‘sandwich’ interaction made by an invariant isoleucine

on one side of the base and two aliphatic residues in the domain-swapped helix on the other

side (Figure 4). In addition, the adenine base engages in Watson-Crick like hydrogen bonding

with the main-chain nitrogen and carbonyl moieties of a residue preceding a conserved

‘positive F’ glycine in the linker between helices 2 and 3 of the ATPase domain

(Supplementary figure 10).

An unresolved question is whether ATP-coupled conformational changes during DNA

translocation are limited to movements of the DNA-binding loops or involve the entire ATPase

domain. Structural superposition of the ATPase domains shows that the DNA-binding loops

of nucleotide-bound MCM6, 4, 7 and 3 are in similar position relative to their ATPase domains

(Figure 5); in contrast, the H2I loop of apo MCM5 at the 3′-end of the DNA occupies a lower

position and its H2I a

is disordered (Figure 5). These observations suggest that DNA

translocation might be achieved by a composite mechanism of whole-domain movements

during the ATP-coupled translocation cycle, and rearrangement of the H2I loop at the end of

the cycle, as H2I detaches itself from the staircase and its ATPase domain re-engages DNA at

the 5′-end.

The ATP status of the ADP-bound MCM2 subunit appears anomalous given its position at the

top of the ring staircase. Several indicators point to MCM2 acting as a ‘seam subunit’ (Eickhoff

et al., 2019) that has only partially engaged with the rest of the C-tier ring: the smaller interface

area with MCM6 (1391 Å

, instead of ~2000 Å

for the other nucleotide-occupied MCM

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interfaces), the disordered conformation of its DNA-binding element H2I a

, and the higher B

value attained during real-space refinement. The ADP moiety of MCM2 is also unusual in the

way it sits in the P-loop, as the nucleotide is shifted so that its b phosphate occupies the position

occupied by the g-phosphate in the ATPgS-bound interfaces (Supplementary figure 11).

Overall, the observations relative to ATP status and DNA binding in the MCM C-tier ring are

consistent with a sequential rotary mechanism of ATP hydrolysis as the basis for translocation

of the human CMG. The structural integrity of the C-tier during translocation is provided by a

domain-swapped helix (Supplementary figure 4) that provides a flexible tether between

contiguous MCM subunits, in a similar fashion as recently described for the replicative gp4

DNA helicase (Gao et al., 2019).

Interaction with AND-1

Analysis of purified human CMG overexpressed in HEK293 cells revealed the co-purification

of sub-stoichiometric amounts of endogenous AND-1, a known replisome factor and the

human orthologue of yeast Ctf4 (Supplementary figure 1). Our previous work had shown that

yeast Ctf4 acts as a recruitment hub for replisome proteins, tethering multiple factors at the

fork via its trimeric structure (Simon et al., 2014; Villa et al., 2016). AND-1 shares its

oligomeric nature with Ctf4, although it appears to have a distinct mechanism of binding to its

replisome partner, Pol a/primase (Kilkenny et al., 2017).

Co-purification of endogenous AND-1 indicated a strong constitutive interaction with the

human CMG complex. We therefore decided to co-express AND-1 together with the

components of the human CMG, and succeeded in purifying a 12-subunit CMG assembly

which we refer to here as CMGA (Supplementary figure 12). CryoEM analysis of CMGA

using Warp for particle picking (Tegunov & Cramer, 2019) and CryoSparc for image

reconstruction (Punjani, Rubinstein, Fleet, & Brubaker, 2017) yielded a 6.77 Å map that was

readily interpretable and permitted the unambiguous docking of the high-resolution structure

of human CMG and the crystal structure of the AND-1 trimer that we reported earlier (Kilkenny

et al., 2017) (Supplementary figure 13).

The structure shows that the disk-shaped AND-1 trimer docks edge-on at a near perpendicular

angle onto the leading face of the CMG (Figure 6). Despite AND-1 being full-length, only the

SepB-like domain of AND-1 is visible in the map, indicating that the N-terminal b-propeller

domain and its extended C-terminal portion spanning the HMG box are flexibly oriented in the

trimeric structure. Relative to the CMG, the trimeric AND-1 disk is arranged so that its N-

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terminal segments are located ahead of the fork and in proximity of the parental double-

stranded DNA. In contrast, the helical structure of the SepB domain and the relative C-terminal

extensions project away from the CMG (Figure 6).

AND-1 binds at the perimeter of the CMG, engaging both Cdc45 and GINS with the first and

last blade of one of its b-propeller domains (Figure 7). The CMG - AND-1 interface is formed

by the B-domain of Psf2 that projects towards the concave surface formed by blades 1 and 6

of AND-1’s b-propeller, as well as the helical portion of Cdc45 that links its two DHH

domains. The interface buries only 1087 Å

, a surprisingly small area for a constitutive

interaction (Figure 7). The limited resolution of our structure is insufficient for unambiguous

identification of interface amino acids. However, we can determine that the interface is of

mixed hydrophobic and hydrophilic nature, and that the tight binding of AND-1 to the CMG

despite the relatively limited interface might be driven by the presence of charge-charge

interactions that become solvent-excluded upon CMGA formation.

DISCUSSION

In this paper, we have used cryoEM to capture a high-resolution view of the human CMG

bound to a fork DNA substrate in the presence of ATPgS. Our map of human CMG-ATPgS-

ssDNA allowed us to visualise unambiguously critical features of the complex, such as its

protein-DNA interface and the nucleotide-binding sites, and to represent them in an accurate

atomic model. We have also reported an intermediate-resolution structure of the CMGA

assembly, which described the mode of interaction of CMG with the core replisome factor

AND-1.

DNA binding

In our structure, all six MCM subunits contact ssDNA, spanning a total of 11 nucleotides. The

footprint of an MCM subunit on ssDNA covers four nucleotides, rather than two as previously

reported (Figure 3) (Eickhoff et al., 2019), with two overlapping nucleotides between

neighbouring subunits. Interaction of MCM6, 4, 7 and 3 with DNA takes place via an identical

set of contacts mediated by both PS1 and H2I DNA-binding loops (Figure 3). A significant

difference is that MCM6 and MCM2 use aromatic residues at a conserved H2I position to

unstack the nucleotides at the 5′-end of the DNA and disrupt the B-form DNA. These aromatic

residues intermesh with consecutive bases much as the teeth of a cogwheel; such contacts

appear well suited to avoid slippage and transmit torque when an MCM subunit engages the

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leading DNA strand emerging from the N-tier, at the top of the staircase. Overall, the

arrangement of PS1 and H2I loops within the C-tier ring follows closely the DNA spiral,

lowering steadily their vertical reach from MCM2 at the top of the binding staircase to MCM5

at the bottom (Figure 2).

Earlier structural work on yeast and fly CMG had shown that DNA can be bound via two

different sets of MCM subunits: MCM6, 4 and 7 (Abid Ali et al., 2016) or MCM2, 3, 5 and 6

(Georgescu et al., 2017; Goswami et al., 2018). A recent cryoEM analysis of fly CMG in the

act of translocating on DNA revealed the existence of several different conformational states,

which appear to encompass and extend the previously described DNA-bound states of the

CMG (Eickhoff et al., 2019). In light of this analysis, our CMG-DNA structure would most

likely correspond to state 2B, in which MCM6, 4, 7 and 3 contact DNA and are bound to ATP,

with MCM2 in the process of exchanging ADP for ATP and re-engaging with DNA at the 5′-

end. Thus, the emerging evidence from this wealth of structural data strongly indicates multiple

modes of asymmetric DNA binding in the C-tier ring as a key feature of DNA translocation by

the CMG.

ATP site occupancy and hydrolysis

The site occupancy and hydrolysis status of ATPgS in our CMG-DNA structure is in general

agreement with the sequential rotary model of ATP utilisation but also reveals some

unexpected findings. Nucleotide occupancy is known to correlate with DNA binding, and in

our structure ATPgS-bound MCM6, 4 and 7 interact with DNA. In the model, ring subunits at

or near the bottom of the DNA-binding staircase have hydrolysed ATP; accordingly, the

MCM3-5 interface has converted the slowly-hydrolysable ATPgS to ADP (Supplementary

figure 8C), whereas MCM5 is in the apo state. These observations are in line with the

sequential rotary model, however they represent an apparent discrepancy with the model of

Eickhoff and colleagues (Eickhoff et al., 2019), in which ATP binding by MCM3, but not its

hydrolysis, is important for asymmetric translocation mediated by a MCM3-5 dimer.

Interestingly though, the ADP-bound MCM3-5 interface remains as extensive as for the three

ATPgS interfaces of MCM6-4, MCM4-7 and MCM7-3 (2078 Å

of buried surface area versus

an average value of 2034 Å

for the ATPgS interfaces).

Furthermore, in our structure the MCM5-2 interface is void of nucleotide and the MCM2-5

gate is open. Given the finding that MCM3 has hydrolysed ATPgS, MCM5 might be one step

ahead in the ATP cycle and may have released its ADP, in preparation for re-joining the C-tier

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ring at the top of the staircase. That MCM3 and MCM5 of all six subunits should have

hydrolysed ATPgS is in agreement with the effect of Walker A K-to-A mutations in fly CMG,

showing that loss of ATP binding by MCM3 and MCM5 caused the largest decrease in ATP

hydrolysis rates (Ilves et al., 2010), and with a four-fold reduction in DNA unwinding caused

by an ‘arginine finger’-to-alanine mutation in fly MCM5 (Eickhoff et al., 2019). Whether the

open state of the apo MCM2-5 interface represents a natural intermediate state in the

translocation cycle, or rather a stalled or paused state of the helicase remains to be established.

At any rate, the unexpected indication of ATPgS hydrolysis provides evidence that the CMG

has engaged productively with the fork DNA substrate and might have undergone a limited

degree of translocation.

The ADP-bound status of MCM2, at the top of the staircase and interacting with the 5′-end of

the ssDNA, is apparently inconsistent with a sequential rotary model of ATP hydrolysis. We

have already described several elements of evidence indicating that the MCM2 appears to

behave as a ‘seam’ subunit (Eickhoff et al., 2019). An intriguing possibility is that the observed

ATPgS hydrolysis by MCM2 might be a clue of translocation with reverse polarity, that could

have been prompted by idling of the helicase upon incubation with slowly-hydrolysable

ATPgS. Evidence that the CMG can backtrack on ssDNA has been provided by recent single-

molecule studies (Burnham, Kose, Hoyle, & Yardimci, 2019).

DNA translocation

The staircasing arrangement of the DNA-binding loops and the ATP-hydrolysis status in the

MCM ring are both supportive of a sequential rotary mechanism of DNA translocation for the

human CMG. Differences, such as the observed ATP-hydrolysis status of MCM3, with the

proposed model of asymmetric translocation (Eickhoff et al., 2019), remain to be explained

and might be species-specific.

Our structure captures a high-resolution snapshot of the CMG trapped on fork DNA with

ATPgS and does not provide conclusive evidence concerning models of asymmetric

translocation. However, insight into asymmetry in MCM2-7 behaviour comes from analysis of

the solvation free-energy for formation of the interfaces between contiguous ATPase domains

G) in the C-tier ring, using the EBI PISA server (Krissinel & Henrick, 2007). The analysis

shows striking differences in D

G values among MCM interfaces. Although the three ATPgS

interfaces, as well as the ADP-bound MCM3-5 interface, bury similar surface areas (between

1975 Å

and 2140 Å

), formation of the MCM4-7

ATPgS

and MCM6-4

ATPgS

interfaces shows

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more than 2-fold higher energy gains and 7-fold lower P-values than for the MCM7-3

ATPgS

and

the MCM3-5

ADP

interfaces (Supplementary figure 14). Thus, the D

G analysis indicates that

the contacts binding together the MCM 4-7 and 6-4 interfaces are much tighter and more

specific than in the other MCM interfaces. These observations might provide a structural basis

for the finding that human MCM4, 6 and 7 can be recovered as a stable heterotrimeric complex

from HeLa cells (Ishimi, 1997). They further suggest that MCM subunits 4, 6 and 7 might

behave as a rigid body in the asymmetric mode of DNA translocation. This would be in

agreement with biochemical evidence that loss-of-ATP-binding K-to-A mutations in fly

MCM6 and 4 causes only modest reductions in DNA unwinding (Ilves et al., 2010), and that

loss of ATP hydrolysis at the MCM6-4 and MCM4-7 interfaces caused by alanine mutation of

the ‘arginine finger’ in MCM4 and 7 has equally modest effects on unwinding (Eickhoff et al.,

2019).

The evolutionary invariance of catalytic residues in all six MCM2-7 ATPases represents a

challenge for models of asymmetric translocation that consider ATP binding and hydrolysis to

be important for a subset of MCM subunits. The following points in this regard can be made:

as far as we can determine in our cryoEM map, all catalytic and sensor MCM residues at each

nucleotide-bound interface engage correctly with the nucleotide and are therefore potentially

capable of catalysis. Furthermore, symmetric translocation is adequate for DNA replication by

homo-hexameric DNA helicases in viruses, bacteria and archaea, implying that it represents

the evolutionary consensus for DNA translocation. Finally, differentiation of the eukaryotic

MCM into six distinct proteins may have evolved to endow the CMG with its unique mode of

loading and activation and possibly termination. Asymmetric translocation might therefore

represent an adaptation to cope with MCM sequence diversification rather than a process of

optimisation. Consequently, the eukaryotic CMG might be capable of considerable

mechanistic flexibility, concerning the role played by each of its MCM subunits during

translocation.

CMGA

Our cryoEM structure of the human CMGA elucidates the interaction mechanism of the

replisome component AND-1 with the CMG. AND-1 does not contact the MCM proteins and

binds to the side of the CMG where Cdc45 and GINS are located. Thus, an important role of

Cdc45 and GINS, in addition to activating the CMG helicase activity, is to mediate CMG’s

interaction with AND-1. Only the trimer of AND-1’s SepB-like domains is visible in the map,

indicating that its N-terminal b-propeller and extended C-terminal region are flexibly arranged

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relative to its central trimeric structure. AND-1 is docked onto the CMG like a rigid body, with

a single b-propeller wedged in between Cdc45 and GINS. This mode of CMG binding is

similar to the mode of interaction that was recently described for yeast Ctf4 with the CMG

(Yuan et al., 2019), suggesting that the resulting architecture of the CMGA is functionally

important and has been evolutionarily preserved.

Because of the high interaction angle of the disk-like AND-1 trimer relative to the plane

containing the leading edge of the CMG, the N-terminal b-propeller domains of AND-1, not

visible in our structure, would be placed in the trajectory of the parental DNA. This striking

feature of the CMGA architecture indicates that AND-1/Ctf4 can in principle contact the

parental DNA ahead of the CMG’s leading edge of translocation. Clearly, these findings point

to further discoveries and unexpected observations that await future structural studies of the

eukaryotic replisome.

We had originally reported that Ctf4 can interact with the CMG via a CIP (Ctf4-Interacting

Peptide) motif present in the N-tail of yeast Sld5 (Simon et al., 2014), as well as in Ctf4’s

multiple protein partners (Villa et al., 2016). In the light of our current observations, we believe

that the mode of AND-1 interaction with the CMG described here and reported for yeast Ctf4

represents the principal mode of AND-1/Ctf4 recruitment to the replisome. Binding by the CIP

of yeast Sld5 might further secure the association of Ctf4 to the yeast CMG, as well as possibly

act as a safety mechanism for keeping Ctf4 anchored to fork when its primary interaction site

with the CMG is disrupted. A CMG-binding motif equivalent to the yeast CIP is not found in

human Sld5 or any other human CMG components, indicating that the Ctf4-Sld5 CIP

interaction is unique to budding yeast.

METHODS

Construct design and preparation. For expression of human CMG, ORFs for full-length

human MCM2 (P49736), MCM3 (EAX04367), MCM4 (P33991), MCM5 (P33992), MCM6

(Q14566), MCM7 (P33993), Cdc45 (AAC67521), Psf1 (Q14691), Psf2 (Q9Y248), Psf3

(Q9BRX5) and Sld5 (Q9BRT9) were synthesised using the GeneArt Gene Synthesis service

(ThermoFisher). GenBank EAX04367 codes for an MCM3 isoform (853 aa) that contains 45

additional residues at the N-terminus relative to Swissprot P25205.

ORFs were codon optimised for overexpression in human cells and designed with flanking

restriction sites for insertion into the ACEMam1 and 2 vectors of the MultiMam transient

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