Mechanisms of replication origin licensing: a structural perspective

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ScienceDirect Mechanisms of replication origin licensing: a structural perspective Franziska Bleichert1 The duplication of chromosomal DNA is a key cell cycle event that involves the controlled, bidirectional assembly of the replicative machinery. In a tightly regulated, multi-step reaction, replicative helicases and other components of the DNA synthesis apparatus are recruited to replication start sites. Although the molecular approaches for assembling this machinery vary between the different domains of life, a common theme revolves around the use of ATP-dependent initiation factors to recognize and remodel origins and to load replicative helicases in a bidirectional manner onto DNA. This review summarizes recent advances in understanding the mechanisms of replication initiation in eukaryotes, focusing on how the replicative helicase is loaded in this system. Address Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Corresponding author: Bleichert, Franziska ([email protected], [email protected]) 1 Affiliation from 01/2020: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United States. Current Opinion in Structural Biology 2019, 59:195–204 This review comes from a themed issue on Protein nucleic acid interactions Edited by Fred Allain and Martin Jinek

https://doi.org/10.1016/j.sbi.2019.08.007 0959-440X/ã 2019 The Author. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

In eukaryotes, replication initiation occurs in two, temporally separate stages. During origin licensing in the late M and G1 phases of the cell cycle, the motor module of the eukaryotic replicative helicase, the minichromosome maintenance 2-7 (Mcm2-7) complex, is loaded onto duplex DNA with the help of the origin recognition complex (ORC), the eukaryotic initiator, and the cofactors Cdc6 and Cdt1 [reviewed in Refs. 3,4,11,12]. A pair of Mcm2-7 is deposited at each origin as an inert head-to-head double hexamer and remains bound until the end of G1 [13–15]. In S phase, Dbf4-dependent and cyclin-dependent kinases (DDK and CDK) promote origin firing and Mcm2-7 activation, whereby the Mcm2-7 double hexamer is converted into two distinct active Cdc45-Mcm2-7-GINS (CMG) helicases [reviewed in Refs. 4,11,12]. Concomitantly, origin DNA is melted and replication factors are recruited to establish bidirectional replisomes [16]. Unraveling the molecular mechanisms of replicative helicase loading and activation has been a major research focus for several decades. With the development of in vitro reconstitution assays for eukaryotic replication initiation and technological advances in structural biology, multi-protein replication initiation intermediates have become tractable targets for near-atomic resolution structural studies [17,18]. At the same time, singlemolecule techniques have made it possible to understand the dynamics of replication initiation [19,20,21]. This review discusses how these recent efforts have helped define models for origin recognition and replicative helicase loading in eukaryotes.

Origin recognition and remodeling by ORC Introduction The timely and accurate replication of cellular genomes is essential for cell and organismal viability. In all life forms, this task is performed by multi-component protein machineries, termed replisomes, which are assembled in accord with cell cycle cues bi-directionally on specialized regions of chromosomal DNA, so-called replication origins [1–5]. Central to the function of replisomes are replicative helicases, ring-shaped motors that are topologically linked to DNA and processively unwind the parental duplex by strand exclusion during replication [6,7]. The controlled recruitment of these helicases to origins is performed by dedicated initiator and loader proteins, which recognize origins and chaperone the helicase onto DNA [8–10]. www.sciencedirect.com

The heterohexameric origin recognition complex (ORC) serves as the replication initiator in many eukaryotic species [22,23]. ORC forms productive interactions with origin DNA in an ATP-dependent manner and primes these sites for replicative helicase loading [13,14,22,24]. The initiator itself is composed of six different subunits (Orc1-6), five of which have a modular organization and are predicated on an ATPases associated with various cellular activities (AAA+) fold, an oligomerization and nucleotide hydrolase domain, as well as a winged-helix (WH) motif [22,23,25–27]. Its sixth component, Orc6, does not follow this scheme but contains two cyclin-box folds that are related to the transcription factor TFIIB [28–30]. The detailed intricacies of ORC’s threedimensional architecture have recently been revealed Current Opinion in Structural Biology 2019, 59:195–204

196 Protein nucleic acid interactions

through crystallographic studies that yielded the first near-atomic resolution structure of a eukaryotic initiator (Figure 1a and b) [31]. This Drosophila ORC structure established that Orc1-5 assemble into a near-planar, two-layered ring of AAA+ ATPase and WH domains, whereby the WH elements domain-swap with the AAA+ regions of adjacent protomers. By contrast, Orc6 does not contribute to ring formation but is tethered to the periphery of the complex through interactions with a domain insert in Orc3 [30,31]. An unexpected feature of Drosophila ORC observed both structurally (crystallographically and by EM) and in solution is a large rotation of the Orc1 AAA+ domain that disrupts the composite ATPase site with its Orc4 partner (Figure 1a and b) [31,32]. ATP binding can stabilize this ATPase center in the Drosophila complex and in ORC from other eukaryotes, suggesting this conformational switch helps modulate ATPdependent initiator functions (Figure 1a and b) [32,33,34]. How ORC engages DNA has been a long-standing question. A recent cryo-EM structure of Saccharomyces cerevisiae ORC in complex with a subregion of an autonomously replicating sequence (ARS) element nicely shows DNA binding in ORC’s central channel and provides detailed insights into the interactions between the initiator and replication origins (Figure 1a and b) [34]. In this structure, several ORC subunits are seen to contact DNA using specialized elements in the AAA+ and WH domains, although the details of these interactions vary. All ORC AAA+ domains, apart from that of Orc4, engage the sugar-phosphate backbone using the initiator specific motif (ISM), a helical insertion present in prokaryotic and eukaryotic initiators (Figure 1c) [26,34,35]. The Orc2-5 WH domains, on the other hand, bind DNA using a b-hairpin region that reaches into the major groove (except for Orc3, which contacts the DNA backbone) (Figure 1d) [34]. Strikingly, additional interactions with DNA are made by several basic patches and one of the cyclin folds within Orc6; together, these elements cooperate with the ISMs and b-hairpins to bend DNA as it exits ORC’s central channel [34]. Drosophila ORC also bends DNA [32,36], suggesting this activity is a conserved property of ORC and likely important for Mcm2-7 loading. In S. cerevisiae and in closely related yeast species, the selective association of ORC with origins is achieved by the read-out of the ARS consensus sequence (ACS) [22]. The DNA-bound structure of budding yeast ORC explains the basis for the sequence-specific DNA recognition [34]. Thymine bases within the ACS are contacted by three ORC subunits: a basic patch in Orc1, the Orc2-ISM, and a yeastspecific helix inserted in the WH b-hairpin of Orc4 (Figure 1e) [34]. Interestingly, the Orc1 basic patch is conserved in metazoans and enhances Drosophila ORC’s Current Opinion in Structural Biology 2019, 59:195–204

affinity for DNA [32], implying it may engage DNA in a similar manner as seen in the yeast complex. However, contrary to S. cerevisiae, ORC targeting to chromosomes in other eukaryotes, including metazoans, does not rely on specific DNA sequences but rather on DNA topology and contextual chromatin cues, and is aided by auxiliary regions in some ORC subunits (for example, the Orc1 bromoadjacent homology domain) [37–39]. Structures of DNAbound metazoan initiator complexes will be important to understand how the different DNA and chromatin binding regions cooperate in ORC during initiator recruitment and helicase loading in the absence of specific DNA sequence motifs. The pentameric ORC ring contains a gap that initially allows DNA entry into ORC’s central channel. After DNA binding, the opening is sealed by the co-loader Cdc6 in an ATP-dependent manner, trapping DNA in the center of the ORC
Cdc6 toroid (Figure 1a) [40,41]. This ORC
Cdc6 complex is now poised for helicase loading. Despite its importance, a high-resolution structure of this initiator
co-loader complex before Mcm2-7 recruitment is still missing and will be necessary to address several remaining questions pertaining to ORC
Cdc6 function. For example, Cdc6 has been reported to induce conformational changes in ORC and to extend the ORC-dependent footprint on origin DNA in S. cerevisiae, yet a structural explanation for these observations is currently lacking [27,40,42–44]. Moreover, since Cdc6 is an AAA+ ATPase, structural information will also be needed to elucidate how conformational changes associated with its ATP hydrolysis cycle regulate Mcm2-7 loading [27,45].

Loading of the first helicase hexamer by ORC
Cdc6 Origin-bound ORC
Cdc6 serves as a landing pad for Mcm2-7 and helps chaperone the helicase onto DNA. Mcm2-7, like ORC and Cdc6, is an AAA+ ATPase [46,47]. Each of the six Mcm2-7 subunits comprises an AAA+ fold, which is appended by an N-terminal domain and a C-terminal WH module (except for Mcm2, which lacks the C-terminal WH region) [48,49]. In the preloaded state, these Mcm2-7 domains co-assemble into a heterohexameric, two-tiered ring that resembles a lefthanded lock-washer with a gap between subunits Mcm2 and Mcm5, although closed rings have also been observed (Figure 2ai and b) [50–54]. S. cerevisiae Mcm2-7 is further found stably in complex with Cdt1, which laterally braces the Mcm2, Mcm4, and Mcm6 subunits, stabilizing the open hexameric ring and potentially preventing ring closure [19,52,53]. Although the opening between Mcm2 and Mcm5 serves as the entry gate for DNA during loading, it is too narrow to allow DNA passage in the free Mcm2-7 and the heptameric Mcm2-7
Cdt1 complexes, rationalizing why these assemblies cannot self-load onto origins in the absence of ORC
Cdc6 [52,54,55]. www.sciencedirect.com

Structural mechanisms for eukaryotic DNA replication initiation Bleichert 197

Figure 1

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autoinhibited ORC

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GT T T T GT AT AT T 5’

C A A A A C A T A T A A 3’

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Orc1BP Current Opinion in Structural Biology

Origin recognition by ORC. (a) Schematic of subunit and domain arrangements in different conformational states of DNA-free ORC and in originbound ORC before and after Cdc6 (red) recruitment. The arrow in the left panel indicates the domain rotation of the Orc1-AAA+ module during ORC activation. (b) Structures corresponding to the different ORC assemblies in A are shown in cartoon representation and transparent surface rendering. Domains or subunits not present in constructs used for structural studies are shown in grey in (a). (c)–(e) DNA engagement by S. cerevisiae ORC. The AAA+ and winged-helix (WH) domains of Orc1-5 (in gray cartoon) form a collar around duplex DNA in (c) and (d), respectively, with many initiator specific motifs (ISMs) and b-hairpin elements directly contacting the DNA duplex. (e) Conserved DNA sequence elements are recognized by three ORC regions, an insertion helix in the WH domain of Orc4 (Orc4IH), a loop in the ISM of Orc2, and a basic patch in Orc1 (Orc1BP). Nucleobases that are contacted by amino acids in these elements are colored dark red. The DNA sequence is shown on the left. Structures presented are derived from PDBs 4XGC [31], 5UJ7 [33], and 5ZR1 [34].

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198 Protein nucleic acid interactions

Figure 2

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Encounter complex

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Current Opinion in Structural Biology

Replicative helicase loading in eukaryotes. (a) Current model for loading of the first Mcm2-7 hexamer by ORC
Cdc6 derived from biochemical and structural studies. Subunit and domain organization of Mcm2-7 is indicated in the left upper panel. The color scheme for ORC and Cdc6 is the same as in Figure 1. (b) Cryo-EM structures of Mcm2-7
Cdt1 (PDB 5XF8 [52]), a loading intermediate containing ORC
Cdc6 and Mcm2-7
Cdt1 (OCCM; PDB 5V8F [57]), and the DNA-loaded Mcm2-7 double hexamer (PDB 5BK4 [61]), shown in cartoon representation with transparent surface rendering. The crack in the helicase ring between Mcm2 and Mcm5 is highlighted by a dashed line in the isolated Mcm2-7
Cdt1 complex. NTD – N-terminal domain. WH – winged-helix domain.

Elucidating the physical basis for ORC
Cdc6-dependent origin recruitment and loading of Mcm2-7 has been a major focus in the field. Biochemical studies have shown that the WH domain in the C-terminus of Mcm3 is Current Opinion in Structural Biology 2019, 59:195–204

essential and sufficient for Mcm2-7 binding to Cdc6 or ORC
Cdc6 [41,56]. In a recent cryo-EM structure of a loading intermediate that contains DNA, ORC
Cdc6, and Mcm2-7
Cdt1 (known as the OCCM complex), the www.sciencedirect.com

Structural mechanisms for eukaryotic DNA replication initiation Bleichert 199

Mcm3-WH domain is seen to directly interact with Cdc6 and the WH domain of Orc2 (Figure 2aii-iv) [57]. These contacts may be the first ones to be established in an encounter complex between the initiator
loader assembly and the helicase and would ensure that Mcm2-7 is only recruited to origins bound by both ORC and Cdc6 (Figure 2aii). In the OCCM, the WH domains of Mcm4, Mcm6, and Mcm7 likewise latch onto ORC
Cdc6, while some of the helicase AAA+ modules stack onto the WH tier of the ORC
Cdc6 ring (Figure 2aiii, iv and b) [57]. Importantly, DNA is already inserted into the central pore of Mcm2-7 in the OCCM structure, indicating this complex resembles a late-stage loading intermediate (Figure 2aiv and b). Comparison of isolated ORC
DNA, Mcm2-7, and Mcm2-7
Cdt1 structures with their counterparts in the OCCM reveals that helicase loading is accompanied by numerous conformational changes in these initiation factors. In the pre-loading state, several of the Mcm27 WH domains occupy positions that would sterically hinder docking of the helicase onto ORC
Cdc6 (Figure 2ai, ii) [52]. Overcoming this inhibitory effect requires Cdt1, which interacts with the Mcm6 WH domain and is likely involved in repositioning this autoinhibitory Mcm6 region during Mcm2-7
Cdt1 recruitment [57,58]. The helicase ring itself is also reconfigured during OCCM formation from a lefthanded helical assembly into a more planar ring with a partially closed Mcm2/5 gate, although binding of Mcm2-7
Cdt1 to ORC
Cdc6 likely first mediates Mcm2/5 gate opening to allow DNA insertion (Figure 2ai,iii, iv and b) [52,57]. Besides conformational changes in protein factors, the DNA itself is also substantially remodeled during OCCM formation. While DNA bending by ORC is expected to facilitate docking of Mcm2-7 onto the ORC
Cdc6 ring and probably helps align the DNA duplex with the Mcm2/5 gate upon helicase recruitment (Figure 2aiii), several interactions that stabilize the bent DNA in ORC
DNA are broken in the OCCM (Figure 2aiv) [34,57]. It is possible that productive contacts between ORC
Cdc6 and Mcm27
Cdt1 rings trigger conformational changes in ORC’s basic patch regions to release their grip on DNA, allowing DNA unbending and duplex insertion into the helicase ring. Ctd1 may also aid in this process by repositioning Orc6, as both proteins have been shown to interact biochemically [59]. Finally, single-molecule and biochemical studies have shown that Cdt1 departs from origins during the loading of the first Mcm2-7 hexamer, an event that is preceded by Cdc6 release and that occurs concomitantly with Mcm2-7 ring closure (Figure 2aiv, v) [19,20,58]. This dynamic association of initiation factors predicts additional loading intermediates, and their visualization at high resolution will be an important task for future studies to fully understand the mechanics of Mcm2-7 loading. www.sciencedirect.com

Loading of the second helicase hexamer — one or two ORCs? Eukaryotic replication origins are licensed by loading two Mcm2-7 rings onto duplex DNA to form head-to-head double hexamers (Figure 2avi and b) [13–15], which are stabilized by extensive interactions between Mcm2-7 N-terminal regions [49,60,61,62,63]. Like the first hexamer, the second Mcm2-7 relies on ORC, Cdc6, Cdt1 and the C-terminus of Mcm3 for successful loading [56,64]. Single-molecule experiments have shown that both Mcm2-7 hexamers are deposited in a sequential manner with different kinetics, and that the licensing reaction involves one ORC but two Cdc6 and Cdt1 molecules (Figures 2a and 3 a) [19,20]. While ORC remains associated with origins until double Mcm2-7 hexamers are formed, Cdc6 and Cdt1 are recruited and released during each of the two loading events (Figure 2a). Together, these observations have led to proposals that both helicases might be deposited by distinct mechanisms[19,20]. The structural details explaining these differences, however, are not understood. Likewise, which interaction interfaces are used to recruit and load the second Mcm2-7 remains to be firmly established. In contrast to the one-ORC licensing model, efficient Mcm2-7 loading has recently been reported to require two inverted ORC binding elements [64]. Interestingly, origins in S. cerevisiae contain several low-affinity ORC sites up to several hundred base pairs away from the primary ACS. These observations suggest that two initiator molecules are involved in loading, with each ORC depositing one of the Mcm2-7 hexamers by a similar mechanism onto DNA (Figure 3a) [64]. In this ‘quasisymmetric’ two-ORC model, both loaded Mcm2-7s are then predicted to slide or translocate on dsDNA towards each other until they meet to form a head-to-head double hexamer. Successful dimerization would require the two helicases to be loaded with the N-termini facing each other. While the sequence-specific DNA binding activity of ORC could ensure the proper orientation of both Mcm2-7 in budding yeast, it is unclear how this outcome would be achieved in the absence of DNA sequencespecific origin recognition in higher eukaryotes. It is possible that the two Mcm2-7 loading events are coordinated (e.g. by looping of the intervening DNA segment or by specific bridging factors) but this premise needs to be tested experimentally. Although at first glance the one-ORC and two-ORC models seem contradictory, they are not mutually exclusive. Hopping of ORC between high- and low affinity sites could account for both findings and would additionally provide a means to couple loading at both sites temporally and spatially [64]. One advantage of the two-ORC model is that symmetric, bidirectional replisome assembly would be a consequence of the ‘quasi-symmetric’ origin organization. However, symmetric initiator binding sites are not a strict Current Opinion in Structural Biology 2019, 59:195–204

200 Protein nucleic acid interactions

Figure 3

(a)

(b)

Eukaryotes One-ORC model Cdt1

Mcm2-7 asymmetric origin

E. coli

Two-ORC model DnaB

ORC

DnaA Cdc6 quasi-symmetric origin

symmetric helicase loading

DnaB

asymmetric origin

symmetric helicase loading Current Opinion in Structural Biology

Models for symmetric helicase loading in eukaryotes and bacteria. (a) In the one-ORC model, one ORC deposits both Mcm2-7 hexamers sequentially onto origins containing an asymmetric ORC binding site. Loading of the second Mcm2-7 complex may be templated by the first loaded Mcm2-7 hexamer. Conversely, in the two-ORC model, origins are quasi-symmetric and harbor two, oppositely oriented ORC binding sites. Each ORC loads one Mcm2-7 hexamer by the same mechanism. The loaded hexamers slide or translocate towards each other to form double hexamers. (b) Symmetric DnaB helicase loading in E. coli on an asymmetric origin. The initiator DnaA forms a filament at origins, binding doublestranded DNA regions and one strand of the melted duplex. DnaB is recruited via different mechanisms to both single DNA strands and loaded in opposite orientations.

The role of ATP binding and hydrolysis in Mcm2-7 loading

double hexamer formation [70,71]. Of the six different ATPase sites formed at the interfaces between subunits in the Mcm2-7 ring, several hydrolyze ATP during the loading reaction, and catalysis is coupled to Cdt1 release and Mcm2/5 gate closure (Figures 2a and 4 b) [19,70,71]. At present, it is not completely understood how ATPase activity is triggered but it is conceivable that DNA insertion into the central Mcm2-7 pore could stimulate the ATPase sites, which in turn may drive changes in Mcm2-7 ring conformation. Notably, the N-terminal and AAA+ tiers of Mcm2-7 rotate with respect to each other during the transition from the OCCM to the double hexamer, which could lead to Cdt1 ejection and Mcm2/5 gate closure [49,57,60,61].

It is well established that Mcm2-7 loading requires binding and hydrolysis of ATP [24,66]. ORC, Cdc6, and Mcm2-7 all contain catalytically-competent ATPase sites (Figure 4) [45,47,55,67–69]; however, the details by which the hydrolytic functions of each of these enzymes affect helicase loading have only recently come to light. In vitro reconstitution of helicase loading using various ATPase-deficient initiation factors has revealed that the catalytic activity of Mcm2-7, but not that of ORC or Cdc6, is strictly needed for

In vivo, the ATPase motifs of ORC and Cdc6 are also important for helicase loading and cell viability [72–77]. These observations appear at odds with recent findings that their ATPase activity is dispensable for double hexamer formation reconstituted in vitro using recombinant proteins [70,71]. In the case of Cdc6, these disparate findings can be explained by a quality control function of the co-loader [56]. Biochemical studies have found that ATP hydrolysis

prerequisite for bidirectional replication onset. Bacterial replisomes, for example in Escherichia coli, assemble in opposite orientations on asymmetric origins, with both DnaB helicase hexamers recruited through different mechanisms by the initiator DnaA (Figure 3b) [2,65]. Additional research is therefore needed to resolve outstanding questions concerning the loading mechanism of the eukaryotic Mcm2-7 replicative helicase, and especially of the second hexamer.

Current Opinion in Structural Biology 2019, 59:195–204

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Structural mechanisms for eukaryotic DNA replication initiation Bleichert 201

Figure 4

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ORC Cdc6 (OCCM) no hydrolysis

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Current Opinion in Structural Biology

Composite ATPase centers in origin licensing factors. (a) ATP binding sites in ORC
Cdc6 (within OCCM) are formed at four different interfaces between subunits, but hydrolysis only occurs at two of these sites [57]. An arginine finger required for stimulating ATP hydrolysis is provided by the adjacent protomer. (b) Mcm2-7 contains six ATPase sites, one at each subunit interface. In the OCCM complex, the Mcm2-7 hexamer is in a pre-catalytic state and four binding sites are occupied with the slowly hydrolysable ATP analog ATPgS. By contrast, in the loaded Mcm2-7 double hexamer, ATP has been hydrolyzed to ADP. The Mcm7/3 and Mcm2/6 ATPase sites are either empty or contain ADP [60,61].

by Cdc6 leads to disassembly of unsuccessfully loaded Mcm2-7 hexamers, which could be important in vivo for recycling of initiation factors that are sequestered in kinetically trapped (dead-end) loading intermediates [54,56,58,70,71]. Although the mechanism that promotes the release of loading factors from origins is not fully defined yet, it is interesting to note that Cdc6 co-association with origin-bound ORC is dependent on ATP (Cdc6 forms a bipartite ATPase site with Orc1) [24,27,42,45]. ATP hydrolysis by Cdc6 is therefore expected to cause the co-loader to disengage from ORC and, because of Cdc6’s interaction with Mcm3, to destabilize any non-loaded Mcm2-7
Cdt1 at origins. In this regard, Cdc6 ATP hydrolysis would act as a molecular timer to define a temporal window in which loading of one hexamer must occur [71].

replication initiation, in particular into ORC and Cdc6mediated Mcm2-7 loading. Nonetheless, our mechanistic understanding of the events that lead to replication onset are far from complete. Key outstanding questions concern not only how the second Mcm2-7 hexamer is loaded, but also how the double hexamer transitions into the active CMG helicase during origin firing. Trapping different activation intermediates and visualizing these at high resolution will be key to resolve the mechanism of double hexamer separation, origin melting and single DNA strand extrusion. These structural snapshots, combined with the continued development of biochemical and single-molecule approaches to dissect the initiation reaction, will undoubtedly uncover many novel and surprising principles that ensure the successful assembly of bidirectional replication machineries.

The role of ORC’s ATPase activity, on the other hand, has remained more mysterious. Within ORC, only Orc1 possesses catalytic activity and forms a composite ATPase site with Orc4 (Figure 4a) [68,69,73]. ATP stabilizes ORC on DNA and regulates productive initiator association with origins, while ATP hydrolysis facilitates repeated rounds of Mcm2-7 loading, possibly by releasing ORC from origins or resetting ORC after double hexamer assembly for a new round of loading [22,68,73]. Consistent with this notion, ORC is seen to disengage from origins upon licensing in single-molecule studies [20]. Whether ORC departure is coupled to Orc1 ATP hydrolysis, however, remains to be experimentally tested.

Conflict of interest statement Nothing declared.

Acknowledgements This work was supported by the Novartis Research Foundation and the European Research Council under the European Union’s Horizon 2020 research and innovation program (ERC-STG-757909).

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29. Liu S, Balasov M, Wang H, Wu L, Chesnokov IN, Liu Y: Structural analysis of human Orc6 protein reveals a homology with transcription factor TFIIB. Proc Natl Acad Sci U S A 2011, 108:7373-7378. 30. Bleichert F, Balasov M, Chesnokov I, Nogales E, Botchan MR, Berger JM: A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation. eLife 2013, 2:e00882. 31. Bleichert F, Botchan MR, Berger JM: Crystal structure of the  eukaryotic origin recognition complex. Nature 2015, 519:321-326 This paper reports the first near-atomic resolution structure of a eukaryotic initiator, obtained by crystallizing Drosophila ORC. The structure shows that five subunits organize into a two-tiered ring with a domainswapped arrangement of AAA+ and winged-helix domains. ORC is seen in an unexpected autoinhibited conformation that may help regulate DNA binding and Cdc6 co-association during replication initiation. 32. Bleichert F, Leitner A, Aebersold R, Botchan MR, Berger JM: Conformational control and DNA-binding mechanism of the metazoan origin recognition complex. Proc Natl Acad Sci U S A 2018, 115:E5906-E5915. 33. Tocilj A, On KF, Yuan Z, Sun J, Elkayam E, Li H, Stillman B, JoshuaTor L: Structure of the active form of human origin recognition complex and its ATPase motor module. eLife 2017, 6:e20818. 34. Li N, Lam WH, Zhai Y, Cheng J, Cheng E, Zhao Y, Gao N, Tye BK:  Structure of the origin recognition complex bound to DNA replication origin. Nature 2018, 559:217-222 Using cryo-EM, the authors reveal at high-resolution how S. cerevisiae ORC interacts with an ARS region, explaining the physical basis for the sequence-specific origin recognition of budding yeast ORC. The structure also reveals a large bend in the DNA that is predicted to help align DNA with the Mcm2-7 gate during helicase loading. 35. Erzberger JP, Mott ML, Berger JM: Structural basis for ATPdependent DnaA assembly and replication-origin remodeling. Nat Struct Mol Biol 2006, 13:676-683. 36. Clarey MG, Botchan M, Nogales E: Single particle EM studies of the Drosophila melanogaster origin recognition complex and evidence for DNA wrapping. J Struct Biol 2008, 164:241-249.

20. Ticau S, Friedman LJ, Ivica NA, Gelles J, Bell SP: Single-molecule  studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading. Cell 2015, 161:513-525 A single-molecule helicase loading assay is developed to study the dynamics and protein stoichiometry of origin licensing. The experiments reveal that both Mcm2-7 hexamer are loaded sequentially and that one ORC but two molecules of Cdc6 and Cdt1 are required for double hexamer formation.

38. Vashee S, Cvetic C, Lu W, Simancek P, Kelly TJ, Walter JC: Sequenceindependent DNA binding and replication initiation by the human origin recognition complex. Genes Dev 2003, 17:1894-1908.

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41. Sun J, Evrin C, Samel SA, Fernandez-Cid A, Riera A, Kawakami H, Stillman B, Speck C, Li H: Cryo-EM structure of a helicase loading intermediate containing ORC-Cdc6-Cdt1-MCM2-7 bound to DNA. Nat Struct Mol Biol 2013, 20:944-951. 42. Mizushima T, Takahashi N, Stillman B: Cdc6p modulates the structure and DNA binding activity of the origin recognition complex in vitro. Genes Dev 2000, 14:1631-1641. 43. Cocker JH, Piatti S, Santocanale C, Nasmyth K, Diffley JF: An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. Nature 1996, 379:180-182. 44. Santocanale C, Diffley JF: ORC- and Cdc6-dependent complexes at active and inactive chromosomal replication origins in Saccharomyces cerevisiae. EMBO J 1996, 15:6671-6679. 45. Randell JC, Bowers JL, Rodriguez HK, Bell SP: Sequential ATP hydrolysis by Cdc6 and ORC directs loading of the Mcm2-7 helicase. Mol Cell 2006, 21:29-39. 46. Schwacha A, Bell SP: Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication. Mol Cell 2001, 8:1093-1104. 47. Ilves I, Petojevic T, Pesavento JJ, Botchan MR: Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. Mol Cell 2010, 37:247-258. 48. Bochman ML, Schwacha A: The Mcm complex: unwinding the mechanism of a replicative helicase. Microbiol Mol Biol Rev 2009, 73:652-683. 49. Li N, Zhai Y, Zhang Y, Li W, Yang M, Lei J, Tye BK, Gao N:  Structure of the eukaryotic MCM complex at 3.8 A. Nature 2015, 524:186-191 A cryo-EM structure of the S. cerevisae Mcm2-7 double hexamer is presented at 3.8 A˚ resolution. This structure provides detailed insights into the arrangement of subunits, into interactions between the two hexamers, and into the general architecture of the ATPase sites. 50. Costa A, Ilves I, Tamberg N, Petojevic T, Nogales E, Botchan MR, Berger JM: The structural basis for MCM2-7 helicase activation by GINS and Cdc45. Nat Struct Mol Biol 2011, 18:471-477.

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56. Frigola J, Remus D, Mehanna A, Diffley JF: ATPase-dependent  quality control of DNA replication origin licensing. Nature 2013, 495:339-343 In this paper, the authors discover a novel quality-control mechanism for licensing that is dependent on the co-loader protein Cdc6. If helicase loading cannot progress due to missing loading factors or phosphorylation of initiator subunits, ATP hydrolysis by Cdc6 promotes Mcm2-7 release and disassembly of loading intermediates. 57. Yuan Z, Riera A, Bai L, Sun J, Nandi S, Spanos C, Chen ZA,  Barbon M, Rappsilber J, Stillman B et al.: Structural basis of Mcm2-7 replicative helicase loading by ORC-Cdc6 and Cdt1. Nat Struct Mol Biol 2017, 24:316-324 This study, together with previous work from the same labs, describes the architecture of an helicase loading intermediate containing origin DNA, ORC, Cdc6, Cdt1, and Mcm2-7 (OCCM). In this 3.9 A˚ resolution cryo-EM structure, Mcm2-7 is already loaded onto DNA but the gate in the helicase ring is still partially open. The structure provides insights into the loading mechanism of the first Mcm2-7 hexamer. www.sciencedirect.com

70. Kang S, Warner MD, Bell SP: Multiple functions for Mcm2-7  ATPase motifs during replication initiation. Mol Cell 2014, 55:655-665 Mutational analyses of conserved motifs within the six ATPase centers in the Mcm2-7 hexamer demonstrates that the different active sites play diverse roles during helicase loading and activation. Similarly to the Diffley group, the authors also find that ATP hydrolysis by Mcm2-7 is essential for reconstituting helicase loading and for in vitro DNA replication initiation, whereas the enzymatic activity of ORC and Cdc6 is not. 71. Coster G, Frigola J, Beuron F, Morris EP, Diffley JF: Origin  licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol Cell 2014, 55:666-677 This study genetically and biochemically analyzes the role of Mcm2-7 ATP binding and hydrolysis during origin licensing. The experimental findings reveal that ATP binding to Mcm2-7 subunits is important for hexamer stability and that ATP hydrolysis by Mcm2-7 subunits, but surprisingly not by ORC and Cdc6, is essential for double hexamer formation. Current Opinion in Structural Biology 2019, 59:195–204

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