REVIEW SUMMARY◥

DNA REPLICATION

Mechanisms for initiating cellularDNA replicationFranziska Bleichert,* Michael R. Botchan,* James M. Berger*

BACKGROUND: Cellular life depends on theability of organisms to accurately duplicate andtransmit genetic information between genera-tions. In bacteria, archaea, and eukaryotes, thisprocess uses sophisticatedmolecularmachineries,termed replisomes, which copy chromosomalDNA through semiconservative replication. Atthe core of these replication factories reside ring-shaped hexameric helicases that assist poly-merases in DNA synthesis by processively

unzipping the parental DNA double helix. Re-plicative helicases are loaded onto DNA by ded-icated initiator, loader, and accessory proteinsduring the initiation of DNA replication in atightly regulated,multistep process. Althoughinitiators and loaders are phylogenetically relatedbetween the bacterial and archaeal/eukaryoticlineages, the molecular choreography of DNAreplication initiation turns out to be surprisinglydiverse across different systems and employs dif-

ferent strategies to prepare replication originsfor subsequent replisome assembly.

ADVANCES: Early work in bacterial systemsestablished that initiator proteins recognizespecific sequence elements (termed replicationorigins). Nucleotide-dependent oligomeriza-tion of the bacterial initiator DnaA at originsfacilitatesDNAmelting and provides an access

point for the replicativehelicase, DnaB, which isrecruited to and loadedonto each of the single-stranded origin regionsby interactions with theinitiator and other loader

proteins to establish bidirectional replicationforks. It has recently become clear that thestrategies for origin recognition, helicase load-ing, and duplex DNAmelting in archaeal andeukaryal systems, as well as the order of thesesteps, deviate considerably from the arche-typal initiation events followed by bacteria.Although nucleotide-dependent interactionsbetween initiator subunits still control earlysteps of replication initiation and regulate theassociation of initiators with origin DNA, theydo not appear to contribute to DNA melting.Instead, archaeal/eukaryotic initiators [Orc inarchaea or themultisubunit origin recognitioncomplex (ORC) in eukaryotes] and helicase co-loading factors cooperate to recruit and loadthe replicative helicase motor, the minichro-mosome maintenance (MCM) complex, ontoduplex DNA in an inactive, double-hexamericform. Helicase activation, DNA melting, andreplisome assembly do not occur until sub-sequent cell cycle phases, preventing pre-mature replication and exposure of melted,single-strandedDNA regions toDNAdamagingfactors.

OUTLOOK: Although the core components ofthe initiation pathway have been identified,enabling reconstitution of the replication ini-tiation cascade in vitro with recombinant pro-teins from bacterial and eukaryotic systems,the mechanisms by which these initiation fac-tors function at the molecular level remain illdefined. Moreover, how replication initiationis temporally and spatially linked to the localchromatin environment and coordinated withother cellular (in bacteria and archaea) ornuclear (in eukaryotes) processes is not wellunderstood. Future studies that integrate ge-netic, biochemical, biophysical, and structuralapproaches will be pivotal for resolving thesequestions.▪

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The list of author affiliations is available in the full article online.*Corresponding author. Email: franziska.bleichert@fmi.ch(F.B.); mbotchan@berkeley.edu (M.R.B.); jmberger@jhmi.edu (J.M.B.)Cite this article as F. Bleichert et al., Science 355, eaah6317(2017). DOI: 10.1126/science.aah6317

Overview of similarities and differences in the initiation of DNA replication across thethree domains of life. Bacteria and archaea/eukaryotes share a requirement for an adenosinetriphosphate (ATP)–dependent initiator factor that helps catalyze the loading of two ring-shaped,hexameric helicases onto a replication origin, thereby nucleating the formation of a bidirectionalreplication fork. The bacterial initiator forms a multimeric assembly that actively melts the originbefore loading single helicase hexamers, whereas in archaea and eukaryotes, the helicase isloaded as an inactive dodecamer that then isomerizes into two active, single hexamers during orfollowing origin melting.

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REVIEW◥

DNA REPLICATION

Mechanisms for initiating cellularDNA replicationFranziska Bleichert,1*† Michael R. Botchan,2* James M. Berger1*

Cellular DNA replication factories depend on ring-shaped hexameric helicases to aid DNAsynthesis by processively unzipping the parental DNA helix. Replicative helicases are loadedonto DNA by dedicated initiator, loader, and accessory proteins during the initiation of DNAreplication in a tightly regulated, multistep process.We discuss here themolecular choreographyof DNA replication initiation across the three domains of life, highlighting similarities anddifferences in the strategies used to deposit replicative helicases onto DNA and to melt the DNAhelix in preparation for replisome assembly. Although initiators and loaders are phylogeneticallyrelated, the mechanisms they use for accomplishing similar tasks have diverged considerablyand in an unpredictable manner.

DNA is expressed as a linear triplet code,with numerous noncoding regulatory re-gions that render the genomes of cellsquite large. For example, in humans, eachof our 1013 cells carries roughly 2 m of

DNA; an individual’s body will synthesize morethan a light year’s worth of DNA from concep-tion to death [1016 cell divisions in ~70 years (1)].At the same time, the error rate by which base-mispairing mutations are accidentally introducedinto newly made strands is vanishingly low, lessthan one mistake per 108 nucleotide-additionevents. How such fidelity is achieved withoutsacrificing speed (a single replication fork cancatalyze 25 to 1000 nucleotide incorporationevents per second) to meet the demands ofcell proliferation remains a frontier questionin biology.The creation of new DNA strands from paren-

tal chromosomes depends on a large, dynamicmul-tiprotein complex termed the replisome,whichusesanarrayofDNAunwinding (helicase) and synthesis(primase/polymerase) functionalities, coupledwithvarious processivity (sliding ring clamps and theirloaders), protective [single-strandedDNA (ssDNA)binding], and scaffolding factors. Despite the clearfunctional conservation of core replisomal activ-ities, the helicases and primase/polymerases usedfor replicating DNA have evolved twice indepen-dently, with bacteria using one class of enzymesand archaea/eukaryotes using another (2). Thisdichotomy agrees well with the proposal that theadoption of DNA as the genetic material of livingorganisms occurred after the prior evolution ofRNA and protein-based self-replicating systems.

Replisome assembly is tightly linked to cell-cyclestatus (3). Dedicated factors known as initiatorsplay a critical role in recognizing replication startsites (termed origins) and in depositing replic-ative helicases onto origins, often with the helpof specialized loading factors. In eukaryotes,chromosome-bound initiator complexes and loadedreplicative helicases outnumber the actual ori-gins that become activated in S phase. Many ofthese dormant origins are required to rescuestalled forks (additional roles for the remaindermay yet be discovered), and the extent to whichthis choice is stochastic or determined by chro-mosomal context is not known (4–6). Timely andaccurate replication onset is extremely impor-tant to cell viability, because aberrant helicaseloading can directly lead to genetic instabilitiesand cellular transformation, as well as geneticdiseases such as primordial dwarfism (7–11).Here, we summarize the state of knowledge

with respect to how initiation factors operateat the molecular level and the extent to whichparticular initiation mechanisms overlap or aredistinct between bacteria, archaea, and eukar-yotes. Given space constraints, we apologize tothose colleagues whose work could not be cited.

Phylogeny and architecture ofinitiation enzymesInitiators/helicase loaders

Cellular replication initiators are closely relatedto one another evolutionarily (12), which initiallyled to speculation that the mechanisms under-lying origin recognition and helicase loadingmightbe largely conserved between different cellularsystems. In fact, the common subunit-level con-servation of initiator structure often belies verydifferent mechanisms of action.Replication initiators share a similar domain

architecture, in which individual protomers arecomposed of a AAA+ [adenosine triphosphatases(ATPases) associated with various cellular activ-ities] nucleotide-binding-and-hydrolysis domain,

coupled to a C-terminal helix-turn-helix (HTH)domain (Fig. 1A). Both domains directly inter-act with origin DNA regions (13–17). Bacteriausually encode a single initiator protein, knownas DnaA, whereas archaea rely on one or moreparalogous copies of an initiator known as Orc.Many eukaryotes use a six-subunit assembly termedthe origin recognition complex (ORC), five sub-units of which are predicated on a AAA+/HTHunit. Duplication and specialization of the an-cestral Orc1 gene of archaea likely allowed forgreater regulatory potential of initiator functionin eukaryotes, where commitment to S phaseinvolves activation of many origins and is linkedto other aspects of cell metabolism. Interesting-ly, only a subset of the ORC components hasbeen identified to date in certain unicellulareukaryotes, such as protists, suggesting that ini-tiators in these systems may be functionally moresimilar to those in the archaeal system than thosein other eukaryotes (18, 19).The sequence similarity of the AAA+ domain

in replication initiators is sufficiently preservedthat the enzymes can be grouped into their ownsubfamily, or clade, within the AAA+ superline-age (12) (Fig. 1B). By contrast, the HTH regionsof these proteins have apparently arisen throughtwo different fusion events: one in bacteria thatused a NarL/FixJ-type HTH fold and another inarchaea/eukaryotes that incorporated a winged-helix (WH) domain (13, 20). The core AAA+/HTHelement of initiators is often appended withdistinct auxiliary folds, which offer additionalfunctionalities that can affect origin recognition,helicase recruitment, and initiator interactions(Fig. 1A).The AAA+ domain plays a central role in con-

trolling higher-order initiator assembly and func-tion (Fig. 1C). AAA+ proteins in general formhomo- and heterooligomers in which the aden-osine triphosphate (ATP)–binding site of onesubunit is complemented by catalytically func-tional amino acids from a partner protomer(notably a basic side chain such as arginine,termed an “arginine finger”) (21). This arrange-ment allows initiators such as ORC and DnaAto assemble into large cracked rings or helicalarrays, respectively (16, 22) (Fig. 1, D and E).The bipartite binding and hydrolysis of ATPfurther allows for allosteric coupling betweensubunits to switch initiators between differentfunctional states (Fig. 1C).Although some initiators appear capable of in-

dependently carrying out both origin recogni-tion and helicase recruitment/deposition [e.g.,Helicobacter pylori and Pseudomonas spp. DnaA(23–25) and SulfolobusOrc1 (26)], many are aidedby specialized helicase-loading factors. Some, suchas eukaryotic Cdt1 (27, 28), are noncatalytic. Others,such asDnaC/DnaI of bacteria andCdc6 of eukary-otes, are AAA+ ATPases and close evolutionarycousins of replication initiators (12, 29) (Fig. 1B).Like DnaA and ORC, ATP-dependent helicaseloading factors can assemble into larger, homo-meric and heteromeric arrays (e.g., DnaC canform a spiral assembly, while Cdc6 appears cap-able of docking into an open ORC ring) (16, 30, 31).

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1Department of Biophysics and Biophysical Chemistry, JohnsHopkins University School of Medicine, Baltimore, MD 21205,USA. 2Department of Molecular and Cell Biology, Universityof California Berkeley, Berkeley, CA 94720, USA.*Corresponding author. Email: franziska.bleichert@fmi.ch (F.B.);mbotchan@berkeley.edu (M.R.B.); jmberger@jhmi.edu (J.M.B.)†Present address: Friedrich Miescher Institute for BiomedicalResearch, Basel, Switzerland.

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Here again, the joint AAA+ ATPase site is criticalfor controlling the activity of these enzymes.

Replicative helicases

Helicases comprise the front end of the repli-some. Tasked with unwinding the entire genome,replicative helicases have evolved to couple highrates of speed (when working alongside cognatepolymerases) with highly processive movement.Cellular replicative helicases universally formdonut-

shaped hexamers and are thought to encircle oneof the two DNA strands during translocation tophysically pry apart a downstream duplex (32, 33)(Fig. 2, A and B). The use of six independent yetcoupled ATPase sites provides a highly coordi-nated means for generating directional movementand the motive force necessary to help separateparental DNA (32).Unlike initiator/loaders, cellular replicative heli-

cases actually derive from two distinct ATPase

branches. One, exemplified by the DnaB-familyhelicases of bacteria, uses a RecA-type ATPasefold (34). The other lineage, the MCMs (mini-chromosomemaintenance proteins), is a AAA+ATPase, albeit from a clade distinct from thatcomprised by replication initiators (12) (Fig. 1B).Although both DnaB- and MCM-class helicasesshare a distant ancestral ATP binding module[the additional strand catalytic glutamate (ASCE)or P-loopATPase fold] (12), their ATPase subunits

Bleichert et al., Science 355, eaah6317 (2017) 24 February 2017 2 of 10

Fig. 1. Phylogenetic and structural kinship of replication initiators. (A) Initiators are multidomain proteins, comprising auxiliary, AAA+ ATPase, andHTH domains. Specific domain functions are indicated. (B) Replication initiators and helicase loaders are closely related evolutionarily, forming aseparate AAA+ superfamily clade (12). AAA+ ATPases involved in DNA replication belonging to other AAA+ clades are also shown (for completeness,clades not containing DNA replication proteins are included in the diagram). The helix 2 insert, SFIII helicase, HCLR (HslU, ClpX, Lon, RuvB), and Pre-sensor 2 insert are separate clades within the Pre-sensor 1 hairpin superclade. (C) ATPase sites in AAA+ oligomers (shown for DnaA) [Protein Data Bank(PDB): 2HCB (22)] are formed at the interface between adjoining protomers and contain characteristic sequence elements for ATP binding andhydrolysis. In some instances (e.g., in MCMs), the Sensor 2 arginine can be provided in trans. (D) Bacterial DnaA [PDB: 2HCB (22)] and (E) eukaryoticORC [PDB: 4XGC (16)] assemble into oligomers stabilized by interactions between the AAA+ and HTH domains of adjacent protomers.

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are oriented in a physically orthogonal way withrespect to one another and to substrate nucleicacid in a hexameric ring (35) (Fig. 2, C and D).Consequently, key functional loops and aminoacids within bacterial and archaeal/eukaryoticreplicative helicases reside on separate secondarystructural elements thathave evolved convergently,and the two motors travel with opposite polarityon DNA (Fig. 2A).The central ATPase elements of both DnaB-

class enzymes and MCMs have been augmentedwith various unrelated folds that serve essentialfunctions during replication initiation and DNAunwinding. For example, the N termini of bothhelicases contain evolutionarily distinct scaffold-ing domains that can serve as binding sites forssDNA and that provide a collar of additionalcontacts that hold the hexameric rings together(36–41). MCMs also retain a highly mobile WHfold C terminal to their central ATPase core ofvariable and enigmatic function (42, 43).

Initiator-dependent origin recognition

Origins are cis-acting DNA sequences that marksites of initiator-dependent replisome assembly.In prokaryotes and budding yeast such as Saccha-romyces cerevisiae, origins are identified by con-served DNA sequence motifs (44–47). By contrast,ORC in multicellular organisms shows no DNAsequence specificity for binding (48, 49). InS. pombe and metazoans, origin specificity isfluid and appears to rely on local context such aschromatin status and/or accessibility, rather thanon sequence. For example, origin recognition infission yeast depends on an A/T hook domainappended to Orc4 (50), whereas in metazoans,chromatin accessibility, epigenetic features, andancillary ORC interactions with other proteinsare influencing factors (51–56).

Bacteria

Bacterial origins are generally specified by asingle locus that contains a number of definedinitiator binding sequences. Some of these se-quences (e.g., DnaA boxes) conform to a fairly uni-form consensus motif that can be occupied byDnaA throughout the cell cycle, whereas othersare degenerate and bound only during initiation(44, 57–59). The DnaA box appears to be recognizedexclusively by the HTH domain of DnaA (13, 60);closely spaced arrays of DnaA boxes in turn pro-mote the ATP-dependent self-assembly of mul-tiple DnaA subunits into a higher-order helicaloligomer through lateralAAA+/AAA+ interactions(22, 61, 62). The precise structure of an assembledDnaA/origin nucleoprotein complex has yet to beestablished, but it likely involves the wrapping ofDnaA box–containing DNA segments about theexterior of the DnaA spiral (Fig. 3A). There is evi-dence in somebacterial species that these extendedDnaA/origin superstructures can accommodateshort local bends or large loops of DNA as well(63–65).

Archaea

Like bacteria, the replication origins of archaeaare generally demarcated by the presence of

Bleichert et al., Science 355, eaah6317 (2017) 24 February 2017 3 of 10

Fig. 2. Bacterial and archaeal/eukaryotic replicative helicases are hexameric motors but belongto different evolutionary ASCE ATPase lineages (12). (A) Strand displacement by replicative helicasemovement (arrows) results in duplex DNA unwinding. (B) Active replicative helicases form hexamersthat encircle ssDNA. E. coli DnaB bound to ssDNA [PDB: 4ESV (156)] and eukaryotic Mcm2-7 (com-plexed with GINS and Cdc45) [PDB: 3JC5 (157)] are shown. (C) Topology diagrams of the motor do-mains of DnaB and MCM highlight the related but distinct folds (RecA versus AAA+, respectively) ofthese two ATPases, with ATP binding/hydrolysis and DNA binding motifs oriented differently with re-spect to each other. (D) The disparate locations of conserved sequence motifs and DNA binding loopscause the RecA-like and AAA+ folds to be positioned almost perpendicular to each other in the contextof the DnaB and Mcm2-7 hexamers (indicated by green arrows) (35). PDBs: DnaB, 4ESV (156); Mcm2-7,3JA8 (40). Only the five central b strands, the Walker A motif with its preceding helix, and the DNAbinding loops are shown and colored as in (C). Motor domains are depicted in gray.

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select sequence elements. These repeats, known asorigin recognition boxes [ORBs (47)], serve as theprincipal means for localizing Orc-family initia-tors to multiple origins in archaeal genomes [al-though not all of them seem to rely on Orc forinitiation (66)]. Unlike DnaA, archaeal Orc pro-teins use a bipartite strategy for ORB recognition,in which the initiator WH domain associates witha conserved origin submotif (known as a mini-ORB), and the outer edge of the central AAA+ATPase fold binds to a second, less-well-conservedorigin segment (15, 17) (Fig. 3B). Multiple ORBscan often be found in close proximity to oneanother in archaeal origins, sometimes in over-lapping pairs, other times in tandem, linear ar-rays. Although Orc proteins have been shown tobind tightly to ORB sequences (47, 67–70), evi-dence for the formation of higher-order, ATP-controlled Orc oligomers (such as those formedbyDnaA orORC) has been limited, althoughmanymembers of this family appear to have all therequisite conserved catalytic amino acids neces-sary to form a canonical, bipartite AAA+-typeATPase site.

Eukaryotes

In many eukaryotes, including fungi, plants, andmetazoans, the hexameric ORC assembly is primar-ily responsible for specifying prospective originsand sites of replicative helicase loading (71, 72).The five subunits of ORC that contain a AAA+domain (Orc1 to 5) form a notched ring, whereasthe sixth (Orc6) binds to the rest of the particleby a short C-terminal a helix that interacts withan auxiliary domain in Orc3 (16, 73). Only one ofORC’s subunits (Orc1) exhibits ATPase activity(74–76). Two others—Orc4 and Orc5—bind butdo not appear to hydrolyze ATP, whereas Orc2and Orc3 seem to have lost the ability to asso-ciate with nucleotide entirely (16, 74–77).Structural evidence indicates that the ORC

ring encircles DNA, likely using the AAA+ andWH folds of the Orc1 to 5 subunits as bindingdeterminants (16, 78) (Fig. 3C). This interactionmay account for the observed coupling betweenORC’s ATPase activity and productive originengagement (49, 71, 75). ATP binding by ORCappears sufficient for helicase loading in vitro,whereas ATP hydrolysis by ORC is inversely cor-related with DNA binding and is required formultiple rounds of helicase loading, suggestingthat ATP hydrolysis helps to reset ORC for sub-sequent loading events (74, 75, 79–81).There is increasing evidence, particularly out-

side of budding yeast, that ORC has multiplemeans of associating with DNA before the for-mation of a helicase-loading competent complexwith DNA. This capacity derives in part fromauxiliary DNA- and chromatin-binding domainssuch as a bromo-adjacent homology (BAH) do-main (found in many Orc1 subunits), a transcrip-tion factor IIB (TFIIB)–like domain (Orc6), and anA/T hook repeat (S. pombe Orc4) (50, 56, 82, 83)(Fig. 3C). Thus, in those eukaryl lineages that relyon the existence of relatively hard-wired originsequences (such as S. cerevisiae), origin speci-fication appears predominantly carried out by

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Fig. 3. Initiators bind origin DNA and mark sites for starting DNA replication. (A) In bacteria, theDnaA initiator is recruited to replication origins by DnaA boxes and other sites using its HTH domain.DnaA binding to duplex DNA facilitates DnaA filament formation and promotes duplex melting; ssDNAis sequestered by the AAA+ domains of a DnaA filament, creating a single-stranded substrate for load-ing two DnaB hexamers onto complementary DNA strands in opposite directions. Structures of DnaAbound to duplex [PDB: 1J1V (13)] and ssDNA [PDB: 3R8F (14)] are shown as insets. (B) The archaealinitiator Orc binds to specific duplex DNA sequence elements at replication origins, termed mini-ORBs, using its WH domain, and to adjacent DNA using its AAA+ fold. A crystal structure of archaealOrc1 bound to DNA is shown at right [PDB: 2QBY (17)]. (C) In eukaryotes, the initiator ORC engagesduplex DNA in a sequence-independent (S. cerevisiae excepted) but ATP-dependent manner, likelyusing DNA binding elements that line the ORC’s central channel. A structural model for DNA-ORC(based on the crystal structure of apo-ORC) [PDB: 4XGC (16)] is shown at right, docked into theelectron microscopy (EM) structure of an ORC-DNA-Cdc6 complex [Electron Microscopy Data Bank(EMDB): 5381 (31)]. Initiator recruitment to chromosomes is facilitated by secondary DNA- or nucleosome-binding elements.

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the AAA+/WH domains of ORC that encircleDNA. By contrast, local chromatin cues that at-tract other ORC regions—many of which remainto be elaborated—probably serve this role in mostother eukaryotes.

From origin melting to helicase loadingand vice versa

Once aDNA region has been selected andmarkedby the appropriate initiator as an origin, the nextstep along the initiation pathway involves a seriesof events that culminate in the placement of twocopies of a replicative helicase around comple-mentary single DNA strands. Insight into howhexameric helicases become loaded ontoDNAhasbeen influenced by landmark studies of polymer-ase sliding clamps and their loaders [reviewed in(84, 85)]. Sliding clamps form rings that encircleDNA and latch onto polymerases to improve theprocessivity of strand synthesis. The clamp load-ers themselves are AAA+ ATPases that descendfrom a clade closely related to that of replicationinitiators andhelicase loaders (12) (Fig. 1B). Clamploaders operate by forming notched rings thatfirst bind to and stabilize an open-ring state ofa sliding clamp and subsequently associate withtarget DNA (which enters through the notch);the loaded clamp is then released in a closed-ringform (Fig. 4A). ATP binding and hydrolysis controlthe clamp binding and release cycle, respectively.Because replicative helicases are also rings

and the ATP-dependent initiator/helicase-loaderproteins are evolutionary cousins of clamp load-ers, it has often been assumed that the mech-anisms for loading the two classes of proteinswould largely parallel each other (78, 86, 87).However, there is substantial variation as towhich replicative helicases favor forming nat-urally open- or closed-ring states andwhich favorforming stable hexamers versus dissociatedmonomers (88). As a consequence, a considerablenumber of divergent, parallel strategies haveevolved for catalyzingwhat is an essential, ancient,and physically challenging process. Even the tim-ing by which helicase loading occurs with respectto origin melting varies in different organisms.

Bacteria

In bacteria, origin melting precedes helicase recruit-ment and loading (Fig. 3A). The formation of theATP-dependent nucleoprotein complex betweenDnaA and duplex originDNA serves as a precursorfor the subsequentmelting of adenosine-thymidine(AT)–rich regions situated adjacent to the primaryassembly locus (89–91). Melting likely occurs bydirect interactions between the AT-rich duplexDNA and the AAA+ ATPase domains of DnaA; oneof the two melted strands is sequestered and sta-bilized by a DNA-stretching mechanism akin tothat used by the RecA homologous recombina-tion protein (14). Precisely how DnaA’s ATPasedomain engages the duplex AT-rich region justbefore melting has yet to be established but mayinvolve the recognition of a repeating trinu-cleotide motif by DnaA’s AAA+ domains that hasbeen shown to be important for subsequent sta-ble ssDNA-DnaA filament formation (92).

Once DnaA has properly opened a replicationorigin, two copies of a DnaB-family helicase arerecruited and loaded onto the ssDNA. Thus far,three varying approaches have emerged bywhich this process occurs (88). In certain Gram-negative bacteria (such as E. coli), six copies ofthe DnaC helicase loader first bind to and crackopen aDnaB hexamer in an ATP-facilitatedman-ner that is highly analogous to how clamp loadersopen a sliding clamp (87) (Fig. 4B). An auxiliaryN-terminal domain onDnaA helps to localize DnaB-DnaC complexes to the melted origin (by bindingto the DnaB N-terminal collar), where one copyof the complex loads onto each of the two singleDNA strands in opposite orientations (93–96);interactions between DnaA and DnaCmay assistthis process (30). The role of ATP hydrolysis andthe precise order of events following DnaB-DnaCloading remain poorly understood, but it hasbeen observed that ATP-DnaC inhibits DnaBhelicase activity, that ATP hydrolysis by DnaCoccurs after DnaB loading, and that RNA primersynthesis by the DnaG primase protein helps toeject DnaC (97, 98). Large conformational changesin the DnaB N-terminal region appear not onlyto help coordinate DnaC release but also to ac-tivate the helicase for productive DNA unwind-ing (37).In comparison with the ring-opening mech-

anism used by E. coli, in some Gram-positivebacteria (such as Bacillus subtilis), the replic-ative helicase appears to be assembled into a

hexamer around ssDNA by both a DnaC-familyhelicase loading factor (known as DnaI) andnoncatalytic cochaperones (99). During the load-ing process, the helicase/helicase-loader complexprobably still progresses through a 6:6 intermed-iate that encircles ssDNA (100, 101), and primasecan bind to this dodecamer before helicase load-er dissociation. However, precisely when certainfactors bind to and release another, or how ATPturnover controls the helicase deposition andrelease cycle, has not been defined.Although the use of a dedicated DnaC/DnaI-

family helicase loader represents the textbookview of replication initiation in bacteria, thereare a number of bacterial species (perhaps amajority) that appear to lack such proteins(23, 24, 102). In such organisms, a recently iden-tified factor, DciA, has been suggested to take theplace of DnaC/I protein in the loading process(103). Alternatively, in H. pylori (which also lacksDnaC/I), the replicative helicase has been foundcapable of assembling into not just a hexamericring but a “head-to-head” (N terminus to N ter-minus) double hexamer (104, 105). Expression ofthis protein in E. coli can overcome a need forDnaC during replication (24), suggesting that itmay independently load onto DNA when thecorrect origin substrate is presented.

Archaea

The exact order of DNA melting and helicaseloading in archaea has yet to be firmly established.

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Fig. 4. The E. coli helicase loader DnaC follows a clamp loader–like strategy for replicative helicaseloading. (A) Clamp loaders are pentameric AAA+ assemblies that help open ring-shaped sliding clamps.An open clamp-loader–clamp complex binds a primer-template junction within its central pore; ATP hydro-lysis by the loader leads to clamp closure and loader dissociation, trapping the clamp on DNA. (B) E. coliDnaC, a monomer in solution, uses its AAA+ domains to self-oligomerize upon binding DnaB (forming aDnaB6-DnaC6 complex), promoting helicase ring opening. ssDNA enters through the crack in the ring tobind the central channel of the DnaBC complex. DnaB ring closure and DnaC release leave the DnaBhelicase loaded on ssDNA. Structures of intermediate states for clamp and DnaB loaders are shown in(A) and (B) [PDB: 3U60 (158); EMDB: 2321 (87)].

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Although early studies detected the ATP-dependentdeformation of origin DNA by archaeal Orc(67, 106), it is unclear whether this activity re-flects bone fide DNA melting (66). Recent workhas reported that Orc prebound to ORB sequencescan promote the loading of pre-opened Mcmhelicases onto duplex DNA; ATP binding to Orcseems to be required for loading, but how nucleo-tide controls initiator activity is not well under-stood (26). Once loaded, structural and biochemicalstudies have found that archaeal Mcms can formdouble hexamers (as are formed in eukaryotes)(107–110) and that archaea have homologs of theeukaryotic Mcm-associated factors GINS (Go, Ichi,Ni, San) and Cdc45 (111–113). Nonetheless, thecurrent data indicate that archaeal Orc does notoperate by a clamp-loader type mechanism, butrather serves more as a recruitment factor forchaperoning and aligning the helicase aroundreplication origins (26). This divergence is consist-ent with the notion that DNA replication evolvedindependently in bacterial and archaeal lineages.

Eukaryotes

In eukaryotes, helicase loading occurs beforeorigin melting, an order opposite to that of bac-teria (Fig. 5). The replicative helicase is firstloaded in an inactive state in G1, whereas DNAopening is coupled to helicase activation in S phase(114, 115). ORC and Cdc6, together with Cdt1,collaborate to deposit two copies of the Mcm2 to7 (Mcm2-7) complex around duplex DNA as adouble hexamer that is stabilized by interactionsbetween theMcm2-7N-terminal domains (116–118).

Mcm2-7 loading occurs when cyclin-dependentkinase (CDK) activity is low, because CDK in-hibits several loading factors by phosphoryl-ation (119–122). During S phase, new rounds ofMcm2-7 loading byORC are blocked, and a secondkinase (theDbf4-dependent kinase, orDDK) phos-phorylates the latent helicase (123), preparingthe double hexamer for activation and eventualseparation (124, 125). Several other noncatalyticproteins (Sld2, Sld3, Sld7, and Dbp11 in buddingyeast), some of which require CDK phosphoryla-tion, thenhelp chaperone twoaccessory/scaffoldingfactors—the GINS tetramer and Cdc45—ontoMcm2-7, forming an 11-subunit complex, the CMG(Cdc45,Mcm2-7, GINS), that encircles ssDNA andis now active for DNA unwinding (126–128).Overall, the coupling of helicase loading and ac-tivation with distinct phases of the cell cycle pre-vents both the premature formation of ssDNA andrereplication, helping to ensure that a given origincan support only one round of initiation.In metazoans, the players in the Mcm2-7

loading and activation process are largely pre-served with that of budding yeast, except thatseveral of the noncatalytic chaperones havediverged considerably. For example, Sld3 canbe found as a domain in a much larger proteinknownasTreslin (129–131); themetazoanorthologof Dbp11 is thought to correspond to TopBP1 (132),a BRCA1 C-terminal (BRCT) domain DNA damageresponse protein; and Sld2 has been proposed toreside in the N terminus of the DNA repairhelicase, RecQ4 (133). Metazoans also have evolveda new factor, Geminin, which is not found in

budding yeast but which binds to Cdt1 to reg-ulate its activity in the Mcm2-7 loading process(134–136).Why are so many different proteins required

simply to establish and open a eukaryotic rep-lication origin? Part of the answer likely lies inthe need for eukaryotes to tightly regulate originfiring to respond to changing chromatin land-scapes (e.g., for progressing through different de-velopmental fates) and to avoid DNAdamage andgenetic instabilities arising from aberrant rerepli-cation (8, 137). However, some of the complexitymay be due to the path eukaryotes took in loadinginactive helicase complexes onto duplex DNA andthen needing to awaken these helicases through acomplex, programmed isomerization event thatleads to origin melting and ssDNA encirclementduring S phase. Given the evolutionary kinshipbetween replication initiators and the knownability of DnaA to open replication origins directly,onemight have assumed that ORCwould serve asa “meltase” that forms the initial replicationbubble. However, it has become clear that theprimary function of the initiator is to help loadthe Mcm2-7 complex onto duplex DNA (116, 117)and that it is the activation ofMcm2-7, combinedwith its own ATPase activity and its metamor-phosis into the CMG, that drives duplex separa-tion (127, 138).How the Mcm2-7 complex is loaded by ORC

onto DNA is a major question in the field. Onemodel for this event invokes a clamp-loader typeof approach, with ORC and Cdc6 collaborating tocrack open an otherwise closed Mcm2-7 ring to

Bleichert et al., Science 355, eaah6317 (2017) 24 February 2017 6 of 10

Fig. 5. Eukaryotic replicativehelicase loading proceedsthrough a mechanism distinctfrom the canonical clamp-loader reaction. ORC binds toDNA and recruits its coloaderCdc6, forming a hexameric AAA+assembly that encircles duplexDNA. One Mcm2-7 hexamer,which can exist in an open-ringstate in solution (139–141), isrecruited to the DNA duplex byORC-Cdc6, aided by anothercoloader, Cdt1. After recruitment,the first Mcm2-7 complex is heldin place by ORC, while Cdc6 andCdt1 dissociate (145). With thehelp of another Cdc6 and Cdt1,ORC facilitates the loading of asecond Mcm2-7 complex ontoduplex DNA to yield a head-to-head Mcm2-7 double hexamer(116, 117). At the beginning ofS phase, helicase activationpromotes duplex DNA melting,CMG formation, and bidirectionalreplisome assembly. Structuresof key intermediate states areshown where available [EMDB:5429 (141) and 5625 (78); PDB:3JA8 (40)].

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deposit the helicase onto DNA (78, 86). However,a distinguishing feature of the clamp-loader re-action is that the loader first binds and opens atarget clamp before it places it around DNA (84)(Fig. 4A). This order of events (which is echoedby the action ofE. coliDnaC onDnaB) (Fig. 4B) issensible, because there is no free DNA end tothread througha closed clamp (or a closedhelicasering) during loading. For ORC-Cdc6, however,there is compelling evidence that these two fac-tors form a stable, closed-ring complex that en-circles duplex DNA before it can associate withMcm2-7 and Cdt1 (16, 31) (Fig. 5).A second difference between clamp-loading

systems and Mcm2-7 loading in eukaryotes isthat Mcm2-7 complexes [like their archaeal rel-atives (26)] readily formpre-opened rings (139–141).In budding yeast, there is evidence that ATP canhelp stabilize a closed form of the Mcm2-7 ring(139); however, nucleotide alone has proven in-sufficient to induce Mcm2-7 hexamer closure inother systems studied to date (140, 141). Althoughthis dichotomy may at first seem contradictory, itlikely reflects a simple difference in the tendencyof free Mcm2-7 hexamers from different orga-nisms to preferentially equilibrate between openversus closed ring forms, rather than to adopt oneor the other state in an all-or-nonemanner. As forCdt1, this protein tightly associates with Mcm2-7in budding yeast (although not in metazoans)(116, 142, 143) and appears to block an innateability ofMcm2-7 to bindDNAon its own (116, 117).How Cdt1 accomplishes this task is not known; itcould either sterically prevent access through theMcm2/5 gate or it may restrain an Mcm2-7 ringin an open state to prevent the helicase fromstably closing around DNA.Collectively, the available data indicate to us

and others (144) that replicative helicase loadingin eukaryotes does not proceed through a con-ventional clamp-loader type of process. Instead,ORC-Cdc6 may act as a combined recruitmentplatform and alignment jig, akin to what hasbeen seen in archaea (26). In this scenario, theATPase cycle of ORC-Cdc6 would not activelyopen a closedMcm2-7 ring but rather would helpfacilitate the timely and/or efficient ejection ofCdt1 from Mcm2-7, either unblocking or dere-straining the Mcm2-7 hexamer to allow it tostably encircle duplex DNA.The mechanism for loading of the second

Mcm2-7 hexamer is highly enigmatic. The ORC-Cdc6 assembly is inherently asymmetric, yet asingle ORC appears to catalyze the formationof a two-fold symmetricMcm2-7 double hexamer(145) (Fig. 5). Not only does ORC remain as-sociated with the first loaded Mcm2-7 ring butalso a new copy of both Cdc6 and Cdt1 is neededto load the second Mcm2-7 hexamer (145). Thesefindings suggest that theORC-Cdc6 complex holdsthe firstMcm2-7 ring (in particular the N-terminalcollar of the helicase) in an unknown conforma-tion that, after the bindingof anewCdc6protomer,recruits a second Cdt1-bound Mcm2-7 hexamerthrough collar-collar interactions (125, 146). Onediscordant aspect of this model is that it is dif-ficult to reconcile with the finding that the C-

terminal region of Mcm3, which directly inter-acts with Cdc6 (78), appears necessary for theloading of both hexamers (147). There is alsoevidence that ATP binding and hydrolysis byMcm2-7 play key, but as yet poorly understood,roles in the loading reaction (81, 138). Nonetheless,the persistence of the initial ORC–Mcm2-7 com-plex would seem useful for preventing an other-wise unstable single Mcm2-7 hexamer from losingits grip and dissociating from the DNA. Theexistence of an ORC–Mcm2-7 intermediate alsoargues against “clamp-closer” types of helicaseloadingmodels [as can be catalyzed by the phageT4 clamp loader (148, 149)], instead implicatingthe stable binding of the secondMcm2-7 hexameras a motivating factor for ring sealing.Once formed, theMcm2-7 double hexamer is a

highly stable particle that persists until the onsetof S phase (116–118). Preparation of the doublehexamer for dissociation is promoted by the ac-tion of DDK, which phosphorylates the extremeN termini of Mcm2, Mcm4, and Mcm6 (150–153);how this event begins to disrupt the stable, highlyinterdigitated structure formed by the twoMcm2-7N-terminal collars is uncertain. Even less is knownabout the next series of events that lead to CMGformation. ATP turnover byMcm2-7 hexamers hasbeen proposed to play a role in untwisting ofduplex DNA (39, 40, 138), but direct evidence forthis action is still lacking. Budding yeast Sld3and Sld2 chaperones (held in proximity to oneanother by Dpb11) have been found to engageGINSandCdc45, respectively, aswell as to interactwith ssDNA (154, 155); these and other findingssuggest that there is a highly coordinated handoffbetween the dissolution of the double hexamer,the timing of origin melting, the opening of thetwo Mcm2-7 rings to allow escape of one singlestrand of DNA from each hexamer, and the trans-fer of GINS/Cdc45 to Mcm2-7.

Concluding remarks

We have highlighted the similarities and differ-ences that nature has used to solve two complextopological problems that occur during the initia-tion of DNA replication: the loading of ring-shaped replicative helicases onto replication originsand the melting of duplex DNA in preparationfor assembling two independent replisomes. Anunanticipated outcome of the data available todate is that eukaryotic and archaeal Orc/Cdc6proteins appear to work in a manner distinctfrom the structurally related clamp-loader com-plexes, which bring processivity factors to DNApolymerases. Likewise, DnaA and ORC, whichserve similar roles as replication initiators andshare both close homology and certain physicalcharacteristics, also operate on origin DNA in afundamentally differentmanner,melting the originin one instance but not the other. These mech-anistic differences are striking and raise thequestion of how and why bidirectional repli-cation is universally conserved. Studies haveshown that the rate-limiting steps for replicationoccur during initiation, underscoring the com-plexity of simultaneously constructing two forks.Only by continuing to study DNA replication in

multiple model systems will we begin to fully ap-preciate the power of evolution and the creativityof nature in their persistent tinkeringwithwhat isone of the fundamental processes of life.Note added in proof: While this review was in

press, three research articles were published thatused single-molecule biochemistry and cryo-electronmicroscopy to illustrate that the Mcm2-7 ring isopen before its engagement of ORC-Cdc6 and thatthe N- and C-terminal tiers of the Mcm2-7 gateclose sequentially around duplex DNA (159–161).These results are consistent with the idea that ORC-Cdc6 does not act through a clamp loader–typemechanism duringMcm2-7 loading. A fourth paperraises important questions about the directionthe CMG faces during DNA translocation (162).

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ACKNOWLEDGMENTS

All authors contributed to the writing and editing of themanuscript. This work was supported by NIH grants GM071747 toJ.M.B. and CA030490 to M.R.B. and J.M.B.

10.1126/science.aah6317

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Mechanisms for initiating cellular DNA replicationFranziska Bleichert, Michael R. Botchan and James M. Berger

originally published online February 16, 2017DOI: 10.1126/science.aah6317 (6327), eaah6317.355Science 

, this issue p. eaah6317Scienceeukaryotes, prokaryotes, and archaea and many outstanding questions to be answered.bidirectional replication initiation. There are key similarities and multiple differences in replication mechanisms betweenand helicase loading. These processes identify potential origins of replication and prepare them for subsequent

review replication initiation across the three domains of life, with a focus on origin selectionet al.assemblies. Bleichert Accurate duplication and transmission of genetic information to the next generation requires complex molecular

Diverse molecular choreography of replication

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