Review Article MCM Paradox: Abundance of Eukaryotic

Review ArticleMCM Paradox Abundance of Eukaryotic ReplicativeHelicases and Genomic Integrity

Mitali Das1 Sunita Singh2 Satyajit Pradhan3 and Gopeshwar Narayan1

1 Cancer Genetics Laboratory Department of Molecular and Human Genetics Banaras Hindu University Varanasi India2Department of Zoology Mahila Mahavidyalaya College Banaras Hindu University Varanasi India3 Department of Radiotherapy amp Radiation Medicine Banaras Hindu University Varanasi India

Correspondence should be addressed to Gopeshwar Narayan gnarayanbhuacin

Received 29 August 2014 Accepted 30 September 2014 Published 19 October 2014

Academic Editor Malayannan B Subramaniam

Copyright copy 2014 Mitali Das et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

As a crucial component of DNA replication licensing system minichromosome maintenance (MCM) 2ndash7 complex acts as theeukaryotic DNA replicative helicase The six related MCM proteins form a heterohexamer and bind with ORC CDC6 and Cdt1 toform the prereplication complex Although theMCMs are well known as replicative helicases their overabundance and distributionpatterns on chromatin present a paradox called the ldquoMCMparadoxrdquo Several approaches had been taken to solve theMCMparadoxand describe the purpose of excess MCMs distributed beyond the replication origins Alternative functions of these MCMs ratherthan a helicase had also been proposed This review focuses on several models and concepts generated to solve the MCM paradoxcoinciding with their helicase function and provides insight into the concept that excess MCMs are meant for licensing dormantorigins as a backup during replication stress Finally we extend our view towards the effect of alteration of MCM level Though anexcess MCM constituent is needed for normal cells to withstand stress there must be a delineation of the threshold level in normaland malignant cells This review also outlooks the future prospects to better understand the MCM biology

1 Introduction

In early 1980s minichromosome maintenance (MCM) pro-teins were first identified in budding yeast Saccharomycescerevisiae [1] These mutant variants were defective in main-tenance of minichromosome a plasmid with a cloned cen-tromere and replication origin [2 3] MCM proteins werethus found to have essential roles in DNA replication [4 5]Subsequent identification of proteins with a particular identi-cal sequence termed ldquoMCMboxrdquo [6] leads to the constitutionof a protein family the MCM family The family consistsof at least 6 homologues MCM 2ndash7 [1] which is directlyinvolved in eukaryotic DNA replication [7] MCMs remain atthe centre of interest of biochemists geneticists and cancerbiologists since 30 years of their identification The studiesprovide good knowledge of MCM structure function par-ticular role in DNA replication and abnormalities leading tocancer On the other hand some paradoxes came forwardwith the advancement of studies regarding the function ofMCMs in DNA replication It has been found that the MCM

hexamers are loaded in excess onto chromatin and dis-tributed on the locations distant from the origins Solving theparadox known as the ldquoMCM paradoxrdquo is challenging to thescientific community In this review we have discussed therole of MCMs in DNA replication concepts generated tosolve the paradoxes their extension to cancer biology andfuture perspective

2 MCMs as a Component ofReplication Origins

MCM 2ndash7 is expressed in all eukaryotes [1] Physical interac-tion betweenMCM 2ndash7 components was determined by sev-eral molecular techniques such as two-hybrid coimmuno-precipitation and biochemical purification [8ndash11] It has beenproposed that MCM 2ndash7 makes a heterohexameric complexin 1 1 1 1 1 1 ratio The core of MCM 2ndash7 complex com-prising MCM 4 6 7 has DNA helicase activity [12ndash14] Theother three components of the complex may play other roles

Hindawi Publishing CorporationMolecular Biology InternationalVolume 2014 Article ID 574850 11 pageshttpdxdoiorg1011552014574850

2 Molecular Biology International

during replication such as MCM2 binds to histones andfacilitate histone redeposition during DNA synthesis [15]However the fact that MCM proteins are essential in ver-tebrate DNA replication was confirmed by the studies inXenopus where MCM depleted egg extracts were unable tosupport further DNA synthesis [16 17]

A parallel concept of ldquoreplication licensingrdquo was emergingin 1980s from the observation that sperm chromatin in anucleus replicated only once in a cell cycle and becamecompetent again inmitosis but a specificDNAsequence is notrequired for replication initiationThis concept hypothesizedthat there must be a positive ldquolicenserdquo to mark the replicatedsequences from the unreplicated ones thus preventing thereinitiation of replication at the already replicated sites [18]The cell cycle dependent change in subcellular localization ofMCMs in yeast and Xenopus proposed these proteins to bereplication licensing factors [8 15 19] However the repli-cation licensing mechanism involves a series of events anda number of proteins The first step of replication licensingmechanism is the formation of prereplication complex (pre-RC) Pre-RC formation involves assembly of a number of pro-teins such as origin recognition complex (ORC) Cdc6 Cdt1and MCM 2ndash7 This in turn leads to a chain of events allow-ing binding of DNA polymerase and other several factorsto chromatin to start DNA replication [7 20ndash22]

3 MCMs Function as Helicases

After the identification of MCMs as replication licensingfactors their biochemical function at the chromatin had beensearched forThe heterohexameric ring structure of MCM 2ndash7 suggests that this protein complex may work as replicativehelicase The entire MCM 2ndash7 complex in fission yeast orMCM (4 6 7)

2complex from the mouse shows a globular

structure with a central channel of 3-4 nm similar to manymultisubunit helicases from all kingdom of life This channelcan encircle either single or double strandedDNA [1] In 2001Labib and Diffley [23] enquired whether MCM 2ndash7 is theeukaryotic DNA replication fork helicase The first evidencecame from the study of Yukio Ishimi where they had shownthat purified MCM 4 6 7 complexes have 31015840 rarr 51015840 DNAunwinding capacity [24] Consequently they have also shownthat mouse MCM 4 6 7 complexes have intrinsic DNA heli-case activity [12] Not only the MCM 4 6 7 subcomplex buta wholeMCM2ndash7 complex had been reported to have in vitrohelicase activity where theMCM 2ndash5 junction acts as an ATPdrive gap or ldquogaterdquo [25] Recent studies have shown that theMCM 2ndash7 proteins make a core of the replicative DNA heli-case and that they are first loaded at replication origins in aninactive formThe helicase is then activated by recruitment ofthe Cdc45 and GINS proteins into a holo-helicase known asCMG(Cdc45MCM2ndash7GINS) [26] Identification of severalinteracting partners and the real action mechanism of thisholo-helicase keeps researchers involved in studies ofMCMsVery recently it has been shown that the human CMG com-plex (hCMG) can unwind DNA duplex regions up to 500 bphydrolyzing ATP and in combination with human DNApolymerase 120576 leads to the formation of oligonucleotide

products up togt10 kb [27]The same group has also identifiedhuman chromosome transmission fidelity 4 (hCTF4) as aninteracting partner of hCMG complex [28]

The helicase activity of MCM proteins was also found inarcheal species [29]Methanobacterium thermoautotrohicumthe bacterium contains only one MCM protein which formsa homohexameric ring The homohexamer contains a posi-tively charged central channel of 2ndash4 nm to bind with DNAand act as a helicase [30ndash33] A single MCM homolog fromSulfolobus solfataricus has been reported to form hexamers insolution and can dissolve 51015840 tailed oligonucleotides by itshelicase activity [34]

MCM2ndash7 proteins are categorized in superfamily 6 (SF6)helicases [35 36] in which each subunit of a hexameric heli-case contains anAAA+domain In these helicases theATPasesites are generated in between two adjacent subunits oneis providing the walker A andwalker Bmotif for ATP bindingand the other partner provides the arginine finger motif forATP hydrolysis MCM 2ndash7 is also related to viral homohex-americ helicases (superfamily 3 (SF3)) including the SV40large T antigen and papilloma virus E1 protein [26]

Boos et al [26] also summarized the evidence indicatingMCM 2ndash7 as the engine of the replicative helicase FirstlyMCM 2ndash7 travels with replication forks forming part of thepurified replisome progression complex (RPC) in yeast [3637] and vertebrates [38ndash41] Secondly acute inactivation ofMCM 2ndash7 subunits during S phase results in rapid replisomeinactivation [42 43] and third purified yeast MCM 2ndash7complex shows in vitro helicase activity [25]

4 The MCM Paradox

Above discussed lines of evidence substantially show thatMCM 2ndash7 complex has helicase activity to unwind doublestranded DNA and thus helps to initiate DNA replicationBut there are some issues that prohibit accepting the role ofMCM 2ndash7 as helicase The issues termed as ldquoMCM paradoxrdquocame from two observations (a) eukaryotic MCM 2ndash7 com-plexes are not localized at the sites of replication like viral andbacterial helicases MCM 2ndash7 complexes in higher eukary-otes are distributed over nonreplicated DNA rather thanon replication forks (b) the paradox is elaborated by anotherobservation that excess MCM heterohexamers are loadedonto chromatin rather than replication origins and ORCs[44] The basic questions were how MCM 2ndash7 complexesperform their function as helicase from a distance away fromthe replication sites andwhat the purpose of the excess boundMCMs onto chromatin more than the origins themselves is

Madine et al [45] have shown by immunofluorescencethat Xenopus MCM3 does not colocalize with sites of DNAreplication The protein is almost uniformly distributed onchromatin and is suddenly lost during replication Same lineof evidence in human cells came from another study in 1996The authors had used high resolution confocal microscopyto determine the subnuclear sites of chromatin-boundMCMproteins in comparison to the sites of replicating DNAThey found MCM proteins to be entirely nuclear in HeLacells at interphase and to exist in nucleoplasmic and chro-matin bound forms The chromatin bound form is present

Molecular Biology International 3

throughout the nucleus in late G1 and early S and atdiscrete subnuclear sites following progression into S phaseInterestingly as the S phase proceeds further the MCMs aredisplaced from their site on replicating chromatin Further-more the chromatin bound fraction was found to be absentin G2 and mitotic nuclei The observation that MCMs arepresent on unreplicated chromatin rather than replicatingone emphasized their role in monitoring the unreplicatedchromatin to ensure only a single round of DNA repli-cation [46] In Chinese hamster ovary (CHO) fibroblastsMCM2 associates with early and late replicating chromatin inlate telophase Subsequently more MCM2 is associated tochromatin throughout the G1 phase and the maximumloading occurs at theG1S transitionMCM2was found to getdissociated from replicons shortly after their initiation [47]In 2005 Masata et al [48] used a robust statistical methodto be more accurate in analyzing the localization of MCMproteins and replication machineries In HeLa cells majorityofMCM2 proteins do not colocalize with replication foci buta very small but significant fraction of MCM2 signals colo-calize with DNA replication foci throughout the S phaseThecolocalization of MCM2 protein with replication foci duringS phase thus explains an active function ofMCM2ndash7 complexin replication Supporting the above findings a recent in vivolicensing assay has proved that the concentration of chro-matin bound MCM2 and MCM4 changes with progressionof G1 phase and they fail to colocalize with other replicationmachinery [49]

Eukaryotic DNA replication starts at multiple sitesthroughout the genome In budding yeast the initiationsites known as autonomously replicating sequences aredistributed all over the genome at an interval of every 40 kbIn somatic cells replication can be initiated once every sim150 kb In embryonic cells of Xenopus laevis DNA replicationinitiation sites are regularly spaced at every 9ndash12 kb withoutsequence specificity [50] In all cases the initiation is firstmarked by the binding of ORC [7] As noted earlier the sec-ond part of MCM paradox concerns the abundance of chro-matin boundORCs andMCM2ndash7 complexes In yeastXeno-pus and highly proliferating human cancer cells HeLa thenumber of MCM 2ndash7 complexes bound to chromatin highlyexceeds the number of origins [10 51ndash55] In Xenopus thenumber of MCM 2ndash7 complexes is found to be 10ndash40 timesmore than the actual initiation events are [56 57] Moreprecisely in Xenopus the minimum length of DNA requiredto recruit ORC and MCM 2ndash7 is sim80 bp The ratio of MCM2ndash7 ORC in this small fragment is perfectly 1 1 With aDNA fragment longer than 80 bp the MCM 2ndash7 ORC ratioincreased up to 40 1 [58]

5 Proposed Models to ResolvelsquolsquoMCM Paradoxrsquorsquo

Several plausible mechanisms have been proposed to explainthe function of MCM 2ndash7 as replication helicase from adistance away from the replication site According to ldquoMCMevictionrdquo mechanism there is always a minor MCM pop-ulation that escapes immunodetection but is associated at

replication forks and the rest major fraction is released fromthe replication forks upon replication initiation [47 59] as aconsequence of chromatin condensation [60] A rotary pumpmodel depicts that those MCM 2ndash7 complexes have alwaysbeen loaded at a distance from an active replication fork andlike rotary motors they translocate DNA along its axis byhelical rotation causing unwinding at the constrained repli-cation fork [61] With little variation of rotary pumpmodel aploughshare model has been proposed where MCM helicaseencircles and translocates on dsDNA Unwinding of DNAdouble strand occurred through a rigid ldquoploughsharerdquo presentat the trailing edge of the helicase that is inserted in betweenDNA double strands and cleaves the duplex The model alsoassumes that multiple pairs of MCM 2ndash7 hexamers bind toeach replicon in G1 phase but only a single pair is activatedto perform as helicase [62] All the models have a fact incommon that there must be at least a few active MCMs at thereplication fork The fraction of these MCMs at replicationforks may be undetectable in comparison to the detectablefraction present on chromatin distant from the active repli-cation sites Together with the ideas and observations thecurrentmodel ofMCMaction has been proposed byAparicioet al [59] The group found that in HeLa cell nuclei theMCM proteins at G1 phase first concentrate on chromatinstructures that subsequently become replication forks at Sphase The concentration of bound MCMs at replicationforks gets reduced after replication initiation leaving a verysmall but detectable fraction still bound to the forks duringthe S phase supposed to be the helicase The findings weresimilar to those of a previous study [63] The authors pro-posed an extension of ldquoMCM evictionrdquo mechanism of MCMdynamics based on the ldquoloop modelrdquo of mammalian replica-tion factories [59 64] According to thismodel at a very earlyonset of G1 the MCM 2ndash7 complexes start to be loaded ontoorigin DNA in an ORC-Cdc6-Cdt1-dependent manner andspread all over the adjacent sequencesThe amount ofMCMsto be loaded on each originmay differ influencing their firingability At G1-S boundary only a fewMCM2ndash7 complexes getactivated by Cdc45 GINS and other protein factors Theseactivated MCMs become able to escape the posttranslationalmodification signals that can cause their dissociation fromchromatin and remain bound to both active and dormantorigins upon initiation of DNA synthesis In a multirepli-con chromatin arrangement several replicon units remainorganized in cohesion-stabilized loops to form a rosettestructure Most of the origins are distributed at the base ofthe loops confining replication event at the central part ofthe rosette The activation of replication forks leads to theMCM-BPmediated dissociation of inactiveMCMs [65] fromchromatin and continuous entry of PCNA A small fractionof MCMs remain always bound to the active site of the DNAreplicationThus the first part of theMCMparadox has beensolved [59]

6 Excess MCMs Are Meant toSurvive Replicative Stress

The second part of the MCM paradox concerns the purposeof quantitative excess of MCMs over origins The common


approach was to reduce the dosage of MCMs in replicatingcells and analyze the posteffects In yeast it has been foundthat depletion of MCM2 does not affect their growth rate buta 50 reduction of the dosage ofMCM2 in yeast decreases thenumber of origins to be used per replication eventThere wasan indication that the excess MCM proteins may be requiredfor successful initiation at all replication origins [10] Insteadof hampering cell cycle depletion ofMCM7 causes activationof checkpoint signalling in human cancer cells suggestingtheir roles in replication checkpoint signalling or activation ofreplication origins at the time of incomplete DNA replication[66 67] It is evident that MCMs may have an alternativerole related toDNA replication however it remained difficultto conclude due to the lack of missing link(s) The conceptof ldquodormantrdquo origins has been proposed to be the missinglink based on the origins that remain suppressed duringnormal DNA replication and can get activated and supportinitiation when replication forks are stalled in response toreplicative stresses [68 69] In Xenopus laevis there are 10dormant origins distributed adjacent to each active originBoth the active and dormant origins are bound by MCM 2ndash7complexes and have potential to carry out replication processBut the dormant origins remain inactivated during a normalreplication event by ATR mediated signalling pathway Ina stressed replication condition such as in presence ofaphidicolin when the ATR signalling is inhibited by caffeinean ATR-kinase inhibitor the dormant origins get activatedand partially rescue the replication process ConsequentlyRNAi-based partial depletion of MCMs in Caenorhabditiselegans does not show any observable effect on cell viabilityor proliferation but becomes lethally hypersensitive to anontoxic dose of replication inhibitor hydroxyurea Theseobservations suggested the role of excess MCM 2ndash7 complexin licensing dormant origins during replication stress [68]Extension of similar studies in human cancer cells shows thatRNAi mediated MCM5 silencing allows the cells to grow at anormal rate but makes these MCM depleted cells vulnerableto any kind of replication stress such as hydroxyurea oraphidicolin The authors suggest that excess MCM 2ndash7 pro-vides the cells safeguards against replicative stress by licensingdormant origins [70] An extensive study using siRNAagainsteach component of MCM 2ndash7 complex in human cancercell lines was concordant with the above findings Acutedownregulation ofMCM2 andMCM3 permits the formationof a limited but sufficient amount of MCM 2ndash7 complexallowing the cells to proliferate But their hypersensitivityto replication stress shows increased chromosomal aberra-tions and eventually raises a checkpoint response to blockmitotic division The acute downregulation of MCM 4ndash7on the other hand may reduce the number of chromatinbound MCM 2ndash7 complexes beyond the threshold and thusbecomes fatal It has been suggested that the excess MCM 2ndash7 complexes license dormant origins as a backup mechanismto maintain genomic integrity during replication stress [71]The consequences of attenuated MCM expression in wholeanimal were studied using mouse model Chaos3 (chromo-somal aberrations occurring spontaneously 3) is a murinechromosomal mutation of MCM4 The viable mutant alleleMCM4Chaos3 produces the polypeptide with a change from

wild type phenylalanine to isoleucine at residue 345 (F345I)producing a destabilized form of MCM4 MCM4Chaos3Chaos3female mice develop mammary adenocarcinoma after 12-month latency Hence mutant MCM4 causes cancer pre-disposition The embryonic fibroblasts (MEFs) from thesemutant mice showed increased rate of DNA breaks aftertreatment of aphidicolin [72] MEFs containing hypomor-phic MCM4Chaos3Chaos3 show approximately 40 reductionof all MCM 2ndash7 expression Depletion of MCM2 -6 or -7in these mutant cells resulted in deleterious effects suchas growth retardation inhibited cell proliferation genomicinstability and cancer predisposition However heterozygouscondition of MCM4Chaos3 with normal MCM3 rescued thenormal phenotype by increased chromatin bound MCMs[73] MEFs from MCM2IRES-CreERT2IRES-CreERT2 mice showdecreased origin use in presence of hydroxyurea and are thushypersensitive to these replicative stresses [74] Both of thestudies indicate that in vivo a cellular high level of MCMsis required for licensing active origins and maintenance ofdormant origins as a backup in distress However excessMCMs are not onlymeant to license dormant origins but alsotake part in recombinational repair under replication stress asreported in fission yeast [75]

It is evident that the replication licensing mechanismensures the nonlicensing of a single new origin at the onsetof S phase to prevent rereplication [76 77] In a subsequent Sphase if the cells face accidental problems such as replicationfork stalling or disassembly they cannot be retrieved Cellstherefore keep backup licensed dormant origins which arenot used normally but are used when replication is at stake[69 78] So the most plausible explanation for the ldquoMCMparadoxrdquo is that cells maintain abundant MCM 2ndash7 com-plexes distributed over the chromatin as a backup licensingmechanism to combat natural replicative stress generatedduring S phase (Figure 1)

7 Effects of MCM Attenuation In Vivo

Thedepleted level ofMCMshas been proved to be deleteriousfor vertebrate systems Positional cloning of mutated MCM5in embryonic cells of Zebra fish leads to impaired retinaldevelopment characterized by deregulated cell cycle andincreased rate of apoptosis The mutation was applied in thedifferentiated cells at the third day of development but theeffect was restricted to those retinal stem cells hamperingdevelopment of retina tectum and hindbrain This indicatesthat different tissues respond differentially to MCM5 deple-tion [79] Mutation in MCM4 was reported for the first timein human population in 2012 A frameshift mutation leadsto the formation of truncated MCM4 protein The mutationoccurs due to the insertion of a single nucleotide prior toexon 2 thus shifting the splice acceptor site This muta-tion is associated with a diseased phenotype called familialglucocorticoid deficiency characterized with poor adrenaldevelopment short stature and NK cell deficiency andincreased DNA breakage [80 81] However there is no clearreport of cancer predisposition in these patients The con-sequences of MCM2 and MCM4 mutations were discussed


Backup origin

Replicon clusterLicensed main origin

Activation of originInhibition of origin activation

Licensed dormant originExternal or internal replication stress

Replication fork stalling

Chk1 signalling

Chk1 signalling

(a) Normal licensing

No backup





Chk1 signalling

Chk1 signalling

(b) Limited licensing

Figure 1 Purpose of excess MCMs to license dormant origins as a backup mechanism for stress (a) prior to S phase the MCMs licensemain origins as well as many dormant origins which remain prevented to be fired by Chk1 signalling At the time of stress when replicationforks get stalled the backup origins get activated and continue the replication process (b) in case of a limited licensing condition at the timeof stress there is no backup to retrieve the replication process hence the cells cannot withstand the stress (adapted from [71])

in previous section [73 74] There is another example ofdominant mutant allele Mcm4D573H which has been foundto be associated with spontaneous dominant leukemia inmice These leukemic mice have a normal level of MCMproteins but MCM4D573H is unable to incorporate intoMCM 2ndash7 complexes causing chromosomal abnormalitiesleading to cancer [82] Mouse embryos homozygous for amutantMCM2 (Mcm2IRES-CreERT2IRES-CreERT2) allele developnormally but can survive only for 10ndash12 weeks They acquiresevere impairment of tissue development due to stem celldeficiency accumulate huge chromosomal abnormalitiesand immediately die of cancer [83] MCM2 depletion causesa high rate of thymic lymphoblastic lymphoma characterizedwith manifold genomic instabilities The most common ofthese chromosomal abnormalities is a deletion of a genomicregion of approximately 05Mb altering expression of severalgenes contributing to the development of cancer [84] Abovediscussed citations exemplify the biochemical cellular andphenotypic consequences of limited licensing in the form ofimpeded MCM loading increased genomic instability and anultimate formation of cancer respectively [69]

8 MCM Expression and Cancer

MCMs are found to be overexpressed in multiple cancerssuch as in prostate cancer [85] colon cancer [86] menin-gioma [87] breast carcinoma [88] medulloblastoma [89]gastric adenocarcinoma [90] cervical cancer [91 92] osteo-sarcoma [93] and esophageal squamous cell carcinoma [94]

Aberrant expression of individual components of MCM 2ndash7 complex makes them suitable biomarkers for malignancies[95] MCM2 is a proposed biomarker in oligodendrioma[96] renal cell carcinoma [97] esophageal squamous cellcarcinoma [98] laryngeal carcinoma [99] breast cancer [100101] large B cell lymphoma [102] oral cancer [103] ovariancancer [104] and gastric cancer [105]MCM3 is a proliferativemarker in papillary thyroid carcinoma [106] MCM4 is aproliferation marker of nonsmall cell lung cancer [107]MCM5 is a prognostic marker of ovarian cancer and prostatecancer [104 108] MCM6 protein level in plasma of hepa-tocellular carcinoma patients may be a novel biomarker forthe malignancy [109] Deregulated expression of MCM7 is apotential biomarker for colorectal cancer [110] small lungadenocarcinoma [111] and oral squamous cell carcinoma[112]

Hence the overexpression of MCM genes can define theproliferative stage of a cell [113] The reason behind deregu-lated expression of these proteins is still an open questionThere are two possible explanations One is that due to aninappropriate action of CDKs the malignant cells lost thepower to differentiate hence continuously divide and expressMCMs MCMs in this case are not directly oncogenic butmark the proliferative status of the cells Another possibility isthat deregulation of replication licensing system itself causesthe development of cancer by inducing relicensing of repli-cated DNA promoting chromosomal instability [114]

Aneuploidy is an obvious consequence of inappropriatereplication licensing The overexpression of MCM2 alongwith other licensing proteins is significantly associated with


Normal cell

Intact genome

Normal MCMconstituent


Depleted MCMlevel

Mutations and other factorsCancer

Genomic instability

Elevated MCMlevel

Figure 2 The unavoidable consequence of change in MCM level is genomic instability which may result in cancer The threshold levelof MCMs for genome maintenance of normal cells is still to be quantified and the factors causing aberrations in MCM level are yet to beidentified

aneuploidy to cause penile cancer [115] The cervical cancercell lines harboring high level of aneuploidy show overex-pression of MCMs [92 116] Dosage alterations of MCMshave vivid cytogenetic backgroundThe 3q and 8q containingMCM2 and MCM4 respectively have been reported toshow genomic amplification and chromosomal gain [117 118]MCMs thus may cytogenetically control their expression bya positive feedback loop participating in the ldquovicious cyclerdquo[119] of aneuploidy and chromosomal instability

Inhibition of MCMs on cancer cells has also been provedto be deleterious A geminin mediated reduction in thechromatin bound form of MCM2 promotes cell type specificcheckpoint response A p53+Rb+ cell U2OS showed anearly S phase arrest by activation of intra-S phase checkpointsOn the other hand p53minusRbminus cell line Saos2 showed anaccumulation in late S and G2-M phase and loss of intra-Sphase checkpoints In both cases the ultimate consequencewas apoptosis In IMR90 primary fibroblasts overexpressionof geminin caused G1 arrest and did not lead to apoptosisA ldquolicensing checkpointrdquo thus may take care of the primarycells with insufficient licensed origins and prevent them fromproceeding into S phase [120] Similar results were reportedwhere inhibition of MCM2 by RNAi produces apoptoticresponse in P53+ and P53minus cancer cell lines but causes G1arrest in normal cells [121] Trichostatin A and siRNA medi-ated downregulation of MCM2 generated apoptotic responsein HCT116 cells [122] Knocking down MCM7 not onlyinhibits cell proliferation in glioblastoma multiforme tumorcells but also prevents tumor growth in mouse models ofglioblastoma multiforme [123]

9 Conclusion and Perspective

It is evident from the above discussion that either elevation ordepletion of MCM level causes genomic instability and thusconsequently cancer (Figure 2) From the concept of MCMparadox and its solution we come to a conclusion that a highlevel of MCMs is needed by a cell normally to maintain thegenomic constitution There is a gap of knowledge as theproper quantification of the threshold level of MCMs belowor above of which is fatal remains to be filled Since cancercells with elevated level of MCMs grow faster there must be

a difference in usage of dormant and main licensed originsenlightenment in this area will help understand the replica-tion mechanism of cancer cells better As noted earlier thatthe licensing checkpoints respond differently in cancer andnormal cells [119] more information is required for the com-plete designing of licensing checkpoint signalling MCMs areplaced downstream to many signal transduction pathways[124 125] and are also reported to be controlled by miRNA[126 127] It will be very interesting to record the crosstalkbetween several signal transduction pathways controllingMCM expression and map a complex signalling network ofDNA replication licensing

Besides DNA replication licensing MCMs are alsoactively involved in transcription processMCM2ndash7 complexcomponents are also identified as the components of RNApolymerase II transcriptional apparatus [128] It has beendemonstrated that MCM5 is indispensable as a signal trans-ducer and activator of transcription 1 (Stat 1) mediated tran-scriptional activation [129] Recently Hubbi et al presentedanother line of evidence suggesting that the abundance ofMCM proteins must be meant for functions other than DNAhelicase activity They demonstrate that MCM 2 3 5 andMCM 7 proteins directly interact with hypoxia-induciblefactor-1 (HIF-1) 120572 subunit to inhibit HIF-1 transcriptionalactivity by oxygen dependent manner Thus MCM proteinsplay important role in regulating transcription during oxida-tive stress [130] Furthermore the existence of filamentousstructure made of excess MCMs located distally from originswith a capacity of chromatin remodeling [131] extends MCMparadox towards the solution with alternative functions ofMCMs in DNA metabolism

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors thankfully acknowledge the financial supportfrom theDepartment of Biotechnology Government of India


(vide Project no BTPR9246Med30172007) to Gopesh-war Narayan Sunita Singh and Satyajit Pradhan BanarasHinduUniversity (Xth andXIth Plan) to GopeshwarNarayanand Sunita Singh and Council of Scientific and IndustrialResearch Government of India for research fellowship toMitali Das

References

[1] S L Forsburg ldquoEukaryotic MCM proteins beyond replicationinitiationrdquoMicrobiology andMolecular Biology Reviews vol 68no 1 pp 109ndash131 2004

[2] G T Maine P Sinha and B W Tye ldquoMutants of S cerevisiaedefective in the maintenance of minichromosomesrdquo Geneticsvol 106 no 3 pp 365ndash385 1984

[3] P Sinha V Chang and B-K Tye ldquoA mutant that affects thefunctions of autonomously replicating sequences in yeastrdquoJournal of Molecular Biology vol 192 no 4 pp 805ndash814 1986

[4] B K Tye ldquoMinichromosomemaintenance as a genetic assay fordefects inDNA replicationrdquoMethods vol 18 no 3 pp 329ndash3341999

[5] B K Tye ldquoMCM proteins in DNA replicationrdquo Annual Reviewof Biochemistry vol 68 pp 649ndash686 1999

[6] H Yan S Gibson and B K Tye ldquoMcm2 and Mcm37 two pro-teins important for ARS activity are related in structure andfunctionrdquoGenes ampDevelopment vol 5 no 6 pp 944ndash957 1991

[7] S P Bell and A Dutta ldquoDNA replication in eukaryotic cellsrdquoAnnual Review of Biochemistry vol 71 pp 333ndash374 2002

[8] J P J ChongHMMahbubani C YKhoo and J J Blow ldquoPuri-fication of an MCM-containing complex as a component of theDNA replication licensing systemrdquo Nature vol 375 no 6530pp 418ndash421 1995

[9] S Dalton and B Hopwood ldquoCharacterization of Cdc47p-minichromosome maintenance complexes in Saccharomycescerevisiae identification of Cdc45p as a subunitrdquoMolecular andCellular Biology vol 17 no 10 pp 5867ndash5875 1997

[10] M Lei Y Kawasaki and B K Tye ldquoPhysical interactions amongMcmproteins and effects ofMcmdosage onDNA replication inSaccharomyces cerevisiaerdquo Molecular and Cellular Biology vol16 no 9 pp 5081ndash5090 1996

[11] Y Adachi J Usukura and M Yanagida ldquoA globular complexformation by Nda1 and the other five members of the MCMprotein family in fission yeastrdquo Genes to Cells vol 2 no 7 pp467ndash479 1997

[12] Z You Y Komamura and Y Ishimi ldquoBiochemical analysis ofthe intrinsicMcm4-Mcm6-Mcm7DNAhelicase activityrdquoMole-cular and Cellular Biology vol 19 no 12 pp 8003ndash8015 1999

[13] Y Ishimi andY Komamura-Kohno ldquoPhosphorylation ofMcm4at specific sites by cyclin -dependent kinase leads to loss ofMcm467 helicase activityrdquo Journal of Biological Chemistry vol276 no 37 pp 34428ndash34433 2001

[14] J-K Lee and J Hurwitz ldquoProcessive DNA helicase activity oftheminichromosomemaintenance proteins 4 6 and 7 complexrequires forked DNA structuresrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 98 no1 pp 54ndash59 2001

[15] M Foltman C Evrin G de Piccoli et al ldquoEukaryotic replisomecomponents cooperate to process histones during chromosomereplicationrdquo Cell Reports vol 3 no 3 pp 892ndash904 2013

[16] M A Madine C-Y Khoo A D Mills and R A LaskeyldquoMCM3 complex required for cell cycle regulation of DNA

replication in vertebrate cellsrdquo Nature vol 375 no 6530 pp421ndash424 1995

[17] Y Kubota S Mimura S-I Nishimoto T Masuda H Nojimaand H Takisawa ldquoLicensing of DNA replication by a multi-protein complex of MCMP1 proteins in Xenopus eggsrdquo TheEMBO Journal vol 16 no 11 pp 3320ndash3331 1997

[18] J J Blow S M Dilworth C Dingwall A D Mills and R ALaskey ldquoChromosome replication in cell-free systems fromXenopus eggsrdquo Philosophical transactions of the Royal Society ofLondon Series B Biological sciences vol 317 no 1187 pp 483ndash494 1987

[19] K M Hennessy C D Clark and D Botstein ldquoSubcellularlocalization of yeast CDC46 varies with the cell cyclerdquo Genesand Development vol 4 no 12 pp 2252ndash2263 1990

[20] R Laskey ldquoThe Croonian Lecture 2001 hunting the antisocialcancer cell MCMproteins and their exploitationrdquo PhilosophicalTransactions of the Royal Society B Biological Sciences vol 360no 1458 pp 1119ndash1132 2005

[21] T J Takara and S P Bell ldquoMultiple Cdt1 molecules act at eachorigin to load replication-competent Mcm2ndash7 helicasesrdquo TheEMBO Journal vol 30 no 24 pp 4885ndash4896 2011

[22] L Wu Y Liu and D Kong ldquoMechanism of chromosomal DNAreplication initiation and replication fork stabilization ineukaryotesrdquo Science China Life Sciences vol 57 no 5 pp 482ndash487 2014

[23] K Labib and J F X Diffley ldquoIs the MCM2-7 complex theeukaryotic DNA replication fork helicaserdquo Current Opinion inGenetics and Development vol 11 no 1 pp 64ndash70 2001

[24] Y Ishimi ldquoADNAhelicase activity is associated with anMCM4-6 and -7 protein complexrdquoThe Journal of Biological Chemistryvol 272 no 39 pp 24508ndash24513 1997

[25] M L Bochman and A Schwacha ldquoTheMcm2-7 complex has invitro helicase activityrdquoMolecular Cell vol 31 no 2 pp 287ndash2932008

[26] D Boos J Frigola and J F X Diffley ldquoActivation of thereplicative DNA helicase breaking up is hard to dordquo CurrentOpinion in Cell Biology vol 24 no 3 pp 423ndash430 2012

[27] Y-H Kang W C Galal A Farina I Tappin and J HurwitzldquoProperties of the human Cdc45Mcm2-7GINS helicase com-plex and its actionwithDNApolymerase 120576 in rolling circleDNAsynthesisrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 109 no 16 pp 6042ndash6047 2012

[28] Y-H Kang A Farina V P Bermudez et al ldquoInteractionbetween human Ctf4 and the Cdc45Mcm2-7GINS (CMG)replicative helicaserdquo Proceedings of the National Academy ofSciences of the United States of America vol 110 no 49 pp19760ndash19765 2013

[29] A Costa and S Onesti ldquoStructural biology of MCM helicasesrdquoCritical Reviews in Biochemistry and Molecular Biology vol 44no 5 pp 326ndash342 2009

[30] Z Kelman J K Lee and J Hurwitz ldquoThe single minichro-mosome maintenance protein ofMethanobacterium thermoau-totrophicum ΔH contains DNA helicase activityrdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 96 no 26 pp 14783ndash14788 1999

[31] D F Shechter C Y Ying and J Gautier ldquoThe intrinsic DNAhelicase activity of Methanobacterium thermoautotrophicumΔH minichromosome maintenance proteinrdquo The Journal ofBiological Chemistry vol 275 no 20 pp 15049ndash15059 2000


[32] J P J Chong M K Hayashi M N Simon R-M Xu and BStillman ldquoA double-hexamer archaeal minichromosome main-tenance protein is an ATP-dependent DNA helicaserdquo Proceed-ings of the National Academy of Sciences of the United States ofAmerica vol 97 no 4 pp 1530ndash1535 2000

[33] R J Fletcher B E Bishop R P Leon RA Sclafani CMOgataand X S Chen ldquoThe structure and function of MCM fromarchaeal M thermoautotrophicumrdquo Nature Structural Biologyvol 10 no 3 pp 160ndash167 2003

[34] F Carpentieri M de Felice M de Falco M Rossi and F MPisani ldquoPhysical and functional interaction between the mini-chromosome maintenance-like DNA helicase and the single-stranded DNA binding protein from the crenarchaeon Sul-folobus solfataricusrdquo The Journal of Biological Chemistry vol277 no 14 pp 12118ndash12127 2002

[35] A Y LyubimovM Strycharska and J M Berger ldquoThe nuts andbolts of ring-translocase structure and mechanismrdquo CurrentOpinion in Structural Biology vol 21 no 2 pp 240ndash248 2011

[36] O M Aparicio D M Weinstein and S P Bell ldquoComponentsand dynamics of DNA replication complexes in S cerevisiaeredistribution of MCM proteins and Cdc45p during S phaserdquoCell vol 91 no 1 pp 59ndash69 1997

[37] AGambus RC Jones A Sanchez-Diaz et al ldquoGINSmaintainsassociation of Cdc45 with MCM in replisome progressioncomplexes at eukaryotic DNA replication forksrdquo Nature CellBiology vol 8 no 4 pp 358ndash366 2006

[38] M Pacek A V Tutter Y Kubota H Takisawa and J C WalterldquoLocalization of MCM2-7 Cdc45 and GINS to the site of DNAunwinding during eukaryotic DNA replicationrdquoMolecular Cellvol 21 no 4 pp 581ndash587 2006

[39] T Aparicio E Guillou J Coloma G Montoya and J MendezldquoThe human GINS complex associates with Cdc45 and MCMand is essential for DNA replicationrdquo Nucleic Acids Researchvol 37 no 7 pp 2087ndash2095 2009

[40] A Gambus G A Khoudoli R C Jones and J J Blow ldquoMCM2-7 form double hexamers at licensed origins in Xenopus eggextractrdquoThe Journal of Biological Chemistry vol 286 no 13 pp11855ndash11864 2011

[41] J-S Im S-H Ki A Farina D-S Jung J Hurwitz and J-KLee ldquoAssembly of the Cdc45-Mcm2-7-GINS complex in humancells requires the Ctf4And-1 RecQL4 and Mcm10 proteinsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 106 no 37 pp 15628ndash15632 2009

[42] K Labib J A Tercero and J F X Diffley ldquoUninterruptedMCH2-7 function required for DNA replication fork progres-sionrdquo Science vol 288 no 5471 pp 1643ndash1647 2000

[43] M Pacek and J C Walter ldquoA requirement for MCM7 andCdc45 in chromosome unwinding during eukaryotic DNAreplicationrdquo The EMBO Journal vol 23 no 18 pp 3667ndash36762004

[44] O Hyrien K Marheineke and A Goldar ldquoParadoxes ofeukaryotic DNA replication MCM proteins and the randomcompletion problemrdquoBioEssays vol 25 no 2 pp 116ndash125 2003

[45] M A Madine A D Mills C Musahl and R A Laskey ldquoThenuclear envelope prevents reinitiation of replication by regulat-ing the binding ofMCM3 to chromatin inXenopus egg extractsrdquoCurrent Biology vol 5 no 11 pp 1270ndash1279 1995

[46] T Krude C Musahl R A Laskey and R Knippers ldquoHumanreplication proteins hCdc21 hCdc46 and P1Mcm3 bind chro-matin uniformly before S-phase and are displaced locally duringDNA replicationrdquo Journal of Cell Science vol 109 no 2 pp 309ndash318 1996

[47] D S Dimitrova I T Todorov T Melendy and D M GilbertldquoMcm2 but not RPA is a component of the mammalian earlyG1-phase prereplication complexrdquo The Journal of Cell Biologyvol 146 no 4 pp 709ndash722 1999

[48] M Masata J Malınsky H Fidlerova E Smirnov and I RaskaldquoDynamics of replication foci in early S phase as visualized bycross-correlation functionrdquo Journal of Structural Biology vol151 no 1 pp 61ndash68 2005

[49] I-E Symeonidou P Kotsantis V Roukos et al ldquoMulti-steploading of human minichromosome maintenance proteins inlive human cellsrdquo The Journal of Biological Chemistry vol 288no 50 pp 35852ndash35867 2013

[50] D M Gilbert ldquoMaking sense of eukaryotic DNA replicationoriginsrdquo Science vol 294 no 5540 pp 96ndash100 2001

[51] H Kimura N Nozaki and K Sugimoto ldquoDNA polymerase 120572associated protein P1 a murine homolog of yeast MCM3changes its intranuclear distribution during the DNA syntheticperiodrdquoThe EMBO Journal vol 13 no 18 pp 4311ndash4320 1994

[52] R Burkhart D Schulte B Hu C Musahl F Gohring and RKnippers ldquoInteractions of humannuclear proteins P1Mcm3 andP1Cdc46rdquo European Journal of Biochemistry vol 228 no 2 pp431ndash438 1995

[53] SDonovan J Harwood L S Drury and J F XDiffley ldquoCdc6p-dependent loading of Mcm proteins onto pre-replicative chro-matin in budding yeastrdquo Proceedings of the National Academy ofSciences of the United States of America vol 94 no 11 pp 5611ndash5616 1997

[54] A Richter and R Knippers ldquoHigh-molecular-mass complexesof human minichromosome-maintenance proteins in mitoticcellsrdquo European Journal of Biochemistry vol 247 no 1 pp 136ndash141 1997

[55] MRitziM Baack CMusahl P Romanowski RA Laskey andR Knippers ldquoHuman minichromosome maintenance proteinsand human origin recognition complex 2 protein on chro-matinrdquoThe Journal of Biological Chemistry vol 273 no 38 pp24543ndash24549 1998

[56] H M Mahbubani J P J Chong S Chevalier P Thommesand J J Blow ldquoCell cycle regulation of the replication licensingsystem involvement of a Cdk-dependent inhibitorrdquoThe Journalof Cell Biology vol 136 no 1 pp 125ndash135 1997

[57] J Walter and J W Newport ldquoRegulation of replicon size inXenopus egg extractsrdquo Science vol 275 no 5302 pp 993ndash9951997

[58] M C Edwards A V Tutter C Cvetic C H Gilbert T AProkhorova and J C Walter ldquoMCM2-7 complexes bind chro-matin in a distributed pattern surrounding the origin recogni-tion complex in Xenopus egg extractsrdquoThe Journal of BiologicalChemistry vol 277 no 36 pp 33049ndash33057 2002

[59] T Aparicio D Megıas and J Mendez ldquoVisualization of theMCM DNA helicase at replication factories before the onset ofDNA synthesisrdquo Chromosoma vol 121 no 5 pp 499ndash507 2012

[60] M A Kuipers T J Stasevich T Sasaki et al ldquoHighly stableloading of Mcm proteins onto chromatin in living cells requiresreplication to unloadrdquo The Journal of Cell Biology vol 192 no1 pp 29ndash41 2011

[61] R A Laskey and M A Madine ldquoA rotary pumping modelfor helicase function of MCM proteins at a distance fromreplication forksrdquo The EMBO Reports vol 4 no 1 pp 26ndash302003

[62] T S Takahashi D B Wigley and J C Walter ldquoPumps para-doxes and ploughshares mechanism of the MCM2-7 DNA


helicaserdquo Trends in Biochemical Sciences vol 30 no 8 pp 437ndash444 2005

[63] M Masata P Juda O Raska M C Cardoso and I Raska ldquoAfraction of MCM 2 proteins remain associated with replicationfoci during a major part of S phaserdquo Folia Biologica vol 57 no1 pp 3ndash11 2011

[64] E Guillou A Ibarra V Coulon et al ldquoCohesin organizeschromatin loops at DNA replication factoriesrdquo Genes andDevelopment vol 24 no 24 pp 2812ndash2822 2010

[65] ANishiyama L Frappier andMMechali ldquoMCM-BP regulatesunloading of the MCM2-7 helicase in late S phaserdquo Genes ampDevelopment vol 25 no 2 pp 165ndash175 2011

[66] D Cortez G Glick and S J Elledge ldquoMinichromosome main-tenance proteins are direct targets of the ATM and ATRcheckpoint kinasesrdquo Proceedings of the National Academy ofSciences of the United States of America vol 101 no 27 pp10078ndash10083 2004

[67] C-C Tsao C Geisen and R T Abraham ldquoInteraction betweenhuman MCM7 and Rad17 proteins is required for replicationcheckpoint signalingrdquo The EMBO Journal vol 23 no 23 pp4660ndash4669 2004

[68] AMWoodward TGohlerMG Luciani et al ldquoExcessMcm2-7 license dormant origins of replication that can be used underconditions of replicative stressrdquoThe Journal of Cell Biology vol173 no 5 pp 673ndash683 2006

[69] R C Alver G S Chadha and J J Blow ldquoThe contribution ofdormant origins to genome stability fromcell biology to humangeneticsrdquo DNA Repair vol 19 no 9 pp 182ndash189 2014

[70] XQGeDA Jackson and J J Blow ldquoDormant origins licensedby excess Mcm2-7 are required for human cells to survivereplicative stressrdquo Genes amp Development vol 21 no 24 pp3331ndash3341 2007

[71] A Ibarra E Schwob and J Mendez ldquoExcess MCM proteinsprotect human cells from replicative stress by licensing backuporigins of replicationrdquo Proceedings of the National Academy ofSciences of the United States of America vol 105 no 26 pp8956ndash8961 2008

[72] N Shima A Alcaraz I Liachko et al ldquoA viable allele of Mcm4causes chromosome instability and mammary adenocarcino-mas in micerdquo Nature Genetics vol 39 no 1 pp 93ndash98 2007

[73] C-H Chuang M D Wallace C Abratte T Southard and JC Schimenti ldquoIncremental genetic perturbations to MCM2-7expression and subcellular distribution reveal exquisite sensi-tivity of mice to DNA replication stressrdquo PLoS Genetics vol 6no 9 Article ID e1001110 2010

[74] D Kunnev M E Rusiniak A Kudla A Freeland G K Cadyand S C Pruitt ldquoDNA damage response and tumorigenesis inMcm2-deficientmicerdquoOncogene vol 29 no 25 pp 3630ndash36382010

[75] K Maki T Inoue A Onaka et al ldquoAbundance of prereplicativecomplexes (Pre-RCs) facilitates recombinational repair underreplication stress in fission yeastrdquo The Journal of BiologicalChemistry vol 286 no 48 pp 41701ndash41710 2011

[76] J J Blow and A Dutta ldquoPreventing re-replication of chromoso-mal DNArdquo Nature Reviews Molecular Cell Biology vol 6 no 6pp 476ndash486 2005

[77] E E Arias and J C Walter ldquoStrength in numbers preventingrereplication via multiple mechanisms in eukaryotic cellsrdquoGenes amp Development vol 21 no 5 pp 497ndash518 2007

[78] J J Blow X Q Ge and D A Jackson ldquoHow dormant originspromote complete genome replicationrdquo Trends in BiochemicalSciences vol 36 no 8 pp 405ndash414 2011

[79] S Ryu J Holzschuh S Erhardt A-K Ettl and W DrieverldquoDepletion of minichromosome maintenance protein 5 inthe zebrafish retina causes cell-cycle defect and apoptosisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 102 no 51 pp 18467ndash18472 2005

[80] C R Hughes L Guasti EMeimaridou et al ldquoMCM4mutationcauses adrenal failure short stature and natural killer celldeficiency in humansrdquoThe Journal of Clinical Investigation vol122 no 3 pp 814ndash820 2012

[81] L Gineau C Cognet N Kara et al ldquoPartial MCM4 deficiencyin patients with growth retardation adrenal insufficiency andnatural killer cell deficiencyrdquo The Journal of Clinical Investiga-tion vol 122 no 3 pp 821ndash832 2012

[82] B N Bagley T M Keane V I Maklakova et al ldquoA dominantlyacting murine allele of Mcm4 causes chromosomal abnormali-ties and promotes tumorigenesisrdquo PLoS Genetics vol 8 no 11Article ID e1003034 2012

[83] S C Pruitt K J Bailey and A Freeland ldquoReduced Mcm2expression results in severe stemprogenitor cell deficiency andcancerrdquo Stem Cells vol 25 no 12 pp 3121ndash3132 2007

[84] M E Rusiniak D Kunnev A Freeland G K Cady and S CPruitt ldquoMcm2 deficiency results in short deletions allowinghigh resolution identification of genes contributing to lympho-blastic lymphomardquo Oncogene vol 31 no 36 pp 4034ndash40442012

[85] B Ren G Yu G C Tseng et al ldquoMCM7 amplification andoverexpression are associatedwith prostate cancer progressionrdquoOncogene vol 25 no 7 pp 1090ndash1098 2006

[86] C Giaginis M Georgiadou K Dimakopoulou et al ldquoClinicalsignificance ofMCM-2 andMCM-5 expression in colon cancerassociation with clinicopathological parameters and tumorproliferative capacityrdquo Digestive Diseases and Sciences vol 54no 2 pp 282ndash291 2009

[87] O Saydam O Senol T B M Schaaij-Visser et al ldquoCompar-ative protein profiling reveals minichromosome maintenance(MCM) proteins as novel potential tumor markers for menin-giomasrdquo Journal of Proteome Research vol 9 no 1 pp 485ndash4942010

[88] U Cobanoglu S Mungan C Gundogdu S Ersoz Y Ozoranand F Aydin ldquoThe expression of MCM-2 in invasive breastcarcinoma a stereologic approachrdquo Bratislavske Lekarske Listyvol 111 no 1 pp 45ndash49 2010

[89] K-M Lau Q K Y Chan J C S Pang et al ldquoMinichromosomemaintenance proteins 2 3 and 7 in medulloblastoma overex-pression and involvement in regulation of cell migration andinvasionrdquo Oncogene vol 29 no 40 pp 5475ndash5489 2010

[90] C Giaginis A Giagini G Tsourouflis et al ldquoMCM-2 andMCM-5 expression in gastric adenocarcinoma clinical sig-nificance and comparison with Ki-67 proliferative markerrdquoDigestive Diseases and Sciences vol 56 no 3 pp 777ndash785 2011

[91] A F Nicol J R Lapa e Silva C B Cunha et al ldquoEvaluationof MCM-2 expression in TMA cervical specimensrdquo PLoS ONEvol 7 no 4 Article ID e32936 2012

[92] M Das S B Prasad S S Yadav et al ldquoOver expression ofmini-chromosome maintenance genes is clinically correlated tocervical carcinogenesisrdquo PLoS ONE vol 8 no 7 Article IDe69607 2013

[93] M L Kuijjer H Rydbeck S H Kresse et al ldquoIdentification ofosteosarcoma driver genes by integrative analysis of copy num-ber and gene expression datardquoGenes Chromosomes and Cancervol 51 no 7 pp 696ndash706 2012


[94] X Zhong X Chen X Guan et al ldquoOverexpressions of G9a andMCM7 in Esophageal squamous cell carcinoma associated withpoor prognosisrdquo Histopathology 2014

[95] A Freeman L S Morris A D Mills et al ldquoMinichromosomemaintenance proteins as biological markers of dysplasia andmalignancyrdquo Clinical Cancer Research vol 5 no 8 pp 2121ndash2132 1999

[96] S B Wharton K K Chan J R Anderson K Stoeber and GHWilliams ldquoReplicativeMcm2 protein as a novel proliferationmarker in oligodendrogliomas and its relationship to Ki67labelling index histological grade and prognosisrdquoNeuropathol-ogy and Applied Neurobiology vol 27 no 4 pp 305ndash313 2001

[97] K Rodins M Cheale N Coleman and S B Fox ldquoMinichro-mosome maintenance protein 2 expression in normal kidneyand renal cell carcinomas relationship to tumor dormancy andpotential clinical utilityrdquo Clinical Cancer Research vol 8 no 4pp 1075ndash1081 2002

[98] HKato TMiyazaki Y Fukai et al ldquoAnewproliferationmarkerminichromosome maintenance protein 2 is associated withtumor aggressiveness in esophageal squamous cell carcinomardquoJournal of Surgical Oncology vol 84 no 1 pp 24ndash30 2003

[99] P Chatrath I S Scott L SMorris et al ldquoAberrant expression ofminichromosomemaintenance protein-2 and Ki67 in laryngealsquamous epithelial lesionsrdquo British Journal of Cancer vol 89no 6 pp 1048ndash1054 2003

[100] M A Gonzalez S E Pinder G Callagy et al ldquoMinichromo-somemaintenance protein 2 is a strong independent prognosticmarker in breast cancerrdquo Journal of Clinical Oncology vol 21 no23 pp 4306ndash4313 2003

[101] C Kobierzycki B Pula A Wojnar M Podhorska-Okolowand P Dziegiel ldquoTissue microarray technique in evaluation ofproliferative activity in invasive ductal breast cancerrdquoAnticancerResearch vol 32 no 3 pp 773ndash777 2012

[102] E C Obermann P Went A Zimpfer et al ldquoExpression ofminichromosomemaintenance protein 2 as a marker for prolif-eration and prognosis in diffuse large B-cell lymphoma a tissuemicroarray and clinico-pathological analysisrdquoBMCCancer vol5 article 162 2005

[103] J Szelachowska P Dziegiel J Jelen-Krzeszewska et al ldquoMcm-2protein expression predicts prognosis better than Ki-67 antigenin oral cavity squamocellular carcinomardquo Anticancer Researchvol 26 no 3 pp 2473ndash2478 2006

[104] H Gakiopoulou P Korkolopoulou G Levidou et al ldquoMini-chromosome maintenance proteins 2 and 5 in non-benignepithelial ovarian tumours relationship with cell cycle regula-tors and prognostic implicationsrdquo British Journal of Cancer vol97 no 8 pp 1124ndash1134 2007

[105] M Liu J-S Li D-P Tian B Huang S Rosqvist and M SuldquoMCM2 expression levels predict diagnosis and prognosis ingastric cardiac cancerrdquoHistology andHistopathology vol 28 no4 pp 481ndash492 2013

[106] Y S Lee S-A Ha H J Kim et al ldquoMinichromosome mainte-nance protein 3 is a candidate proliferation marker in papillarythyroid carcinomardquo Experimental andMolecular Pathology vol88 no 1 pp 138ndash142 2010

[107] J Kikuchi I Kinoshita Y Shimizu et al ldquoMinichromosomemaintenance (MCM) protein 4 as amarker for proliferation andits clinical and clinicopathological significance in non-small celllung cancerrdquo Lung Cancer vol 72 no 2 pp 229ndash237 2011

[108] T J Dudderidge J D Kelly A Wollenschlaeger et al ldquoDiag-nosis of prostate cancer by detection of minichromosome

maintenance 5 protein in urine sedimentsrdquo The British Journalof Cancer vol 103 no 5 pp 701ndash707 2010

[109] T Zheng M Chen S Han et al ldquoPlasma minichromosomemaintenance complex component 6 is a novel biomarker forhepatocellular carcinoma patientsrdquo Hepatology Research 2014

[110] K Nishihara K Shomori S Fujioka et al ldquoMinichromosomemaintenance protein 7 in colorectal cancer implication ofprognostic significancerdquo International Journal of Oncology vol33 no 2 pp 245ndash251 2008

[111] S Fujioka K Shomori K Nishihara et al ldquoExpression of mini-chromosomemaintenance 7 (MCM7) in small lung adenocarci-nomas (pT1) prognostic implicationrdquo Lung Cancer vol 65 no2 pp 223ndash229 2009

[112] T Tamura K Shomori T Haruki et al ldquoMinichromosomemaintenance-7 and geminin are reliable prognostic markersin patients with oral squamous cell carcinoma Immunohisto-chemical studyrdquo Journal of Oral Pathology andMedicine vol 39no 4 pp 328ndash334 2010

[113] J J Blow and B Hodgson ldquoReplication licensingmdashdefining theproliferative staterdquo Trends in Cell Biology vol 12 no 2 pp 72ndash78 2002

[114] J J Blow and P J Gillespie ldquoReplication licensing and cancermdasha fatal entanglementrdquoNature Reviews Cancer vol 8 no 10 pp799ndash806 2008

[115] O J KayesM Loddo N Patel et al ldquoDNA replication licensingfactors and aneuploidy are linked to tumor cell cycle state andclinical outcome in penile carcinomardquoClinical Cancer Researchvol 15 no 23 pp 7335ndash7344 2009

[116] C P Harris X Y Lu G Narayan B Singh V V V S Murtyand P H Rao ldquoComprehensive molecular cytogenetic charac-terization of cervical cancer cell linesrdquoGenes Chromosomes andCancer vol 36 no 3 pp 233ndash241 2003

[117] PH RaoHArias-Pulido X-Y Lu et al ldquoChromosomal ampli-fications 3q gain and deletions of 2q33-q37 are the frequentgenetic changes incervical carcinomardquo BMC Cancer vol 4article 5 2004

[118] G Narayan V Bourdon S Chaganti et al ldquoGene dosage alter-ations revealed by cDNAmicroarray analysis in cervical canceridentification of candidate amplified and overexpressed genesrdquoGenes Chromosomes and Cancer vol 46 no 4 pp 373ndash3842007

[119] T A Potapova J Zhu andR Li ldquoAneuploidy and chromosomalinstability a vicious cycle driving cellular evolution and cancergenome chaosrdquo Cancer and Metastasis Reviews vol 32 no 3-4pp 377ndash389 2013

[120] S Shreeram A Sparks D P Lane and J J Blow ldquoCell type-specific responses of human cells to inhibition of replicationlicensingrdquo Oncogene vol 21 no 43 pp 6624ndash6632 2002

[121] D Feng Z Tu WWu and C Liang ldquoInhibiting the expressionof DNA replication-initiation proteins induces apoptosis inhuman cancer cellsrdquo Cancer Research vol 63 no 21 pp 7356ndash7364 2003

[122] Y Liu G He YWang X Guan X Pang and B Zhang ldquoMCM-2 is a therapeutic target of Trichostatin A in colon cancer cellsrdquoToxicology Letters vol 221 no 1 pp 23ndash30 2013

[123] E P Erkan T Strobel G Lewandrowski et al ldquoDepletion ofminichromosome maintenance protein 7 inhibits glioblastomamultiforme tumor growth in vivordquoOncogene vol 33 pp 4778ndash4785 2013

[124] T J Dudderidge S R McCracken M Loddo et al ldquoMitogenicgrowth signalling DNA replication licensing and survival are


linked in prostate cancerrdquo British Journal of Cancer vol 96 no9 pp 1384ndash1393 2007

[125] T Valovka M Schonfeld P Raffeiner et al ldquoTranscriptionalcontrol of DNA replication licensing byMycrdquo Scientific Reportsvol 3 article 3444 2013

[126] S Majid A A Dar S Saini et al ldquoRegulation of minichro-mosome maintenance gene family by MicroRNA-1296 andgenistein in prostate cancerrdquo Cancer Research vol 70 no 7 pp2809ndash2818 2010

[127] J H Luo ldquoOncogenic activity of MCM7 transforming clusterrdquoWorld Journal of Clinical Oncology vol 2 no 2 pp 120ndash1242011

[128] K Yankulov I Todorov P Romanowski et al ldquoMCM proteinsare associated with RNA polymerase II holoenzymerdquoMolecularand Cellular Biology vol 19 no 9 pp 6154ndash6163 1999

[129] M Snyder W He and J J Zhang ldquoThe DNA replication factorMCM5 is essential for Stat1-mediated transcriptional activa-tionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 102 no 41 pp 14539ndash14544 2005

[130] M E Hubbi W Luo J H Baek and G L Semenza ldquoMCMproteins are negative regulators of hypoxia-inducible factor 1rdquoMolecular Cell vol 42 no 5 pp 700ndash712 2011

[131] I M Slaymaker Y Fu D B Toso et al ldquoMini-chromosomemaintenance complexes form a filament to remodel DNAstructure and topologyrdquo Nucleic Acids Research vol 41 no 5pp 3446ndash3456 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Microbiology


during replication such as MCM2 binds to histones andfacilitate histone redeposition during DNA synthesis [15]However the fact that MCM proteins are essential in ver-tebrate DNA replication was confirmed by the studies inXenopus where MCM depleted egg extracts were unable tosupport further DNA synthesis [16 17]

A parallel concept of ldquoreplication licensingrdquo was emergingin 1980s from the observation that sperm chromatin in anucleus replicated only once in a cell cycle and becamecompetent again inmitosis but a specificDNAsequence is notrequired for replication initiationThis concept hypothesizedthat there must be a positive ldquolicenserdquo to mark the replicatedsequences from the unreplicated ones thus preventing thereinitiation of replication at the already replicated sites [18]The cell cycle dependent change in subcellular localization ofMCMs in yeast and Xenopus proposed these proteins to bereplication licensing factors [8 15 19] However the repli-cation licensing mechanism involves a series of events anda number of proteins The first step of replication licensingmechanism is the formation of prereplication complex (pre-RC) Pre-RC formation involves assembly of a number of pro-teins such as origin recognition complex (ORC) Cdc6 Cdt1and MCM 2ndash7 This in turn leads to a chain of events allow-ing binding of DNA polymerase and other several factorsto chromatin to start DNA replication [7 20ndash22]

3 MCMs Function as Helicases

After the identification of MCMs as replication licensingfactors their biochemical function at the chromatin had beensearched forThe heterohexameric ring structure of MCM 2ndash7 suggests that this protein complex may work as replicativehelicase The entire MCM 2ndash7 complex in fission yeast orMCM (4 6 7)

2complex from the mouse shows a globular

structure with a central channel of 3-4 nm similar to manymultisubunit helicases from all kingdom of life This channelcan encircle either single or double strandedDNA [1] In 2001Labib and Diffley [23] enquired whether MCM 2ndash7 is theeukaryotic DNA replication fork helicase The first evidencecame from the study of Yukio Ishimi where they had shownthat purified MCM 4 6 7 complexes have 31015840 rarr 51015840 DNAunwinding capacity [24] Consequently they have also shownthat mouse MCM 4 6 7 complexes have intrinsic DNA heli-case activity [12] Not only the MCM 4 6 7 subcomplex buta wholeMCM2ndash7 complex had been reported to have in vitrohelicase activity where theMCM 2ndash5 junction acts as an ATPdrive gap or ldquogaterdquo [25] Recent studies have shown that theMCM 2ndash7 proteins make a core of the replicative DNA heli-case and that they are first loaded at replication origins in aninactive formThe helicase is then activated by recruitment ofthe Cdc45 and GINS proteins into a holo-helicase known asCMG(Cdc45MCM2ndash7GINS) [26] Identification of severalinteracting partners and the real action mechanism of thisholo-helicase keeps researchers involved in studies ofMCMsVery recently it has been shown that the human CMG com-plex (hCMG) can unwind DNA duplex regions up to 500 bphydrolyzing ATP and in combination with human DNApolymerase 120576 leads to the formation of oligonucleotide

products up togt10 kb [27]The same group has also identifiedhuman chromosome transmission fidelity 4 (hCTF4) as aninteracting partner of hCMG complex [28]

The helicase activity of MCM proteins was also found inarcheal species [29]Methanobacterium thermoautotrohicumthe bacterium contains only one MCM protein which formsa homohexameric ring The homohexamer contains a posi-tively charged central channel of 2ndash4 nm to bind with DNAand act as a helicase [30ndash33] A single MCM homolog fromSulfolobus solfataricus has been reported to form hexamers insolution and can dissolve 51015840 tailed oligonucleotides by itshelicase activity [34]

MCM2ndash7 proteins are categorized in superfamily 6 (SF6)helicases [35 36] in which each subunit of a hexameric heli-case contains anAAA+domain In these helicases theATPasesites are generated in between two adjacent subunits oneis providing the walker A andwalker Bmotif for ATP bindingand the other partner provides the arginine finger motif forATP hydrolysis MCM 2ndash7 is also related to viral homohex-americ helicases (superfamily 3 (SF3)) including the SV40large T antigen and papilloma virus E1 protein [26]

Boos et al [26] also summarized the evidence indicatingMCM 2ndash7 as the engine of the replicative helicase FirstlyMCM 2ndash7 travels with replication forks forming part of thepurified replisome progression complex (RPC) in yeast [3637] and vertebrates [38ndash41] Secondly acute inactivation ofMCM 2ndash7 subunits during S phase results in rapid replisomeinactivation [42 43] and third purified yeast MCM 2ndash7complex shows in vitro helicase activity [25]

4 The MCM Paradox

Above discussed lines of evidence substantially show thatMCM 2ndash7 complex has helicase activity to unwind doublestranded DNA and thus helps to initiate DNA replicationBut there are some issues that prohibit accepting the role ofMCM 2ndash7 as helicase The issues termed as ldquoMCM paradoxrdquocame from two observations (a) eukaryotic MCM 2ndash7 com-plexes are not localized at the sites of replication like viral andbacterial helicases MCM 2ndash7 complexes in higher eukary-otes are distributed over nonreplicated DNA rather thanon replication forks (b) the paradox is elaborated by anotherobservation that excess MCM heterohexamers are loadedonto chromatin rather than replication origins and ORCs[44] The basic questions were how MCM 2ndash7 complexesperform their function as helicase from a distance away fromthe replication sites andwhat the purpose of the excess boundMCMs onto chromatin more than the origins themselves is

Madine et al [45] have shown by immunofluorescencethat Xenopus MCM3 does not colocalize with sites of DNAreplication The protein is almost uniformly distributed onchromatin and is suddenly lost during replication Same lineof evidence in human cells came from another study in 1996The authors had used high resolution confocal microscopyto determine the subnuclear sites of chromatin-boundMCMproteins in comparison to the sites of replicating DNAThey found MCM proteins to be entirely nuclear in HeLacells at interphase and to exist in nucleoplasmic and chro-matin bound forms The chromatin bound form is present
















Backup origin





Chk1 signalling

Chk1 signalling


No backup





Chk1 signalling

Chk1 signalling










Normal cell

Intact genome



Depleted MCMlevel


Genomic instability

Elevated MCMlevel










Acknowledgments




References



















































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
















Backup origin





Chk1 signalling

Chk1 signalling


No backup





Chk1 signalling

Chk1 signalling










Normal cell

Intact genome



Depleted MCMlevel


Genomic instability

Elevated MCMlevel










Acknowledgments




References



















































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Backup origin





Chk1 signalling

Chk1 signalling


No backup





Chk1 signalling

Chk1 signalling










Normal cell

Intact genome



Depleted MCMlevel


Genomic instability

Elevated MCMlevel










Acknowledgments




References



















































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Backup origin





Chk1 signalling

Chk1 signalling


No backup





Chk1 signalling

Chk1 signalling










Normal cell

Intact genome



Depleted MCMlevel


Genomic instability

Elevated MCMlevel










Acknowledgments




References



















































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Normal cell

Intact genome



Depleted MCMlevel


Genomic instability

Elevated MCMlevel










Acknowledgments




References



















































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



References



















































































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Review Article MCM Paradox: Abundance of Eukaryotic