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Review

Helicase loading: How to build a MCM2-7 double-hexamer Alberto Riera, Silvia Tognetti, Christian Speck ∗ DNA Replication Group, Faculty of Medicine, Institute of Clinical Sciences, Imperial College, Hammersmith Hospital Campus, Du Cane Road, London W12 0NN, UK

a r t i c l e

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Article history: Available online xxx Keywords: MCM2-7 DNA replication Licensing Pre-RC Helicase

a b s t r a c t A central step in eukaryotic initiation of DNA replication is the loading of the helicase at replication origins, misregulation of this reaction leads to DNA damage and genome instability. Here we discuss how the helicase becomes recruited to origins and loaded into a double-hexamer around double-stranded DNA. We specifically describe the individual steps in complex assembly and explain how this process is regulated to maintain genome stability. Structural analysis of the helicase loader and the helicase has provided key insights into the process of double-hexamer formation. A structural comparison of the bacterial and eukaryotic system suggests a mechanism of helicase loading. © 2014 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA replicon model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA replication origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recruitment of the MCM2-7 helicase to the replication origin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The role of ATP-hydrolysis during pre-RC formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Helicase loading – what do we know? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The recruitment of the second MCM2-7 hexamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ORC/Cdc6 function as a MCM2-7 chaperone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The MCM2-7 double hexamer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Precise duplication of the genome is essential for genomic stability and organism survival. As such, cells have evolved highly regulated mechanisms that control DNA replication guaranteeing the faithful replication of the genome. In all living organisms the replication process is initiated at origins of DNA replication. Eukaryotes employ a six-subunit origin-recognition complex (ORC), shown in Saccharomyces cerevisiae to bind to replication origins [1]. ORC is chromatin bound throughout the cell cycle; however in late M phase Cdc6 binds to ORC to form the ORC/Cdc6 complex [2]. ORC/Cdc6 functions together with Cdt1 to

∗ Corresponding author. Tel.: +44 020 8383 3387. E-mail address: [email protected] (C. Speck).

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load the replicative helicase MCM2-7 onto DNA. During helicase loading, also termed pre-replicative complex (pre-RC) formation or DNA licensing, two MCM2-7 hexamers are loaded in an ATPhydrolysis dependent process into a MCM2-7 double-hexamer around double-stranded DNA [3,4]. Interestingly, this complex is not functional as a helicase and still requires activation in S-phase. Numerous protein factors and kinases, including cyclin-dependent kinase (CDK) and Dbf4 dependent kinase (DDK), act together to promote the formation of a Cdc45/MCM2-7/GINS (CMG) complex, which represents the active form of the replicative helicase [5]. During helicase activation the MCM2-7 double-hexamer splits and its ATPase motor becomes activated [6,7]. Within the CMG, MCM2-7 encircles only one strand of DNA, while the other strand is thought to pass through Cdc45 and the four subunit GINS complex, thus enabling the helicase to split the two DNA strands [7,8]. The CMG represents the basis for the replication fork, with DNA polymerases

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and many other factors latching onto this complex during DNA synthesis. Interestingly, multiple factors, including CDK, readily inhibit helicase loading; therefore, once the helicase becomes active in S-phase, no further helicase loading can occur [9,10]. This mechanism guarantees that each piece of DNA becomes replicated during each cell cycle once and only once. Helicase activation is a highly complex reaction of which we have a very limited understanding. On the other hand, the recent success in reconstituting pre-RC formation in vitro with purified proteins allowed its biochemical and structural analysis. This work identified crucial mechanisms in helicase recruitment, detailed specific steps in double-hexamer formation and suggested a mechanism of helicase loading onto DNA, which we review below. 2. DNA replicon model In 1963, the replicon theory was formulated by Francois Jacob, Sydney Brenner, and Francois Cuzin [11], which postulated that a initiator acts in trans at a replication origin to initiate DNA replication. Based on this concept two questions should be addressed: who is the initiator and what function does the initiator have? In bacteria, the DnaA protein recognises the origin and also controls the limiting steps in initiation of DNA replication: DNA unwinding and helicase loading [12,13]. Thus, as predicted in the replicon theory, DnaA has all initiator functions combined within a single protein. Importantly, the steps following DnaA mediated unwinding are energetically favoured, since these are propelled by ATP-hydrolysis, while DnaA mediated DNA unwinding occurs in the absence of ATP-hydrolysis [14,15]. The activity of DnaA is controlled by the nucleotide status of the protein. While ADP-DnaA cannot unwind DNA [16], ATP-DnaA is competent to form multimers on DNA [17,18], which in turn leads to DNA unwinding. Several factors feed into the DnaA nucleotide status and thus help to integrate information from different regulatory loops, defining when DNA replication initiates [13]. Once DNA replication is initiated, protein factors promote DnaA ATP-hydrolysis, which inactivates the DnaA protein and hinder re-replication [13]. In eukaryotes DNA replication is controlled in a fundamentally different way. Here, the helicase becomes loaded on DNA in G1-phase and becomes activated only later in S-phase. Since helicase loading is blocked at the beginning of S-phase, the loading reaction has to be completed prior to the onset of DNA synthesis, otherwise stretches of DNA would be left unreplicated. To avoid under-replication, eukaryotes load the helicase in a very efficient process at a large number of replication origins. During helicase activation in S-phase, limiting factors control the number of active replication forks [5,19]. This staged activation process is vital; otherwise nucleotide/histone pools could be depleted resulting in DNA damage [20]. In analogy to the bacterial system, the helicase loading factors ORC and Cdc6 could be called the initiator, as these factors recognise the origin, function similarly to DnaA in helicase loading and limit the potential of helicase activation in S-phase. The main difference is that the loaded DNA helicase in eukaryotes needs further activation in S-phase, while the bacterial helicase is active right away. 3. DNA replication origins In bacteria, a single DNA replication origin is used for replication of the entire chromosome, while in eukaryotes hundreds to thousands of origins are used for each chromosome [21]. Eukaryotic origins have been genetically defined in S. cerevisiae, they contain an essential A element and several important B elements [22]. The A and B1 elements represent DNA binding sites for ORC [1,23]. Interestingly, budding yeast Cdc6 only forms a stable complex with ORC on the correct DNA template, while origins carrying mutations in

the A and B1 elements induce Cdc6 ATP-hydrolysis and complex disassembly [24]. Thus, Cdc6 serves as a specificity factor; however, it is currently unclear whether this is also true for human Cdc6. In higher eukaryotes, specific origin sequences do not exist, instead, protein factors, epigenetic signatures or DNA structures target ORC to origins of replication [25]. Interestingly, ORC binding sites are partially linked with patterns of gene expression [25,26]. One reason is that both transcriptional active promoters and DNA replication origins require a stretch of nucleosome-free DNA [27–29]. AT- or G-rich sequences promote nucleosome exclusion and are enriched at origins. Origins are also enriched for specific histone variants, such as H3.3 or H2AZ, while chromatin modifiers, such as histone acetylase HBO1, and histone methyl transferase PRSet7, or the chromatin-remodelling complex SNF2H can promote pre-RC assembly [25]. Moreover, it was recently shown that DNA structures, involving DNA supercoiling can facilitate ORC recruitment to DNA [30], although ORC also has affinity for G-quadruplex structures [31]. It remains unclear whether these structural features are required for helicase loading. Protein factors like HMGA or ORCA/Lrwd1, which bind to ORC, facilitate recruitment of ORC to DNA and in part can promote pre-RC assembly [32–35]. Interestingly, even tethering of ORC to non-origin DNA is sufficient to create an artificial origin of DNA replication [36]. Consistent with this, it was found in yeast that ORC/Cdc6 can use non-origin DNA for helicase loading in vitro [3,4], indicating that this reaction has no intrinsic DNA sequence requirement. Thus it appears entirely possible that any DNA sequence could support helicase loading. However, the site specific recruitment of ORC to DNA generates an even spacing of replication origins. Remarkably, only some of the origins are used by the cell every cell cycle, while the other ones can be activated in case a replication fork becomes blocked due to severe DNA damage [37].

4. Recruitment of the MCM2-7 helicase to the replication origin ORC binds DNA and represents a platform for pre-RC assembly. This process is best understood in budding yeast. Here, ORC binds in an ATP dependent manner to the origin [1]. Indeed, Orc1-5 exhibit homology to AAA+ ATPases [23], however, only Orc1, Orc4 (in metazoans) and Orc5 bind ATP [38]. The ATPase activity of Orc1 is essential in vivo; interestingly, a conserved arginine finger in Orc4 is thought to activate Orc1 ATPase [39,40]. On the other hand, Orc6 has similarities with the transcription factor TFIIB [41], but the relevance of this finding is not yet known. The interaction of ORC with origin DNA promotes a structural change in Orc1 and Orc4 [42] and suppresses the ATPase activity of the complex [1,38]. This complex is competent to recruit Cdc6, another AAA+ ATPase, to the replication origin. ORC and Cdc6 form a stable complex on origin DNA, however, on non-origin DNA, the Cdc6 ATPase becomes activated leading to complex disassembly [23,24]. While ORC has a crescent shape, Cdc6 binding gives the complex a circular shape, with DNA likely passing through the centre [42,43]. This is in agreement with biochemical studies, which showed that a DNase I hypersensitive site within the ORC/DNA complex became protected upon interaction with Cdc6 [23]. Within ORC/Cdc6 the subunits are arranged in the following order (clockwise): Cdc6, Orc1, Orc4, Orc5, Orc2 and Orc3, while Orc6 binds to Orc2 [42] (Fig. 1). Remarkably, this ring shaped structure has a similar size and shape as the MCM2-7 helicase, therefore, it was suggested that the two rings interact with each other for pre-RC assembly [23,42]. The MCM2-7 complex consists of six AAA+ ATPase proteins that assume a toroidal two-tiered structure, where the smaller ring is made up of the N-terminal section and the larger ring of the C-terminal ATPase domains.

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Fig. 1. Cryo EM structure of the ORC/Cdc6 complex. The Orc1-5 and Cdc6 subunit location is indicated. While the location of DNA is not known, we indicate a potential location based on the OCCM structure (Fig. 2). This figure is based on EMD-5381.

In budding yeast, the MCM2-7 helicase needs to be imported into the nucleus prior to pre-RC formation and this reaction is dependent on the Cdt1 protein (Fig. 2) [44]. However, for MCM2-7 recruitment to origin DNA, ORC, Cdc6 and Cdt1

Fig. 2. Cdt1 and Mcm3 regulate ORC/Cdc6/Cdt1/MCM2-7 complex formation. A Mcm6-Cdt1 interaction is essential for nuclear import of MCM2-7 and OCCM formation. The Mcm3 C-terminus interacts with Cdc6 and is required for helicase recruitment by ORC/Cdc6. The Cdt1–Mcm6 interaction is essential for Orc1/Cdc6 ATP-hydrolysis, which in turn promotes Cdt1 release and OCM formation.

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Fig. 3. Cryo EM structure of the ORC/Cdc6/Cdt1/MCM2-7 complex. (A) Cryo-EM structure of the OCCM. The location of the Mcm3 and Cdc6 subunits is indicated. The DNA exit site from MCM2-7 is not known; a potential site is indicated. This figure is based on EMD-5625. (B) EM structure of Escherichia coli DnaB/DnaC. This figure is based on EMD-2321.

are required [3,4,45]. In the absence of ATP-hydrolysis an ORC/Cdc6/Cdt1/MCM2-7 (OCCM) complex is formed [46]. Although all Mcm subunits are highly homologous, containing a C-terminal AAA+ and an N-terminal domain, individual Mcm subunits have specialised functions. A C-terminal extension in Mcm6 contains an inhibitory domain, which blocks OCCM formation in the absence of Cdt1 (Fig. 2) [47]. Indeed, MCM2-7-C6, which is missing a conserved Mcm6 C-terminus, can bind to ORC/Cdc6 in a Cdt1-independent manner, while point mutants that interfere with the specific Cdt1–Mcm6 interactions block OCCM formation. Furthermore, deletion of the conserved Mcm3 C-terminus [43] or even a C-terminal V972R point mutation in Mcm3 abrogates binding of Cdt1/MCM2-7 to ORC/Cdc6 [43,45]. Thus, Cdt1 and Mcm3 are central for MCM2-7 recruitment to ORC/Cdc6 (Fig. 2). A recent cryo-EM structure of the OCCM complex revealed how ORC/Cdc6 and Cdt1/MCM2-7 interact with each other (Fig. 3A). The C-terminal section of MCM2-7 latches on the AAA+ ATPase domains of ORC/Cdc6, which leaves the MCM2-7 N-termini available to interact with a second MCM2-7 hexamer, suggesting a route for MCM2-7 double-hexamer assembly. Especially ORC/Cdc6 undergo significant structural changes upon OCCM formation. While ORC/Cdc6 is nearly flat, ORC/Cdc6 within the OCCM adopt a dome shape with the AAA+ domains now pointing towards the MCM2-7 C-termini [42,43]. Interestingly, the structure reveals that Mcm3 and Cdc6 are positioned next to each other, although they do not interact directly. Thus, the Mcm3 C-terminus becomes potentially rearranged during initial ORC/Cdc6–Cdt1/MCM2-7 interaction. Most contacts between ORC/Cdc6 and Cdt1/MCM2-7 are on one side of the complex (Fig. 3A). It remains unknown whether these interactions are required for helicase loading or alternatively function to remodel the complex for MCM2-7 double-hexamer formation.

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5. The role of ATP-hydrolysis during pre-RC formation Once the OCCM is formed, Cdt1 is released to produce a ORC/Cdc6/MCM2-7 (OCM) complex containing a single copy of each protein. This is a very fast reaction [47,48] and involves two ATPases, Orc1 and Cdc6. Initial work using extract-based assays identified that Cdc6 ATPase is of major importance for Cdt1 release [48], while a reconstituted system employing purified proteins showed that Orc1 ATP-hydrolysis also participates in Cdt1 release [47]. Moreover, direct pre-RC ATPase measurements showed that Cdc6 and Orc1 contribute equally to ATP-hydrolysis during Cdt1 release [47]. The analysis of MCM2-7 ATP-hydrolysis during preRC formation is ongoing. Initial work in Xenopus laevis using Mcm6 and Mcm7 mutants, indicated that MCM2-7 ATP-hydrolysis is not important for pre-RC formation, but for DNA synthesis [49]. While a study in budding yeast, employing a Mcm3 arginine finger ATP-hydrolysis mutant, suggested that MCM2-7 ATP-hydrolysis is not required for Cdt1 release, OCM formation or salt stable MCM27 double-hexamer formation. Further analysis of Mcm2, Mcm4 and Mcm5 ATP-hydrolysis mutants could reveal additional functions of MCM2-7 ATP-hydrolysis during pre-RC formation. Interestingly, the two factors that facilitate OCCM formation, Cdt1 and Mcm3, are also involved in ATP-hydrolysis during preRC formation. Specifically, the Cdt1–Mcm6 interaction is essential to promote Cdc6/Orc1 ATP-hydrolysis during pre-RC formation [47]. On the other hand, the Mcm3 C-terminus induces ORC/Cdc6 ATP-hydrolysis [45]. One possibility is that during OCCM formation a specific structural state is sensed, for example, a change in Cdt1 or MCM2-7, which then induces Orc1/Cdc6 ATP-hydrolysis and Cdt1 release. More detailed biochemical and structural analysis is required to fully understand how ATP-hydrolysis is initiated during pre-RC formation. ATP-hydrolysis dependent OCM formation is an important quality control step during pre-RC formation [45,47]. ORC, when phosphorylated by CDK, still supports OCCM formation; however, following Orc1/Cdc6 ATP-hydrolysis, the complex disassembles (Fig. 4). Thus helicase loading is blocked once CDK dependent helicase activation is initiated during S-phase. Similarly, an Orc1-5 complex, missing Orc6, supports OCCM formation, but falls apart in response to ATP-hydrolysis, suggesting that Orc6 could be located at the interface of ORC and MCM2-7. The ATP-hydrolysis driven disassembly guarantees that non-functional pre-RC complexes, which could block transcription or DNA replication, get actively removed from DNA.

Fig. 4. A pre-RC assembly model. (1) ORC is bound to DNA. (2) Cdc6 associates with ORC. (3) Cdt1/MCM2-7 associates with ORC/Cdc6. (4) Rapid ATP-hydrolysis by Cdc6 and Orc1 leads to Cdt1 release and OCM formation. This reaction is blocked by CDK. (5) OCM formation results in a structural change in MCM2-7 to generate a dimerization competent state of the MCM2-7 helicase and recruitment of a second MCM2-7 complex. The recruitment of the second MCM2-7 hexamer is a slow reaction and may require another ORC/Cdc6 complex. (6) The MCM2-7 loading finishes with the formation of a stable double-hexamer encircling dsDNA.

could break open the MCM2-7 ring and may promote helicase loading with a similar mechanism as observed in bacteria [43,52,53]. Consistent with that model, a density was found inside the centre of the MCM2-7 ring, which was tentatively assigned as DNA. If that density is in fact confirmed to be DNA, then MCM2-7 within the OCCM could represent a loaded form of the helicase. A more detailed structural or biochemical analysis is required to resolve this issue.

6. Helicase loading – what do we know?

7. The recruitment of the second MCM2-7 hexamer

The mechanism of eukaryotic helicase loading on DNA is of biological significance, but we are only beginning to understand how this reaction occurs. In bacteria helicase loading has been analysed in detail. Here DnaA unwinds a short stretch of origin DNA and then DnaC loads the hexameric DnaB helicase onto DNA [50]. Recent structural work detailed how six DnaC molecules associate with the N-terminal section of DnaB during helicase loading (Fig. 3B) [51]. While DnaB is a closed circular ring, the DnaC/DnaB complex has a right handed spiral shape, with an opening in the ring – adopting a lock washer conformation. DnaB ring opening allows DNA insertion, which is followed by DnaC ATP-hydrolysis and closing of the DnaB ring around single stranded DNA. In eukaryotes the helicase becomes loaded on double-stranded DNA. A recent cryoEM structure of the OCCM has shed light on this reaction [43]. It was found that DNA enters ORC/Cdc6 complex through a central hole. Moreover, while ORC/Cdc6 forms a near planar ring [42], the complex adopts a spiral shape within the OCCM [43]. The pitch of the spiral matches that of B-form DNA. Thus, an ORC/Cdc6 spiral

Several models have been proposed for MCM2-7 doublehexamer formation [53]. Initially it was suggested that MCM2-7 double-hexamer formation occurs in a concerted manner [4]. More recent work identified a stepwise model [46,47]. Indeed, the OCCM contains only a single hexamer and does not support binding of a second MCM2-7 hexamer [47]. However, the OCM is capable to recruit the second MCM2-7 hexamer [47,54]. Thus, Orc1/Cdc6 ATPhydrolysis and Cdt1 release must remodel the MCM2-7 helicase to establish this competence. Within the MCM2-7 double-hexamer each hexamer is connected to the other via an N-terminal interface [4]. Mutations in this interface do not interfere with OCM formation and even allow recruitment of a second MCM2-7 hexamer [54]. However, the interface mutants do not support saltstable double-hexamer formation, and instead arrest with two MCM2-7 hexamers still attached to ORC/Cdc6. Thus, structural changes at the N-terminal MCM2-7 hexamer interface appear to be required for the successful release of ORC/Cdc6 and the formation of a salt stable double-hexamer.

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One big question is: How is the second MCM2-7 hexamer recruited to the replication origin [53]? Two recent publications addressed this issue [45,47]. As OCM formation is a rapid reaction (seconds) and MCM2-7 double-hexamer formation is slower (minutes) it is possible to separate OCM formation and MCM2-7 double-hexamer formation into two steps. Using this stepwise helicase loading assay it was found that the presence of Cdt1 is a requirement for the first and second MCM2-7 hexamer [47]. Moreover, it was shown that the Mcm3 C-terminus is required for the recruitment of the first and second MCM2-7 hexamer, suggesting that each hexamer needs to interact with ORC/Cdc6 [45]. However, hard proof for the function of two ORC/Cdc6 complexes during pre-RC formation is necessary. In some way it appears unlikely that two ORC/Cdc6 complexes function during helicase loading, as most budding yeast origins contain only a single ORC/Cdc6 binding site [55]. But then again, how the second MCM2-7 hexamer becomes opened during pre-RC formation, if it does not involve ORC/Cdc6, is equally unclear. Although several models can be conceived – the two models discussed here (Fig. 4, step 5) appear most likely. 8. ORC/Cdc6 function as a MCM2-7 chaperone During pre-RC formation, ORC/Cdc6 promotes several structural changes in MCM2-7. After OCCM formation, Orc1 and Cdc6 ATP-hydrolysis remodels the MCM2-7 complex, which yields an OCM complex that is competent to recruit a second MCM2-7 hexamer. Importantly, ORC/Cdc6 stays attached to MCM2-7, until the MCM2-7 double-hexamer is formed. These activities are reminiscent of chaperones, which modify the structure of their client proteins. Chaperones, such as HSP70, are AAA+ ATPases, which use ATP binding and hydrolysis for their function. ORC/Cdc6 is very similar to chaperones, as they consist of AAA+ proteins, where ATP binding and hydrolysis being essential for their function. Nowadays, the term chaperone has been adopted more widely to describe proteins that interact with a partner-protein, helping the partner to acquire a new functionally active conformation before releasing the partner [56]. In that sense ORC serves as a MCM2-7-chaperone. ORC in cooperation with Cdc6 and Cdt1 specifically interact with MCM2-7; Cdc6 and Cdt1 may be considered as co-chaperones of ORC. Cdt1 is released from this initial complex and ORC, assisted by Cdc6, promotes a structural change in MCM2-7 that allows the binding of a second MCM2-7 hexamer (MCM2-7 dimerization). The dimerization competent state represents a new active conformation relevant for double-hexamer formation. The initial MCM2-7 dimer is in direct interaction with ORC/Cdc6 [54]. However, successful MCM2-7 dimerisation promotes the reorganisation of the helicase into the MCM2-7 double-hexamer triggering the release of ORC. Certain chaperones also function by preventing the formation of unwanted structures, e.g. histone chaperones [57]. Similarly, ORC functions during preRC formation to prevent the release of single-hexameric MCM2-7, which otherwise could generate a replication-competent helicase in G1 phase with detrimental effects on regulated DNA replication. These chaperone-like functions of ORC/Cdc6 likely evolved as a requirement for MCM2-7 double-hexamer formation.

5

MCM2-7 hexamers are orientated to each other, although it is clear that both hexamers encircle double-stranded DNA [3,4]. In this configuration, MCM2-7 can slide on double-stranded DNA in an ATP-hydrolysis independent manner, highlighting that DNA passes right through the centre of the MCM2-7 ring. This allows MCM2-7 to slide on chromatin. As multiple MCM2-7 doublehexamers become loaded at each replication origin, the ability for MCM2-7 to slide away appears important; otherwise, incoming MCM2-7 hexamer could not gain access the origin DNA. Then again – if sliding is a passive process, can MCM2-7 slide past nucleosomes? This appears energetically unfavourable – thus other energy consuming enzymes must push MCM2-7 double-hexamers along DNA. Consistent with this replication origins are frequently found near promoters [25]; therefore RNA-polymerases could clear the MCM2-7 double-hexamers away. However, other helicases or histone chaperones could function in a similar way. In principle, one double-hexamer per origin is sufficient to establish a bi-directional replication fork at each origin. Nevertheless, several more MCM2-7 double-hexamers are loaded, with only 1 in 10 MCM2-7 double-hexamers being used in an unperturbed S-phase [37,58]. Studies in Xenopus and human cells have revealed that these additional MCM2-7 complexes safeguard the cell in case of DNA damage [59,60]. If a replication fork becomes terminally blocked, the additional MCM2-7 complexes can become activated to re-establish a functional fork. It remains unclear how this process is targeted to the site of the blocked fork. 10. Conclusion The eukaryotic MCM2-7 helicase becomes loaded on doublestranded DNA. This is exceptional, as replicative helicases in bacteria or viruses become loaded on single-stranded DNA. Hence, the MCM2-7 double-hexamer needs to be activated in S-phase so that an active CMG helicase encircling single-stranded DNA is formed [6,7]. To hinder re-replication, helicase loading cannot occur after helicase activation [61–63]. Therefore it is essential that all DNA replication origins become licensed efficiently, with multiple MCM2-7 double-hexamers loaded on DNA. Inhibition of helicase loading is surveyed by a pre-RC/licensing checkpoint [64,65]. Accordingly, cells that fail to load enough MCM2-7 doublehexamers do not initiate DNA replication. Cancer cells frequently lose this checkpoint, making them vulnerable to helicase loading inhibition. The detailed analysis of the helicase loading mechanisms has generated a framework of knowledge that will propel the development of pre-RC inhibitors. These inhibitors hold the promise to kill cancer cells, while normal cells should be unharmed and progress from the licensing checkpoint upon removal of the inhibitor. Thus, studying helicase loading is of great interest to both basic scientists and clinicians. Acknowledgements We would like to thank Aloys Schepers and Michael Weinreich for comments on the manuscript and the Medical Research Council (MC U120085811) for funding. References

9. The MCM2-7 double hexamer The MCM2-7 double-hexamer is the final product of pre-RC formation. An electron microscopy analysis of the complex showed that two MCM2-7 hexamers are organised in a head-to-head orientation [4]. As the six MCM N-termini and C-termini assemble into two ring structures (Fig. 4, step 6), the overall complex is composed of four rings. It is currently unknown how the two

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Please cite this article in press as: Riera A, et al. Helicase loading: How to build a MCM2-7 double-hexamer. Semin Cell Dev Biol (2014), http://dx.doi.org/10.1016/j.semcdb.2014.03.008