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148 nature structural biology • volume 10 number 3 • march 2003

DNA polymerases replicate chromosomalDNA during the S phase of the cell cycle.To duplicate DNA, they require access to asingle stranded DNA (ssDNA) template,which necessitates separation of the DNAstrands. This task is handled by DNA heli-cases, a group of enzymes that catalyze theunwinding of duplex DNA. They use theenergy derived from nucleoside triphos-phate hydrolysis to translocate along onestrand of the DNA duplex, thus displacingthe complementary strand1. A subgroupof DNA helicases form hexameric rings atreplication forks, specific DNA structuresformed at the origin of replication.

Although hexameric helicases fromdiverse organisms have been extensivelystudied, only limited high-resolutionstructural information is available forthese proteins1. The high-resolutionstructures of the N-terminal half of anarchaeal homolog of the the minichromo-some maintenance (MCM) complex, aputative eukaryotic replicative helicase, isdescribed on page 160 of this issue ofNature Structural Biology2. The structuresreveal a unique architecture at the N ter-minus of the archaeal MCM protein thatis likely conserved in all MCM proteins.

In eukarya, the MCM complex consistsof six essential proteins (MCM2–7) thateach contain conserved C-terminal heli-case domains and divergent N-terminalsequences3–6. The MCM proteins are com-ponents of the pre-replicative complexthat assembles on replication originsbefore S phase. In vivo experiments indi-cate that all six proteins are required forthe initiation and elongation steps of repli-cation7. During DNA replication, all sixproteins appear to travel with the replica-tion fork8. In addition to forming a hetero-hexamer, in vivo and in vitro studies haverevealed the existence of several MCMsubcomplexes5,9–11. Biochemical studieswith these subcomplexes, purified fromyeast and mammalian sources, have shownthat a dimer of the MCM4,6,7 het-erotrimer contains 3′→5′ DNA helicaseactivity (Fig. 1), and its interaction with

either MCM2 or the MCM3,5 dimericcomplex modulates the helicase activi-ty10,11. It is therefore thought that theMCM4,6,7 complex is the eukaryoticreplicative helicase and the other MCMpolypeptides may have regulatory func-tions. Thus far, however, helicase activityhas not been observed with the six subunitMCM2–7 complex4,5,7. It has been postu-lated that additional modifications of thecomplex lead to the activation of its heli-case activity4.

MCM homologs have also been identi-fied in archaea, the third domain of life. Atleast one MCM homolog has been identi-fied in all archaea for which the completegenome is known12. Biochemical studieswith the single MCM protein fromMethanothermobacter thermautotrophicus(mtMCM)13–16 and Sulfolobus solfatari-cus17 have shown that they possess proper-ties similar to those of the eukaryoticMCM4,6,7 complex, including 3′→5′helicase activity (Fig. 1). In vivo studieshave shown that the archaeon Pyrococcusabysii MCM is associated with chromatinonly in replicating, but not arrested,

cells18. Thus, it was suggested that thearchaeal MCM protein is the replicativehelicase12 (Fig. 2).

The MCM structureEM studies have shown that numeroushelicases are hexameric in structure1. Thestudies revealed toroidal structures of120–140 Å in diameter, with a central cav-ity (20–40 Å) large enough to accommo-date ssDNA or double-stranded DNA(dsDNA)1. Because helicases bind ssDNA,it was proposed that they may encirclessDNA (Figs. 1, 2), with the ssDNA located within the central cavity.Movement into the duplex leads to thepositioning of the complementary strandoutside the channel and duplex unwind-ing. EM studies with the humanMCM4,6,7 complex revealed a similarring-shaped hexamer with a diameter of110–120 Å and a central cavity of 20–30 Å(ref. 19).

In archaea, however, the picture is morecomplex. Although the single MCMhomolog from S. solfataricus was shownto form hexamers in solution17, the

Structural lessons in DNA replication fromthe third domain of lifeZvi Kelman and Jerard Hurwitz

The high-resolution structure of the N-terminal half of an archaeal MCM protein domain sheds light on theenzyme’s helicase activity and its role in DNA replication.

Eukarya(MCM 4,6,7)

3´ 3´ 3´

3´ 3´ 3´

5´ 5´ 5´

5´ 5´ 5´

Archaea(MCM)

Bacteria(DnaB)

Fig. 1 Schematic comparison of the helicase activities of the three replicative helicases in theabsence of a helicase loader. The eukaryotic MCM4,6,7 complex can assemble around closed circu-lar DNA, translocate 3′→5′ and form double hexamers only in the presence of fork structures. Thearchaeal MCM protein is a double hexamer, translocates 3′→5′ and can assemble around closedcircular DNA. DnaB cannot assemble around closed circular DNA and translocates 5′→3′.

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nature structural biology • volume 10 number 3 • march 2003 149

mtMCM enzyme appears to form doublehexamers2,13–16 (Figs. 1, 2). This is reminis-cent of the SV40 large T antigen helicase(Tag), which forms double hexamers onthe origin of SV40 DNA20. The eukaryoticMCM4,6,7 complex was also suggested toform double hexamers, but only withforked DNA structures21 (Fig. 1).

The mtMCM protein contains 667amino acids. Its N-terminal region wasshown to be involved in protein multi-merization and ssDNA binding, whereasthe C-terminal portion contains the cat-alytic domains14–16. In this issue, Fletcheret al.2 present the 3.1 Å crystal structure ofthe N-terminal 286 amino acids ofmtMCM (N-mtMCM). The study revealsa unique dumbbell-shaped double-hexamer architecture for N-mtMCM,slim in the center with large bulges at theends. Each monomer folds into three dis-tinct domains. Domain A, at the N termi-nus, is mostly α-helical. Domain B hasthree β-strands and an additional strandheld by a Zn atom. Domain C, which con-nects to the catalytic domain, contains fiveβ-strands and is positioned betweendomains A and B. The dodecamer is 120 Åin diameter, with a long central channel(118 Å), along its 6-fold axis and 23–47 Åin diameter at the narrowest and widestpoints, respectively. The surface of thecentral channel has a high density of posi-

tively charged residues, which may berequired for MCMs to interact withdsDNA. This three-dimensional structurediffers from an EM study of the full-length mtMCM, however, which showedthat the mtMCM protein forms hep-tameric rings, 150 Å wide and 100 Ålong22. Future crystallographic studieswith the full-length mtMCM protein andwith other archael MCMs may shed lighton this discrepancy and identify anypotential functional differences betweenthe two oligomeric forms.

The role of Zn2+

All archaeal16,17 and most eukaryal2,4

MCM polypeptides contain zinc-fingermotifs that are important for their func-tion. In eukarya, a zinc-finger mutant ofMCM2 could not support cell growth23

and a mutation in a putative zinc-fingerdomain of MCM5 (ref. 2) resulted in athermolabile MCM complex24. AnMCM4,6,7 complex containing a zinc-finger mutation in MCM4 showed areduced ability to interact with ssDNAand formed unstable hexamers25. A zinc-finger mutant of mtMCM that did notbind DNA was impaired in ssDNA-bind-ing activity and was devoid of helicaseactivity16.

The three-dimensional structure of N-mtMCM likely explains the impor-

tance of these zinc-fingers in MCM func-tion. A Zn atom in the mtMCM structureis part of domain B, forming a C4-typezinc-finger domain (CX2CXnCX18C, C4).The structure illustrates that domain B,and especially the zinc-finger, plays animportant role in N-mtMCM dodecamer-ization by mediating hexamer–hexamerinteractions2. It is likely, however, thatadditional regions may mediate hexam-er–hexamer interactions because a zinc-finger mutation in full-length mtMCMdid not affect its dodecameric structure16.

DNA bindingAll replicative helicases interact withssDNA and translocate either in the 3′→5′or 5′→3′ direction1. Besides bindingssDNA, many hexameric helicases binddsDNA, but with much lower affinity1.Until now, high-affinity dsDNA bindingby replicative helicases has been reportedonly for virus-encoded proteins. SV40Tag20 and papillomavirus E1 proteins26,for example, both bind to viral replicationorigins and locally melt the DNA duplexto initiate DNA synthesis. In eukarya, andprobably also in archaea, the MCM pro-teins lack origin recognition and meltingfunctions7,12 (Fig. 2). It was recently pro-posed that the eukaryotic MCM complexmay translocate along duplex DNA withboth strands located within its centralchannel, acting as a rotary pump in itsfunction as a replicative helicase27. Thismode of translocation through duplexDNA has recently been observed with thehexameric, ring-shaped replicating heli-cases DnaB and T7 phage gp4 (ref. 28).Clearly, it will be important to determinewhether the MCM helicases can bindand/or translocate along dsDNA.

The structure of the N-mtMCMrevealed a central cavity large enough to

Fig. 2 Models of DNA replication initiation ineukarya and archaea. a, Initiation of replica-tion in eukarya begins when the origin recogni-tion complex (ORC) binds to the origin. CDT1and ATP-bound CDC6 proteins associate withORC and the MCM helicase at the origin. ATPhydrolysis in CDC6 causes conformationalchanges postulated to load the helicase ontoDNA7. This process is likely aided by additionalfactors, but they have been omitted from thefigure for the purpose of clarity. Melting nearthe origin region likely occurs after the loadingof CDC45. A more detailed model can be foundin ref. 7. b, Postulated initiation of replicationin archaea. The CDC6/ORC1 homolog recog-nizes and binds to the origin. Although thefunction/s of the archaeal homologs ofCDC6/ORC1 has not yet been determined, it isbelieved to function similarly to both eukaryot-ic ORC and CDC6. ATP-bound CDC6/ORC1 is pos-tulated to interact with the MCM helicase,leading to its loading on DNA. Details can befound in ref. 12.

CDC6CDT1

ORC

MCM

Eukarya Archaea

CDC6/ORC1

CDC6/ORC1

MCM

CDC45

a b

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150 nature structural biology • volume 10 number 3 • march 2003

accommodate dsDNA2. Indeed, the trun-cated enzyme binds both dsDNA2 andssDNA (X. Chen, pers. comm.), mediated,at least in part, via a β-hairpin fingerlocated in domain C. The finger containsfive positively charged residues in a nine-amino acid stretch, and mutations in twoof these residues abolish ss- and dsDNAbinding. Appreciable dsDNA bindingrequired high protein-to-DNA ratios2,whereas interactions between the full-length protein and ssDNA required con-siderably lower ratios13,16. The eukaryoticMCM4,6,7 complex binds dsDNA onlyweakly21. The high protein concentrationsrequired for dsDNA binding by N-mtMCM may result from the use of atruncated protein, as the catalytic domainmay also interact with DNA. Further stud-ies will be important to determine the rel-ative affinities of MCM proteins for bothssDNA and dsDNA. dsDNA binding maybe important for the translocation of theMCM complex in the rotary pumpmodel27 but not in the proposed ssDNAencirclement helicase model. An impor-tant role for dsDNA binding does not fitwith the findings that replicative helicasesrequire ssDNA for NTPase activity, a stepcritical for their translocation1.

Cdc7 bypassThe action of the CDC7-DBF4 kinase isessential for transition of the pre-replica-tive complex to the pre-initiation com-plex. In the absence of this kinaseactivity, the chromatin loading of pro-teins essential for this step is blocked7. Inyeast, an MCM5 mutation known as bob1bypasses this need29. It was postulatedthat this mutation leads to structuralchanges in the MCM complex mediatedby the CDC7–DBF4 complex. The structure of a N-mtMCM mutant with amutation equivalent to that found in the MCM5-bob1 gene, revealed subtlechanges in the interactions between

domains A and C2. Experiments in yeastwith a variety of MCM5 mutants supportthe notion that structural changesobserved in mtMCM contribute to theCDC7-DBF4 bypass mechanism2. Thesefindings suggest that experiments on thehelicase activity of an MCM complexcontaining this MCM5 mutation shouldbe performed. Our predictions are thatthis change will not lead to activation.More likely, the structural changes result-ing from this mutation contribute to thestable loading of the MCM complex ontoDNA and its interactions with other pro-teins at the fork.

PerspectiveThe structure of the N-mtMCM shouldlead to further structure-function studiesthat will contribute to our understandingof eukaryotic MCM enzymes. Also, it willbe important to determine the structuresof the full-length MCM proteins fromM. thermautotrophicus and from otherarchaea. For such studies, a protein thatforms only single hexamers may be moresuitable (from S. solfataricus, for example)because of the large size of dodecamericcomplexes. Though the N-mtMCM struc-ture has provided us with important leadson the interactions between eukaryoticMCM subunits, the precise roles of theMCM complex as the replicative helicaseremain unanswered. Possibly CDC45,which, similar to MCM, travels with thereplication fork8, and/or as yet unknownproteins are required to activate the heli-case activity of the MCM2–7 complex.Hopefully, it will not be too long until ananswer to these questions is found.

AcknowledgmentsWe would like to thank J. Aarons for the artwork ofFigs. 1 and 2.

Zvi Kelman is in the Center for AdvancedResearch in Biotechnology, University of

Maryland Biotechnology Institute, 9600Gudelsky Drive, Rockville, Maryland 20850,USA. Jerard Hurwitz is in the Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue,Box 97, New York, New York 10021, USA. e-mail: kelman@umbi.umd.edu

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