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Termination of Eukaryotic Replication ForksGambus, Agnieszka

DOI:10.1007/978-981-10-6955-0_8

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Citation for published version (Harvard):Gambus, A 2018, Termination of Eukaryotic Replication Forks. in DNA Replication: From Old Principles to NewDiscoveries. vol. 1042, Advances in Experimental Medicine and Biology, vol. 1042, Springer, pp. 163-187.https://doi.org/10.1007/978-981-10-6955-0_8

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Download date: 23. Jul. 2020

1

Terminationofeukaryoticreplicationforks

AgnieszkaGambus

InstituteofCancerandGenomicSciences,UniversityofBirmingham,

VincentDrive,Birmingham,B152TT,UK

a.gambus@bham.ac.uk

Abstract

TerminationofDNAreplicationforkstakesplacewhentworeplication

forkscomingfromneighbouringoriginsmeeteachotherusuallyinthemidpoint

ofthereplicon.Atthisstage,theremainingfragmentsofDNAhavetobe

unwound,allremainingDNAreplicatedandnewlysynthesisedstrandsligated

toproducecontinuoussisterchromatids.Finally,thereplicationmachineryhas

tobetakenoffchromatinandentwistedsisterchromatidsresolved

topologically.

Overthelastfewdecadeswehavelearnedalotabouttheassemblyofthe

helicaseandreplisomeandtheinitiationstageofDNAreplication.Wealsoknow

muchmoreabouttheabilityofforkstocopewithreplicationstress.However,

onlywithintherecentfewyearswegainedthefirstglimpseofthemechanismof

replicationforktermination.InthischapterIwillsummarisetherecentfindings

onreplicationtermination,weighthisagainstthepastliteratureanddiscuss

relevantconsequencesandviewsforthefuture.

Keywords

EukaryoticDNAreplication,terminationofDNAreplication,ubiquitin,

Cdc48p97segregase,cullins,

2

1.Introduction

TomaintaingenomicstabilityitisessentialthateverystepofDNA

replicationisfaultlesslyexecuted.Mistakesduringeukaryoticreplicationthat

arenotefficientlyrepairedcanleadtomutationsandgenomerearrangements

thatpromotechangesleadingtodevelopmentofcancerandotherdisorders.

DNAreplicationcanbedividedintothreestages:initiation,elongationand

termination.InitiationofDNAreplicationhappenswhenlicensedoriginsof

replicationfirecreatingtwoDNAreplicationforks,whichmoveinopposite

directions.Theelongationstageinvolvestheprogressionofreplicationforksas

theyunwindandreplicateDNA.Finally,terminationhappenswhentwo

replicationforksfromneighbouringoriginsconvergeandtheduplicationof

remainingfragmentofDNAisneatlycompleted.Overtheyearswehavelearnta

lotaboutthemechanismsofDNAreplicationinitiationandelongation(briefly

explainedbelow),butuntilrecentlyourknowledgeofreplicationtermination

wasveryrestricted.Thelastfewyearshavebroughtabreakthroughinour

understandingofmechanismsofreplicationtermination:wehavelearntthat

convergingreplicationforkscanpasseachotherwhenterminatingandwehave

alsounravelledtheworkingsofdisassemblyofterminatedreplisomes.

2.ReplicationforkterminationoccursthroughoutSphase

Whendoesreplicationterminationtakeplace?Inourmindreplication

terminationshouldhappenmostlyattheendofthewholereplicationprocess,

sointhetermsofcellcyclestages-attheendofS-phase.Inrealityhowever,

DNAreplicationforksencounterforksfromneighbouringoriginsthroughoutthe

entireS-phase.ForksemanatingfromoriginclustersfiringinearlyS-phasewill

alsoterminateinearlyS-phase;withaveragerepliconsizeof31kbp(Morenoet

al.,2016;Picardetal.,2014)andanaverageforkspeedof1.5kb/min(Contiet

al.,2007)ittakesabout10minutesforthetwoneighbouringforkstoreachone

another.InfactthereislikelymoreterminationoccurringinmidS-phasethanin

lateS-phaseasthestrictreplicationtimingprogrammedrivingreplicationin

3

eachcellmeansthatonlydifficult-to-replicateregionsarereplicatedinlateS-

phase(Gilbert,2010).

3.Replicationinitiationandelongation

Toensurethatallofthelargeeukaryoticgenomesareduplicatedinfull

beforeeachcelldivision,eukaryoticDNAreplicationstartsfrommultipleorigins

ofreplication.Humancellshaveonaverageabout50thousandofthemspread

throughoutthegenome.ItisalsoessentialthatDNAisreplicatedjustonceper

cellcycleasre-replicationofpartsofthegenomeisathreatforthemaintenance

ofgenomeintegrity.Toachievethis,thereplicativehelicase(proteincomplex,

whichunwindsdoublestrandedDNAduringreplication)canbeloadedonto

DNAonlybeforetheonsetofS-phasewhenCDKactivityislow,andcanbe

activatedonlyduringS-phasewhenCDKactivityishigh.Originsofreplication

aretherefore“licensed”inlateMandG1stagesofthecellcycle,byloadingofthe

coreofthereplicativehelicase:Mcm2-7(Minichromosomemaintenance

2,3,4,5,6,7)complexes.DoublehexamersoftheMcm2-7complexareloaded

ontooriginsthroughtheconcertedactionofORC(OriginRecognitionComplex),

Cdc6andCdt1factors.Thesedoublehexamersencirclethedoublestranded

DNAandarearrangedinN-terminustoN-terminusorientationwiththeC-

terminalhelicasedomainsontheoutside.TherearemultipleMcm2-7double

hexamersloadedaroundeachoriginofreplication,whichmaybefacilitatedby

theirabilitytoslideondoublestrandedDNA(Evrinetal.,2009;Gambusetal.,

2011;Remusetal.,2009).

TheinitiationofDNAreplicationrequirestheactivityoftwoS-phase

kinases:Cdc7/Dbf4(DDK–Dbf4dependentkinase)andCdk/cyclin(CDK–

Cyclindependentkinase).DDKphosphorylatesdoublehexamersofMcm2-7

whileCDKdrivesassociationofGINS(Go-Ichi-Ni-San,complexofSld5,Psf1,Psf2

andPsf3orGINS1,2,3,4)andCdc45withtheMcm2-7complexes,formingthe

CMGcomplex(Cdc45/Mcm2-7/GINS),whichisanactivereplicativehelicase

(Ilvesetal.,2010;Moyeretal.,2006;Simonetal.,2016).Theinitiationprocess

leadstorearrangementofMcm2-7complexes:thedoublehexamerssplitinto

4

twoCMGsandeachofthemnowlikelyencirclesjustsinglestrandedDNA(Costa

etal.,2011;Gambusetal.,2006;Yardimcietal.,2010).

DuringtheelongationstageofDNAreplicationthehelicase(CMGcomplex)

travelsatthetipofthereplicationfork,unwindingthedoublestrandedDNAand

exposingsinglestrandsthatcanactasatemplateforDNAsynthesisbyDNA

replicationpolymerases.TheMCMmotorofCMGbelongstothesuperfamilyof

AAA+ATPasesandisa3’-5’DNAtranslocase,whichencirclestheleadingstrand

ofthereplicationfork(reviewedin(PellegriniandCosta,2016)).ThePola-

primasecomplexinitiatesDNAsynthesiswithashortRNAprimerthatisthen

elongatedforanother20-ntbyPolapolymeraseactivity.Theleadingstrandis

believedtobesynthesisedmainlybyDNAPole(DNApolymeraseepsilon)ina

continuousmanner,whilethelaggingstrandisthoughttobecompletedbyDNA

Pold(DNApolymerasedelta)(Daigakuetal.,2015;Georgescuetal.,2015;

Pavlovetal.,2006).ThelattersynthesisesshortOkazakifragmentsinthe

oppositedirectiontothemovementoftheforkandthesefragmentsneedto

thereforebeprocessedandligatedtoproducethecontinuousDNAstrand

(maturationofOkazakifragments).DNAPoleisthereforefollowingthehelicase

andindeedhasanumberofconnectionslinkingitdirectlytothehelicaseto

facilitatethesmoothprogressionofthefork(seebelowandreviewedin

(PellegriniandCosta,2016)).

DNAunwindinggeneratesacompensatoryincreaseintheintertwiningof

parentalstrands,whichcanbeconvertedintohelicaloverwinding(positive

supercoiling)oftheunreplicatedportionsoftheDNAaheadoftheforks(Postow

etal.,2001;Wang,2002).Thismechanicalstraincanbetransmittedto

replicatedDNAbyrotationatthebranchingpointofthereplicationfork,thus

generatingintertwiningofthedaughterduplexes(knownasprecatenates)

(BeenandChampoux,1980)(Figure1).Recentresearchinbuddingyeasthas

shownhoweverthatduringnormalprogressionofreplicationforks,fork

rotationandprecatenationareactivelyinhibitedbycomponentsofthe

replisomeTimeless/Tof1andTipin/Csm3(Schalbetteretal.,2015).Instead,

supercoilsgeneratedduringreplicationelongationcanberelaxedbybothtypeI

andtypeIItopoisomerases(Wang,2002).Indeed,thecurrentviewassumesthat

positivesupercoilingismainlyrelaxedbytypeIenzymes(TopoI,S.cerevisiae

5

Top1)anywhereintheunreplicatedregion(Postowetal.,2001).Thereplisome

progressioncomplex(RPC)builtaroundCMGatthetipoftheforkcontains

Top1,positioningitperfectlyforitsfunctionaheadofthefork(Gambusetal.,

2006)(Figure1).Interestingly,yeastcellswithoutTop1andalsotop2mutants

canreplicateDNA,butreplicationisnotpossiblewhenbothproteinsare

defective(Bermejoetal.,2007;Brilletal.,1987).

Importantly,replicationforksdonotmovethroughnakedDNAbut

throughachromatinstructure.Nucleosomesthereforeneedtobedismantled

aheadoftheforksandrebuiltbehindtheforks.Theefficientrepositioningof

parentalhistonesisessentialforfullreconstitutionofepigeneticmarkings

throughoutthereplicatinggenome.StudiesofSV40replicationforksprovided

evidencefortheexistenceofonly200-300bpofapparentlynucleosome-free

DNAbehindthereplicationfork(Gasseretal.,1996)andthenucleosomesin

yeastwereshownrecentlytobepositionedimmediatelyaftertheforkpassage

andrestrictOkazakifragmentssizes(SmithandWhitehouse,2012).Progressing

replicationforksneedalsotoremoveotherproteinsattachedtoDNA,for

exampletheunfiredMcm2-7doublehexamers,which,loadedinexcess,serveas

dormantoriginsreadytorescuecollapsedforks.Finally,sisterchromatidsare

topologicallyembracedandheldtogetheruntilmitosisbycohesinring

complexes.ThiscohesionisestablishedduringDNAreplicationasforks

progress(reviewedby(Uhlmann,2009)).

4.Wheredoesterminationofeukaryoticreplicationforkshappen?

Thesimplestanswertothisquestionis:whereverthetwoneighbouring

forksmeeteachother.Recentanalysisofgenome-widereplicationprofilesin

buddingyeast,boththroughhigh-resolutionreplicationprofiling(Hawkinset

al.,2013)andthroughdeepsequencingofOkazakifragments(McGuffeeetal.,

2013),showedthatterminationgenerallyoccursmidwaybetweentwoadjacent

replicationorigins.Theprecisepositionofterminationdependsontherelative

activationtimeofeachoftheoriginsandtheirvariableefficiency.Okazaki

fragmentmappinginhumancells(HeLaandGM06990)alsoconfirmedsuch

mid-pointlocalisation(Petryketal.,2016).

6

Eukaryotesnotonlyhavespecificspatialpatternsbutalsopossess

temporalpatternsofgenomereplication,whichareexecutedbyregulated

activationofreplicationoriginsthroughoutS-phase.High-throughput

experimentsallowedtheidentificationofagenome-widetemporalorderof

replication(Gilbert,2010).InearlyS-phase,“active”chromatinisreplicated

withoriginsofreplicationlocatedingeneralin-betweenthegenes.Not

surprisinglytherefore,manyterminationeventsinearlyS-phasewerefoundto

overlapwithtranscribedgenes.InlateS-phasehowever,whenheterochromatin

isreplicated,manyterminationzoneswerefoundinlargenon-expressed

regionsofDNA(Petryketal.,2016).

Thissequenceindependentlocalisationofterminationsitesisinsharp

contrasttotheorganisationofterminationeventsinE.colichromosomewhere

terminationtakesplacewithinabroadregioncontainingseveralspecialised

forkbarriersi.e.Tus-TERcomplexes,whichconfineforkfusiontoasiteof270

kb(reviewedin(Dimudeetal.,2016)).Duetothesedefinedprokaryotic

TerminationRegions(TER),foranumberofyearsterminationofeukaryotic

replicationforkswasstudiedonlyattheexistingfewlociwithintheeukaryotic

genome,whichcontainspecialisedreplicationforkbarriers(RFBs).Thebest

characterisedofsuchsitesaretheRTS1siteinS.pombe,whichregulatesmating

typeswitching(BrewerandFangman,1988),andtherDNAlocuswithin

ribosomalDNArepeatsofmetazoaandyeasts(DalgaardandKlar,2000).The

RFBbarriersareabletoarrestoneofthetwoneighbouringforksandtherefore

createspecificterminationsites(BastiaandZaman,2014;Dalgaardetal.,2009).

TominimiseforkpausingatRFBstheproteindisplacementhelicaseRrm3helps

todisplacethebarrierstoallowreplicationpassageandisrequiredforfork

terminationatthesesites.InyeastlackingRrm3tenfoldaccumulationof

terminationstructures(“X”shapedDNAstructuresin2DDNAgels)was

observed,whileonlytwofoldaccumulationofpausedforksatthebarrier(Ivessa

etal.,2003;Ivessaetal.,2000).ButRrm3isnotrequiredforbulkreplisome

unloadingduringnormaltermination(Maricetal.,2014),soitisneededonlyfor

forkconvergenceatraresituationswhenoneforkispaused.

In2010,Fachinettietal,identified71terminationregions(TERs)in

buddingyeast,throughacombinationofChromatinimmunoprecipitation(ChiP)

7

andBrdUincorporationexperiments.Theirworkfoundthatthemajorityof

theseregionscontainforkpausingelements,suchastranscriptionclusters,and

thatefficientterminationattheidentifiedsitesrequiresactivityofRrm3and

Top2(Fachinettietal.,2010).However,themorerecenthigh-resolution

approachessuggestthattheseTERsactuallyrepresentsiteswithahigherthan

averageprobabilityofterminationastheyareflankedbyearly-firingefficient

origins.Importantly,changesoforiginfiringpatternmovedthetermination

positioningbothinnon-TERandTERreplicons,indicatingthatitisthetiming

andefficiencyoforiginfiringandnotforkpausingelementsthatdictatethe

preciseplaceofreplicationforkconvergence(Hawkinsetal.,2013;McGuffeeet

al.,2013).

5.Howdoreplicationforksconverge?

Figure2summarisesourcurrentmodelofreplicationforktermination.To

allowconvergenceoftwoapproachingDNAreplicationforksalloftheproteins

boundtoDNAbetweenthemmustbeevicted(Figure2A).Unwindingoffinal

stretchesofDNAcanpresentaproblemfortheforksasthetorsionalstress

createdaheadoftheforkcannotbeeasilyreleasedduetolackofaccessforTopI

(seebelowformoredetails)andhastobetranslatedintoprecatenates,which

accumulatebehindthefork(Figure2B).Twoconvergingforkspresenttwolarge

proteinmachineriesapproachingoneanotherandheadingforhead-oncollision

whileunwindingtheremainingDNAbetweenthem(Figure2C).Afterforks

converge,alloftheremainingDNAneedstobereplicatedandtheRNA-DNA

primerofthelastOkazakifragmentonthelaggingstrandneedstobeprocessed

(Figure2D,E).Oncethisiscomplete,DNAneedstobeligatedintoacontinuous

strandandreplisomesneedtobedisassembled(Figure2E).Finally,the

entangledsisterchromatidsneedtoberesolvedintotwoseparatestrands

(Figure2G).

Recentyearshavebroughtabreakthroughinourunderstandingofthe

aboveprocesses.BeautifulworkfromProfJohannesWalter’slabshedlighton

themechanismbywhichforksconvergeandterminationisresolved(Dewar

8

Walter2015).Tosynchroniseterminationeventsandfacilitatetheiranalysis,

theyconstructedplasmidswithanarrayoflacrepressors(LacRs)boundtolac

operators(LacOs),whichcanbedisruptedbyIPTG.Suchplasmidsreplicatedin

cell-freeXenopuslaeviseggextractaccumulatedblockedforksattheedgesofthe

array.TheblockedforkswerethenreleasedbyadditionofIPTG,andproceeded

toterminatewithintheDNAfragmentcomprisingthearray.Usingthissystem,

Dewaretal.couldmonitor:unwindingofDNAasforksapproacheachother,

synthesisofDNA,ligationofthereplicatedDNAanddecatenationofdaughter

molecules.Strikingly,therateofDNAsynthesiswithinthearraywasalmost

perfectlylinearafterIPTGadditionandresembledtheforkprogressionspeed

reportedinthesameextracts.Itsuggeststhereforethatconvergingforksdonot

slowsignificantlybeforetheymeet;theydonotcollidewitheachotherorstall

butratherpasseachother(Dewaretal.,2015)(Figure2CandD).Suchpassage

canbepossibleasCMGsencircletheleadingstrandofthereplicationforkand

thereforeapproacheachotheronoppositestrandswhenconvergingat

termination(Alietal.,2016;Costaetal.,2011;Fuetal.,2011).Interestingly,

however,recentreportssuggestthatlargeproteinbarriersonthelaggingstrand

canindeedslowdownprogressionofthefork(Duxinetal.,2014;Langstonand

O'Donnell,2017).Doestheapproachingneighbourreplisome,whichisonthe

laggingstrand,notpresentsuchabarrier?Isthereanactivemechanism

regulatingthesmoothpassageofthereplisomes?Orarethereplisomesidlingat

theedgeofthebarrier,intheattempttounwindit,especiallypreparedtodeal

withbarrierslayingaheadandhencebetteratpassingeachothersmoothly?

Moreworkisneededtoanswerthesequestions.

TheresultspresentedbyDewaretal.alsosuggest,atleastinthecontextof

theplasmidtemplate,thattorsionalstressbuildingupaheadoftheforksdoes

notslowdownforkconvergence(Dewaretal.,2015)(seealsobelowforroleof

topoisomerases)(Figure2B).Theremovalofproteins(nucleosomes)aheadof

theforkcouldnotbedirectlyaddressedinthissetupduetotheartificial

“clearingup”ofchromatinaheadoftheforkduetoremovalofthelacarray.

Interestingly,thereconstitutionofeukaryoticDNAreplicationinvitrowith

purifiedbuddingyeastproteinsrevealedinfactthatnucleosomalpackaging

doesappeartoinhibitreplicationtermination.Astheelongationstageofthe

9

reactionwasefficient,butterminationalonewasblocked,itsuggeststhatthe

terminationstagemaybeespeciallysensitivetothepresenceofchromatin

structure(Devbhandarietal.,2017)(Figure2A).

6.ThecompletionofDNAsynthesis

DataprovidedbyDewaratal,suggestthatleadingstrandDNAisreplicated

uptoafewbasesawayfromtheendofthelastOkazakifragmentofthe

encounteredlaggingstrand(Figure2DandE).Thereisnoevidencefor

persistentgapsbetweenthesestrandsupontermination(Dewaretal.,2015).

Thesedata,however,donotexplainwhichpolymerasecarriesonsynthesisof

lastfragmentsofDNAandmaturationofthelastOkazakifragment.TheRNA-

DNAprimerofeachOkazakifragmentonthelaggingstrandisremovedby

concertedactionofDNAPoldandFen1endonuclease(reviewedin

(BalakrishnanandBambara,2013)).DNAPoldcansupportstrand-displacement

re-synthesisoftheDNApreviouslysynthesisedbyPolaandindoingsocan

progressuntilitencountersthenucleosomeoranotherDNAbindingprotein,

bothofwhichareefficientlyrepositionedbehindthereplicationfork(Smithand

Whitehouse,2012).Interestingly,fragmentsofDNAsynthesisedbyPolacanbe

detectedinmaturegenomemostlyatthejunctionsofOkazakifragments,usually

atthenucleosomemidpoint(dyadposition).Intotalabout1.5%ofmature

genomewasshowntobesynthesisedbyPola(Reijnsetal.,2015).

IsthelastOkazakifragmentmaturedbyDNAPole?Theholoenzymeof

DNAPoleisunabletocarryonextendedstranddisplacementsynthesisinin

vitroreconstitutionexperiments,unlessits3’-5’exonucleaseactivityisremoved,

anditcannotmatureOkazakifragmentsonlaggingstrand(Devbhandarietal.,

2017;Ganaietal.,2016).However,DNAPoleinthecontextofthereplisome

tightlyassociateswiththeCMGcomplexthroughtheDpb2subunitofPoleand

GINSandformsafunctionalunit(Langstonetal.,2014;Muramatsuetal.,2010;

Senguptaetal.,2013).Arecentnegative-stainelectronmicroscopy

reconstructionofaCMG-Polecomplexvisualisedthecloseassociationofthis

complex(PellegriniandCosta,2016;Sunetal.,2015)andwefoundthatthe

10

post-replicationreplisomeinbothC.elegansandX.laevisinteractswithPoleand

notPold(Sonnevilleetal.,2017).ThisinteractionofPolewiththereplisome

likelyactsasadditionalprocessivityfactorforPole,inadditiontoactionofPCNA

(Kangetal.,2012;Langstonetal.,2014;Yeelesetal.,2017).Itwouldbe

interestingtoinvestigatePolestrand-displacementactivityinthecontextofthe

replisome.InsupportofthePoleroleattermination,analysisofthegenome-

widelocationofribonucleotidesincorporatedintoDNAbymutantsofPoldand

Poleespeciallypronetosuchmis-incorporationsdiscoveredasubstantialbias

towardPoldproximaltooriginswhichdeclinedtowardthecentreofthe

repliconswherePolesynthesiswasmoreevident(Daigakuetal.,2015).This

wouldsuggestthatPolecarriesoutthereplicationatsitesoftermination.

CanPolematurethelastOkazakifragment?Canitsustainstrand

displacementsynthesiswhensupportedbybothPCNAandtheCMG?Itremains

tobeunravelled.Importantly,DNAPoleonitsowndoesnotinteractwithFen1

(Gargetal.,2004),thereforeanotherprocessingmechanismwouldberequired

tocompletematurationofthelastOkazakifragment,unlessFen1isbroughtin

byadifferentcomponentoftheterminatingreplisome.Alternatively,Polecan

slidealongthelastOkazakifragmenttogetherwiththepost-termination

replisome,makingroomforPoldtodisplaceandmaturethelastRNA/DNA

primer.MuchistobediscoveredabouttheabilityoftheterminatedCMGto

moveawayfromtheterminationsiteespeciallyinthecontextofre-established

nucleosomes.However,Poldhasbeenshownpreviouslytoplayaroleinleading

strandsynthesisinvivo(Daigakuetal.,2015;Johnsonetal.,2015;Wagaetal.,

2001).Moreover,recentdataobtainedfromthebuddingyeastinvitro

reconstitutionsystemofreplicationusingpurifiedproteinsrevealedthat

polymeraseswitchingmaybemorecommonthanexpected.Poldcanplayan

importantroleinestablishingleading-strandsynthesis(Yeelesetal.,2017)and

PoldassembledattheleadingstrandwasshowntobedisplacedifPolewas

addedafterDNAsynthesishasinitiated(Georgescuetal.,2014).Moreresearch

isrequiredtoshowwhichofthepolymerasesfinishesthereplicationjob.

7.RoleoftopoisomerasesduringDNAreplicationtermination

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Theabilityoftopoisomerasestoactaheadofthereplicationforksbecomes

verylimitedastworeplicationforksconverge(SundinandVarshavsky,1980).

Inthiscircumstance,forkrotationandprecatenationbecometheprimary

pathwayofDNArelaxingaheadofthefork.Catenated,doublestrandedDNA

(intertwinedsisterchromatids)canonlyberesolvedbytypeIItopoisomerases

(TopoII,S.cerevisiaeTop2)(Figure2B).ExperimentswithTopoIIinhibitorsin

XenopuseggextractshowedthatTopoIIcanbetrappedbehind,butnotinfront

oftheforks,andresolvesreplicationintermediatesinanon-redundantmanner

withTopoI(Hyrien,2009;Lucasetal.,2001).Interestingly,Top2depletionin

yeastdoesnotstopcellsfromcompletingDNAreplication,norpassingthrough

mitosis,althoughtheydodramaticallymis-segregateandbreaktheir

chromosomesduetosisterchromatidcatenation.Ontheotherhand,inhibition

ofTop2enzymaticactivityinawaythatTop2isstillabletobindDNAbutunable

tocatalysestrandbreakage,causesincompleteDNAreplicationandinduces

G2/Mcellcyclearrest(BaxterandDiffley,2008).Similarly,inhibitionofTopoII

activityinhighereukaryoteswiththesmallmoleculeinhibitorICRF-193was

showntoblockterminationofDNAreplicationinXenopuseggextractand

induceG2arrestinhumancellswithoutthehighlevelofDNAstrandbreaks

associatedwithTopoIIpoisons(Cuvieretal.,2008;Downesetal.,1994;

Skoufiasetal.,2004).ICRF-193trapsTopoIIontheDNAintheformofanon-

covalentintermediatenamedtheclosedclamp(Rocaetal.,1994).Itisunclear

thereforewhetherreplicationterminationdefectsobserveduponadditionof

ICRF-193toXenopuseggextractsisduetoinhibitionofTopoIIactivityorsome

othereffectoftheclosedclamps,suchaschangestonucleosomespacingand

chromatinstructure(Gaggiolietal.,2013;GermeandHyrien,2005).

InagreementwiththeroleofTopoIIinreplicationforktermination,post-

terminationreplisomesfromC.elegansandX.laeviscontainTopoII,unlikethe

buddingyeastReplisomeProgressionComplex,whichrepresentsactivehelicase

andcontainsTop1(Gambusetal.,2006;Sonnevilleetal.,2017).Moreover,

Dewaret.al.reportedthatsite-specificterminationplasmids(describedabove)

requireTopoIIfordecatenationofdaughterplasmids,butTopoIIactivityisnot

12

neededforforkconvergenceandDNAligation(Dewaretal.,2015)(Figure2B

andF).

8.Replisomedisassembly

ThedatapresentedbyDewaratal.suggestthatthedissolutionofthe

replisomeintheplasmid-basedsystemisthelaststageofreplicationfork

termination,executedafterligationofleadingandlaggingstrands(Dewaretal.,

2015)(Figure2E).WorkinbuddingyeastandXenopuslaeviseggextract

discoveredthefirstelementsofthisdissolutionmechanism,whichwasfoundto

beahighlyevolutionaryconservedprocess(Maricetal.,2014;Morenoetal.,

2014)(Figure3AandB).InbothmodelorganismstheMcm7subunitoftheCMG

complexbecomespolyubiquitylatedwhenforksterminate.Theubiquitinchains

attachedtoMcm7arelinkedthroughlysine48(K48)butubiquitylatedMcm7is

notdegradeddirectlyonchromatinasinhibitionofproteasomalactivitydoes

notinhibitCMGdisassembly.Instead,aproteinremodellerCdc48(p97,VCP,

segregase)recognisestheubiquitylatedCMGandthroughitsATPaseactivity

removestheCMGcomplexesfromchromatin(Maricetal.,2014;Morenoetal.,

2014).ItisunclearatpresentwhethertheubiquitylatedMcm7isdegraded

uponremovalfromchromatinorde-ubiquitylated.ArecentreportbyFullbright

etal.suggeststhatduringunperturbedDNAreplicationinXenopuseggextract

ubiquitylatedMcm7islikelytobede-ubiquitylated(Fullbrightetal.,2016).

Interestingly,ubiquitylationofhumanMcm7(bothendogenousand

exogenouslyexpressedincells)wasreportedinthepast,butthefateofthe

ubiquitylatedformofMcm7andthefunctionoftheubiquitylationwasnotclear

(Buchsbaumetal.,2007;KuhneandBanks,1998).

8.1SCFDia2ubiquitinligaseinbuddingyeast.

Inbuddingyeasttheubiquitinligase,whichubiquitylatesMcm7isSCFDia2

(Maricetal.,2014).SCFDia2isamultisubunitligasebuiltaroundaCdc53cullin

scaffold(homologueofCullin1inhighereukaryotes)(Figure3A).Dia2isthe

substratespecificreceptor,F-boxprotein,whichbindsthroughthesubstrate

adaptor(Skp1)totheN-terminalpartofCdc53.TheC-terminusofCdc53,onthe

13

otherhand,bindsRINGdomainfactorHrt1,connectingtheligasetothe

ubiquitinconjugatingenzyme(E2)Cdc34(SCF=Skp1+Cullin1+F-box)(Figure

4B).SCFDia2wasshowntobeessentialforMcm7ubiquitylation,specificallyin

thecontextofCMGduringS-phase–bothinvitroandinvivo.Moreovercells

lackingDia2(dia2D)retainCMGcomplexesonchromatinafterS-phaseuntilthe

nextG1stageofthecellcycle(Maricetal.,2014).Notsurprisinglybuddingyeast

cellslackingDia2,althoughviable,aredefectiveinS-phaseprogressionand

presenthighratesofendogenousDNAdamageandgenomeinstability.Theyare

alsounabletogrowatlowtemperaturesandaresensitivetoDNA-damaging

agentsthataffectreplicationforkprogression(Blakeetal.,2006;Koeppetal.,

2006).

Dia2containsaprotein-proteininteractionN-terminaltetratricopeptide

repeat(TPR)domain,nuclearlocalisationsignal(NLS),anF-boxthatconnectsit

totherestoftheSCFligaseandaC-terminalsubstraterecognitiondomain

comprisingofleucine-richrepeats(LRR).TheTPRdomainofDia2wasshownto

interactwithMrc1andTof1componentsofthereplisomeprogressioncomplex

(RPC)builtaroundtheCMGhelicase(Gambusetal.,2006;Morohashietal.,

2009).Asaresult,Dia2wasdetectedinteractingwithRPCinS-phaseandthis

interactionwaspreservedwhencellsweretreatedwithhydroxyurea(HU)to

stallprogressingreplicationforks(Morohashietal.,2009).Interestinglycells

lackingtheTPRdomainwithinDia2(dia2-DTPR)donotpresentthesevere

phenotypeofdia2Dcells–withtheexceptionofsyntheticlethalitywithrrm3D

(ahelicasesupportingpassageofforkspastprotein-DNAbarriers).Cellslacking

theTPRdomaininDia2were,however,shownconsequentlytohaveapartial

defectinMcm7ubiquitylationandCMGdisassembly(Maculinsetal.,2015).It

seemsthatattachingSCFDia2tothereplisomeviatheTPRdomainincreasesthe

efficiencyofCMGubiquitylation.ItmaynotbeessentialfornormalCMG

disassemblyastheLRRdomaincanstillrecogniseitssubstrateevenwithoutthe

tethering,buttheremaybesituationswhenthisstabilisedinteractionwiththe

replisomeismorevital–forexamplewhenforksstruggletopassDNA-protein

barriersintheabsenceofRrm3.

8.2.CRL2Lrr1ubiquitinligaseinhighereukaryotes

14

Recentresearchfromourandtwoothergroupsdiscoveredthatinhigher

eukaryotestheubiquitinligaseubiquitylatingMcm7atterminationof

replicationforksisnotanSCFbutaCullin2-basedubiquitinligasewitha

LeucineRichRepeat1protein(Lrr1)asasubstratereceptor(Cullin-RingLigase

2withLrr1=CRL2Lrr1)(Dewaretal.,2017;Sonnevilleetal.,2017)(Figure4C).

BothinXenopuseggextractandinC.elegansembryos,inhibitionor

downregulationofCullin1ligaseactivitydidnotinfluenceMcm7ubiquitylation

norhelicasedisassemblyduringS-phase((Sonnevilleetal.,2017)andour

unpublisheddata).OntheotherhandsiRNAdownregulationofCUL-2/LRR-1

complexinC.elegansembryosandimmunodepletionofCRL2Lrr1ineggextract

blockedbothphenotypes(Dewaretal.,2017;Sonnevilleetal.,2017)(Figure

3B).CRL2Lrr1wasalsoshowntobetheonlycullintypeubiquitinligasethat

interactswithpost-terminationreplisomesinXenopuseggextractandC.elegans

embryosandaccumulatesatthesitesofterminationinplasmid-based

terminationsystemdescribedabove(Dewaretal.,2017;Sonnevilleetal.,2017).

Importantly,bothstudiesfoundthatCRL2Lrr1interactsspecificallywith

terminatingCMGandnotwithactivelyunwindinghelicasenordoubleMcm2-7

hexamersofdormantorigins.TheregulatedbindingofCRL2Lrr1topost-

terminationreplisomerepresentsthereforethefirstknownstepofreplisome

disassembly.Finally,theubiquitinligaseactivityofCRL2Lrr1isnecessaryforthe

Mcm7ubiquitylationandhelicasedisassembly,asamutantofCul2-Rbx1

complex,whichcannotbeactivatedbyneddylation,isunabletorescuethe

CRL2Lrr1immunodepletedeggextractunlikeawildtypefullyfunctioning

complex(Sonnevilleetal.,2017).

WhatisCRL2Lrr1?PreviousworkhasshownthatC.elegansLRR-1isan

essentialgene(Pianoetal.,2002).LRR-1isrequiredforembryonicdevelopment

butmaternalrescueallowsanalysisoflrr-1lossoffunctioninadulttissues.lrr-1

mutantsaresterileowingtoseveredefectsingermcellproliferation(Merletet

al.,2010;Starostinaetal.,2010).Inactivationoflrr-1inducesDNAdamage,

whichmayariseduetoDNAre-replicationproblems(ssDNA/RPA-1foci

accumulateinlrr-1germcells,whichalsocontaingreaterthan4NDNAcontent).

ThisinturnleadstohyperactivationofATL-1/CHK-1pathway(ATR/Chk1

pathwayinvertebrates),whichdelaysmitoticentryandresultsinembryonic

15

lethality.InactivationofATL-1/CHK-1checkpointcomponentssupressesthe

proliferationdefectandfullyrestoreslrr-1mutantfertility(Burgeretal.,2013;

Merletetal.,2010).Howthere-replication/DNAdamageisinducedinlrr-1

wormsisnotasyetdetermined.Interestingly,anRNAi-basedsuppressorscreen

oflrr-1andcul-2mutantsidentifiedtwogenesencodingcomponentsoftheGINS

complex,aswellasCDC-7andMUS-101,whichareneededforCMGactivation

(Ossareh-Nazarietal.,2016).ThesedatasuggestthatreducingCMGlevelson

chromatincansupresstheDNAdamagecreatedinlrr-1mutantsandsupress

theirlethality.ThisisinagreementwithLRR-1’sroleinMcm7ubiquitylationas

lowerlevelsofCMGonchromatinwouldcompensateforadefectinCMG

unloading.

Ontheotherhand,anotherstudyfoundthatC.eleganslrr-1mutantsgerm

cellsarrestwith2CDNAcontent,whichmaybeduetoaccumulationofCDK

inhibitorCKI-1asdeletionofonecopyofCKI-1orcki-1RNAitreatmentcan

rescuelrr-1mutantgermcellsnumbers.InsupportoftheCUL-2/LRR-1rolein

targetingCKI-1fordegradation,studyinhumancellsfoundthatoverexpressed

CKI-1wasdegradedfasterwhenLRR-1wasalsooverexpressed(Starostinaet

al.,2010).Interestingly,LRR1orCUL2knockdowninHeLacellsdidnotinducea

strongcellcyclearrestandLRR1wasshowntobeimportanttoregulatelevels

ofcytoplasmicp21(humanCKI)tocontrolactincytoskeletonremodelling

(Starostinaetal.,2010).Furtherstudiesarerequiredtoanalyseindepththerole

ofLRR1inhumancellsandtheinterplaybetweendifferentsubstratesofthis

ubiquitinligase.

Severalquestionsremain–whatisthesignalforpolyubiquitylationof

Mcm7andremovalofhelicase?HowareCMGsprotectedfromubiquitylation

duringelongationandefficientlyubiquitylatedattermination(Figure2)?Dewar

etalhypothesisethatitmaybeconformationalchangeswithinCMGupon

transitionfromencirclingsingle-strandedDNAtodouble-strandedDNAoflast

Okazakifragmentthatprovidethispost-terminationspecificity(Dewaretal.,

2015).Insupportofthishypothesis,itwasshownthatCMGisindeedableto

slideondouble-strandedDNA(Kangetal.,2012).

16

Weshouldalsokeepinmindthatmanysubstratespecificreceptorsof

CRLsrecognisetheirsubstratesonlywhentheyarepost-translationally

modifiede.g.F-boxreceptorsofSCFoftenrecognisephosphorylatedproteins,

VHLinteractingwithCRL2recognisesHif1auponitshydroxylation.Itis

possiblethereforethatterminatingCMGisfirstmodifiedinayetundiscovered

mannerbeforebeingubiquitylated.BuddingyeastMcm2-7complexhasbeen

recentlyshowntobeSUMOylateduponloadingatoriginsinG1stageofcell

cyclebeforeMcm2-7phosphorylation.ThelevelofMcm2-6SUMOylation

decreasesduringS-phaseasMCMbecomesphosphorylatedandactivated,with

exceptionofMcm7,whichSUMOylationwasretainedduringS-phase(Weiand

Zhao,2016).Additionally,deubiquitylatingenzymeUsp7wasdescribedrecently

asaSUMO-specificDUB,removingubiquitinfromSUMOylatedproteinsand

maintaininghighSUMO/lowubiquitinratioatreplicationforks(Leconaetal.,

2016;Lopez-Contrerasetal.,2013).Atheorywasthereforeproposedthat

SUMO-drivenubiquitylationcouldactasasignalfortheterminationofDNA

replication(LeconaandFernandez-Capetillo,2016).Usp7wasalsopreviously

showntointeractwithMCMbindingproteinMCM-BPandtocooperatewithitto

unloadtheMcm2-7complexesfromchromatinattheendofS-phase

(Jagannathanetal.,2014;Nishiyamaetal.,2011).IsUsp7DUBactivityfor

SUMOylatedproteinslinkedwithitsMCM-BPinteraction?IsMcm7inhigher

eukaryotesmodifiedbySUMO?IsSUMOylationofMcm7regulatingits

ubiquitylationatterminationevents?Moreworkisneededtounderstandfully

thiscomplexprocess.

Anotherpossibilityinneedofinvestigationisinvolvementofpriming

ubiquitinligase.IndeedARIH1,anAriadnefamilyRing-Between-Ring(RBR)

ubiquitinligase,wasshownrecentlytointeractwithanumberofCRLsincluding

CRL2sandprimetheirsubstrates(Scottetal.,2016).Itisprobabletherefore,

thatsuchaprimingligaserecognisestheterminatinghelicaseandCRL2Lrr1only

actsonprimedsubstrate.

8.3.Theroleofp97segregaseinreplisomedisassembly

p97,alsoknownasVCPinmetazoans,CDC-48inC.elegans,Cdc48inyeast

andTer94ininsects,isaubiquitin-dependentsegregasethatplaysacentralrole

17

intheregulationofproteinhomeostasis.Onceboundtoubiquitylated

substrates,thisconservedhexamericAAA+ATPaseutilisestheenergyreleased

fromATPhydrolysistoundergoaconformationalchangeacrossitshexamer

structurecalledinterprotomermotiontransmissionmechanism(Huangetal.,

2012;Lietal.,2012).Thismovementallowsp97toremovesubstratesfrom

differentcellularlocationsandcomplexes,likelybysubstratetranslocation

throughp97’snarrowcentralpore(Tonddast-NavaeiandStan,2013).The

separatedorunfoldedsubstratescanthenbedirectedtotheproteasomeand

degradedorde-ubiquitylatedandrecycledwiththehelpofDUBsassociating

withp97.p97carriesonthissegregase/unfoldaseactivityonamyriadof

substratesparticipatinginalargevarietyofcellularprocesses.Notsurprisingly,

knockdownofbothp97allelescausesearlyembryoniclethalityinmiceand

siRNA-depletionofp97incellscausesapoptosis(Mulleretal.,2007;Wojciket

al.,2004).

Theinteractionofp97withitsmanydifferentsubstratesismediatedbya

groupofabout30adaptorproteinsthatspecificallyrecruitubiquitylated

proteins(Meyeretal.,2012;Yeungetal.,2008).Thecofactorsusuallybindto

theN-terminaldomainofp97usingp97interactingmotifs.Thebest

characterisedmajorp97cofactorsincludeUfd1/Npl4heterodimerandp47,

whichbindtothep97inmutuallyexclusivemanner(Brudereretal.,2004).

Further,minorcofactorssuchasFAF1orUBXD7canthenassociatetothep97

complexwithamajorcofactor(Hanzelmannetal.,2011).Someofthecofactors,

suchasUBXD7,canalsointeractwithvariousubiquitinligasesandstreamline

theprocessofubiquitin-dependentsubstrateremoval/degradation(reviewedin

(Meyeretal.,2012)).

Theroleofp97duringDNAreplicationwasfirstsuggestedinC.elegans

embryos.RNAi-mediateddepletionoftheCDC-48complexleadtoadefectincell

division:mitoticentrywasdelayedasaresultoftheactivationoftheDNA

damagecheckpoint.Theseverechromatindefectsobservedinembryosaswell

asmitoticcellsofthegonadsincludedmitoticbridgesandaccumulatedfociof

RAD-51DNArepairprotein.Moreover,embryoslackingCDC-48,UFD-1orNPL-

4arestronglyreducedinDNAcontent(Deichseletal.,2009;Mouyssetetal.,

2008).ItwassubsequentlyshownthatembryoslackingCDC-48orUFD1/NPL-4

18

cofactorsaccumulateoriginlicensingfactorCDT-1onmitoticchromatinand

presentpersistentchromatinassociationofCDC-45/GINSafterS-phaseis

completed(Franzetal.,2011).Thisprocessinvolvesanotherp97cofactor

UBXN-3/FAF1(Franzetal.,2016).Interestingly,inhibitionofCDT-1degradation

anditsaccumulationonchromatininembryoslackingCDC-48orUFD1/NPL-4

doesnotleadtore-replicationphenotypeintheseembryosbutratherastrong

reductionintheirDNAcontent.

Inthecaseofreplisomedisassembly,thesegregasefunctionwasshownto

beessentialtodisassembleubiquitylatedpost-terminationCMGinbudding

yeast,C.elegansembryosandXenopuseggextract(Maricetal.,2014;Morenoet

al.,2014;Sonnevilleetal.,2017).TheATPaseactivityofp97isessentialforthis

disassemblyfunctionasthereplisomecanbeblockedonchromatinwhentwo

ATPasedomainsofp97(D1andD2)aremutatedortheactivityofp97is

blockedwithasmallmoleculeinhibitorNMS973(Dewaretal.,2017;Morenoet

al.,2014;Sonnevilleetal.,2017).Thisreplisomedisassemblydefectphenotype

isnotdriventhroughCdt1de-regulation,norrepresentsnovelbindingof

GINS/Cdc45tomitoticchromosomes(Morenoetal.,2014;Sonnevilleetal.,

2017).Inwormembryos,RNAidirectedinactivationofufd-1andnpl-4leadstoa

defectinreplisomeunloadingandtheUfd1/Npl4heterodimerisfoundto

interactwiththepost-terminationreplisomeinXenopuseggextracts(Dewaret

al.,2017;Sonnevilleetal.,2017).Moreover,plasmidswithaccumulated

terminatingforkscontainenrichedUbxn7andDvc1/SPRTNboundtothem

(Dewaretal.,2017).Futureworkwillshowwhethertheseadditionalco-factors

playaroleinreplisomedisassembly.

8.4.Back-uppathwayforreplisomedisassembly

Importantly,workinC.elegansembryosrevealedthatiftheremovalof

CMGcomplexesisnotaccomplishedduringS-phaseduetodefectiveCRL2Lrr1

thentheycanberemovedfromchromatinatthebeginningofmitosis,inlate

prophase(Sonnevilleetal.,2017)(Figure3C).Thisback-upmitoticpathwayof

replisomedisassemblyalsorequiresp97/Ufd1/Npl4(wormCDC-48/UFD-

1/NPL-4)segregase,buttoaccomplishitp97requiresyetanothercofactor:Fas-

associatedfactor1FAF1(wormUBXN-3)(Sonnevilleetal.,2017).FAF1isan

19

evolutionarilyconservedproapoptoticfactorthatcontainsmultipleprotein-

interactiondomains:ubiquitin-associatedUBA,ubiquitin-likeUBL1andUBL2,

Fas-interactingdomainFID,deatheffectordomain-interactingdomainDEDID,

Ubiquitin-associatedUAS,ubiquitinregulatoryXUBX(Leeetal.,2013;Menges

etal.,2009).FAF1isanessentialgene(Adhametal.,2008),anestablished

modulatorofapoptosis,regulatesNFkBandisinvolvedinubiquitin-mediated

proteinturnover(reviewedin(Mengesetal.,2009)).FAF1wasalsoshownto

bindtop97-Ufd1-Npl4complexviatheUBXdomainandpolyubiquitylated

proteinsviatheUBAdomaintopromoteendoplasmicreticulumassociated

degradationERAD(Leeetal.,2013).Finally,recentworkfromtheThorsten

HoppelabshowedthatFAF-1/UBXN-3isrequiredforcellcycleprogressionin

C.elegansembryoduetotheproblemwithCDT-1degradationandits

inappropriatemaintenanceonchromatinduringmitosis,togetherwithCDC-45

andGINS(Franzetal.,2016).Moreover,Franzetal.hasshownthat

downregulationofFAF1bysiRNAinhumancellscausesapronounced

replicationstressphenotype:defectiveforkprogression,forkstalling,dormant

originfiringandactivationofbothS-phasecheckpoint(ATR/Chk1)andDNA

damagecheckpoint(ATM/Chk2)(Franzetal.,2016).Itremainstobe

investigatedwhetherthisobservedreplicationstressistheresultofCdt1

inducedre-replication,adefectinunloadingofthepost-terminationreplisomes

oroneofthemanyotherFAF1functions.

Intriguingly,theback-upmitoticpathwayofreplisomedisassemblyin

C.elegansembryosismodulatedbytheactivityoftheSUMOproteaseULP-4:co-

depletionofULP-4withLRR-1delayedthereleaseofCMGcomponentsfrom

chromatin(Sonnevilleetal.,2017).ULP-4isamajormitoticSUMOproteasein

wormsandispresentatmitoticchromosomesandatthespindlemidzone

(Pelischetal.,2014).TheULP-4analogousproteasesinhumancellsareSENP6-

7.ItremainstobeunravelledwhetherSUMOplaysaregulatoryroleintheback-

upprocessorwhetherULP-4functionsinanotherwaye.g.bybridgingsome

importantinteractionsandallowingp97complexrecruitment.Itwouldbevery

interestingtoinvestigatetheexistenceofsuchapotentialback-uppathwayin

humansomaticcells.

20

9.Theimportanceoffaultlesstermination

Doesderegulationofterminationcontributetogenomicinstabilityand

humandisease?Cancerchromosomalinstability(CIN)isobservedinmostsolid

tumoursandisassociatedwithpoorprognosisanddrugresistance

(McGranahanetal.,2012).CINleadstoincreasedrateofchangesin

chromosomalnumbersandstructure,andgeneratesintra-tumour

heterogeneity.Recentdataimplicateacentralroleforreplicationstressinthe

generationofCIN(Burrelletal.,2013).Canfaultyterminationprovideasource

ofreplicationstress,whichthencontributestothegenerationofgenomic

instabilityandCIN?Whatarethewaysinwhichproblemsduringreplication

forkterminationcouldleadtogenomicinstability?Atpresentwehaverestricted

experimentaldataonconsequencesofproblemswithreplicationfork

terminationbutwecanspeculatebasedonwhatweknow.

Weknowthatfailuretodecatenatenewlyreplicatedsisterchromatids

uponterminationofreplicationforksdoesnottendtobedetectedbyG2/M

checkpointbutleadstodramaticmissegregationofchromosomesduring

mitosis(BaxterandDiffley,2008).Whataboutotherstagesoftermination

process?

Whatwouldhappenifforkscannotconvergeproperly?Whatiftheir

passingeachotherattheterminationstageisblocked?Wecanimaginethat

problemsduringconvergenceofreplicationforkscouldleadtosimilartorsional

stressesasthesecreatedbylackofTopoisomeraseIduringelongation.

InhibitionofTopoIactivityinhumancells,mouseembryonicfibroblastsand

Xenopuslaeviseggextractfrequentlyinducesreplicationforkreversal(reviewed

in(NeelsenandLopes,2015)).Forkreversalcanhavephysiologicalrolesduring

replicationbutcanalsohavepathologicalconsequences,contributetogenome

instabilityinneurogenerativesyndromesandcancer.Asmallbutreproducible

numberofreversedforkswasdetectedalsoinvariousunchallengedhumancell

lines,whilstderegulationofpoly(ADP-ribose)metabolism,whichregulatesfork

reversalandrestart,induceshighlevelofreversedforksevenintheabsenceof

genotoxicreplicationstress(reviewedin(NeelsenandLopes,2015)).Fork

reversalisalsoveryfrequentinmouseembryonicstemcells(Ahujaetal.,2016).

Wheredothesereversedforkscomefrom?Couldproblemswithterminationof

21

replicationforksbeoneofthesourcesofsuchreversedforks?Interestingly,

transientover-replication,forkreversalandend-processingbyexonucleases

wererecentlyassociatedwithcompletionofreplicationterminationinE.coli

(Wendeletal.,2014).Moreresearchandvisualisationofconvergingforkseither

unchallengedoruponterminationperturbationsisneededtoelucidatethe

possibilityofforkreversalatsitesoftroubledreplicationforktermination.

CanfailuretocompleteDNAsynthesisatterminationsitescreategenome

instabilities?Ithasbeenshownrecently,thatnotalloftheDNAisalways

replicatedinhumancellsduringS-phase–unreplicatedsegmentsresultingfrom

doubleforkstallinginlargerepliconsarefrequentlypresentinG2.Theycanbe

partiallyresolvedduringmitosis,createultrafinebridgesduringsegregationin

mitosisandaresubsequentlyrecognisedintheG1stageofthecellcyclebyDNA

repairproteinp53-bindingprotein1(53BP1)toberesolvedinthisnewcell

cycle(Morenoetal.,2016).FailuretocompleteofDNAsynthesisattermination

siteswouldlikelyleadtoasimilarscenario.

Whataboutinhibitingdisassemblyofthereplisome?Thisisthepartofthe

terminationprocessthatweunderstandbestatpresent.Ifdisassemblyofthe

replisomeconstitutesthelaststepofreplicationtermination,thenthefailureto

removeitshouldnotleaveunligatedDNAnorunusualDNAstructures(Dewaret

al.,2015).ItwouldleavehoweveraDNAhelicaseonaDNAsubstrate.Testedon

syntheticinvitrosubstratesCMGcantranslocateondoublestrandedDNAand

thenstartunwindingDNAifaforkstructureispresent(Kangetal.,2012).One

canimaginethereforethatthesecond-to-lastOkazakifragment,whichmaybein

amid-maturationstagewithaflapcreatedbyPold,couldbesuchasubstratefor

theapproachingpost-terminationCMGtostartde-novounwinding.Inbacteria,

recentdatasuggestthatinterminationzones3’ssDNAflapsarecreatedthat,if

notremovedbyRecGnuclease(inRecGmutants),canprovidesubstratesforde

novoreplication,leadingtore-replicationandcreatingpathologicalDNA

structures.Tusterminationsequenceslimittheextentofsuchre-replication

initiatedinterminationzones(Rudolphetal.,2013).Whatabouteukaryotic

cells?TheydonothaveTusterminatingsequences.Canfaultyterminationof

replicationforksinitiatere-replication?

22

Moreover,CMGcomplexesleftbehindonchromatinwoulddisturbproper

chromatinre-establishmentandposeaproblemtoprocessesforwhichDNAisa

substrate,suchastranscriptionandnextreplication.Asmentionedabove,CMGs

cantranslocateondoublestrandedDNA(Kangetal.,2012),bymovingalong

DNAtheycoulddisplaceotherproteinsboundtoDNA.Atpresentwedonot

knowwhetherCMGslidingondsDNAcandisplacenucleosomesoriftheywillbe

trappedbythem.

Afinalpotentialproblemarisingfromlackofefficientdisassemblyofthe

CMGcomplexesattheterminationofreplicationforksisentrapmentofCdc45

andGINSwithinthesepost-terminationcomplexes.Cdc45wasshowntobea

ratelimitingfactorforDNAreplicationinmammaliancells.Itwasproposedthat

regulatedexpressionlevelsofCdc45enforcesreutilisationofexistingCdc45

duringS-phase,whichinturncanlimitandstaggeroriginactivationthroughout

theS-phase(Kohleretal.,2016;Wongetal.,2011).AlackofCdc45availablefor

recyclingcanthereforepotentiallyslowS-phaseprogressionandinhibitDNA

synthesis.PrimaryuntransformedhumancellswithreducedlevelsofGINS

componentspresentallthephenotypesofreplicationstressandaccumulationof

DNAdamage(Barkleyetal.,2009).Futurestudiesofreplisomedisassemblyin

humansomaticcellsisessentialtoshedlightatthispossibilityassofarthis

processwasinvestigatedonlyinembryonicsystems(Xenopuslaeviseggextract

andC.elegansembryos)whichhavehigherlevelsofCdc45andGINS.

Isthereexperimentalevidencethatfaultydisassemblyofthereplisome

canleadtogenomeinstability?S.cerevisiaecellslackingDia2,whichareunable

toremovepost-terminationCMGfromchromatin,areviablebutpresentvery

highlevelsofgenomicinstabilities(describedabove).LRR-1–theCRL2Mcm7

specificreceptorinhighereukaryotesisanessentialgeneinC.elegans,most

likelyduetoalsootherthanMcm7substrates,asCMGbecomesunloadedbya

back-upsysteminlrr-1embryos.However,partialdownregulationofLRR-1

togetherwithdownregulationoftheback-uppathwayfactors:FAF-1/UBXN-3or

ULP4resultsinsyntheticlethality,suggestingthatinhibitionofCMGremovalby

partialblockingofbothpathwaysresultsinnon-viableworms(Sonnevilleetal.,

2017).FAF1itselfisafactoroftendownregulatedormutatedinmultiple

cancers.Itmaybeitsproapoptoticfunctionthatdrivesthisdownregulation,but

23

inconsequencethesecancerscouldexhibithigherlevelsofgenomicinstability

duetotheirreplicationforkterminationproblems.Itiscrucialthereforethatwe

investigatetheprocessofreplisomedisassemblyinhumancellstoconfirmits

analogy.

FactorsthatdrivereplicationinitiationandtheassemblyofCMG,suchas

Cdc7kinaseandTopBP1(Cut5)initiationfactor,arecurrentlybeingexploredas

potentialanti-cancertherapytargetsintumoursthatpresentdefectsin

chromosomereplication(Chowdhuryetal.,2014;Montagnolietal.,2010).Can

CMGdisassemblyalsoserveasapotentialtargetforfuturetherapies?Couldwe

targettheS-phasepathwayofCMGdisassemblyincancerswithmutatedor

downregulatedFAF1?ForthisweneedtounderstandtheCMGdisassembly

processinmuchmoredetailandcruciallyconfirmitsconservationinhuman

cells.ItseemslikelythatubiquitylationisratelimitingforCMGdisassembly,

althoughitneedstobedemonstratedbymappingtheubiquitylationsitesand

creatinganun-modifiablemutant.Itisclear,however,thatMcm7ubiquitylation

isregulatedinaprecisefashiononmanylevels,bothspatiallyandtemporally.

Finally,manyfactorsimplicatedinDNAreplicationforkterminationand

replisomedisassembly,suchasp97segregaseandUsp7arealsotargetsofsmall

moleculeinhibitorsusedorbeingtestedforantitumourtherapies(Magnaghiet

al.,2013;Reverdyetal.,2012).AbetterunderstandingofCMGdisassembly

pathwayandreplicationforkterminationinhumancellsmighthelpusto

explainthemodeofactionoftheseinhibitorsinclinic.

Figurelegends

Figure1.Topoisomerasesatreplicationfork.TopoIrelaxesthepositive

supercoilingbuildingupaheadofthefork.Sometimesthissupercoilingcanlead

torotationoftheforkandintertwiningofthedaughterstrandsofDNAbehind

thefork(precatenates).TheseareresolvedbyTopoII.

Figure2.Modelofterminationofeukaryoticreplicationforks.Whentwo

neighbouringreplicationforksapproacheachotherfromoppositedirections,all

oftheproteinsorganisingDNAinbetweentheforks(nucleosomesandothers)

24

havetoberemoved,whileTopoIrelaxesthetorsionalstress(positive

supercoiling)(A).WhentwoterminatingforksconvergethesupercoilingofDNA

betweenthemcannotberesoledbyTopoIduetolackofspaceforittoact.

Instead,terminatingforksdependontransmissionofthistorsionalstress

behindtheforkscreatingprecatenatesresolvedbyTopoII(B).During

convergencetworeplisomeapproacheachothermovingonoppositestrandsof

DNA(leadingstrandofeachfork)(C).Thereplisomescanpasseachotherand

mostlikelyCMGslidesontothedoublestrandedDNAoflastOkazakifragment

(D).ThesynthesisofDNAneedstobecompleted,thelastOkazakifragment

maturedandDNAligated.Replisomeisthenubiquitylatedandremovedby

p97/VCP/Cdc48segregase(E).Intertwinedsisterchromatidsneedtobe

resolvedbyTopoII(F).Thefinalproduct:twoindividualsisterchromatidswith

reconsititutedchromatinstructure(G).

Figure3.Modelofreplisomedisassemblyattheterminationofreplication

forks.InbuddingyeastS.cerevisiaetheMcm7subunitoftheterminating

replisomeisubiquitylatedbySCFDia2andremovedfromchromatinbyCdc48

segregase(A).InXenopuseggextractandC.elegansembryosCRL2Lrr1

ubiquitylatesMcm7duringterminationofreplicationforksandCDC-48/p97

segragaseremovesitfromchromatinwithhelpofUfd1/Npl4cofactors(B).If

themechanismofremovalofthereplisomeduringterminationofforksinS-

phasedoesnotworkC.elegansembryoshaveaback-upmechanismremoving

replisomesinprophaseinmitosis.ThismechanismrequiresCDC-48/p97and

Npl4/Ufd1butalsoUBXN-3/FAF1cofactorandisregulatedbyULP-4/Senp6,7

(C).

Figure4.ModelofcullinligasesubiquitylatingMcm7duringtermination

ofreplicationforks.Generalmodeloforganisationofcullinfamilymembers(A).

ModelofSCFDia2ubiquitylatingMcm7inS.cerevisiae(B).ModelofCRL2Lrr1

ubiquitylatingMcm7inC.elegansembryosandXenopuseggextract(C).

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Topo I

Pol

PolCdc45

Pol

GI NS

Topo II

Figure 1. Gambus

Pol

RPARPA RPA

RPARPA

RPA

Pol

RPARPA RPA

RPARPA

RPA

PolCdc45

Pol

GI NS

Pol

RPARPARPA

RPARPA

RPA

Pol Cdc45

Pol IG

SN

PolCdc45GI NS

uu

u

Pol ε Cdc45

IG

SN

cullin

K48

Pol

p97VCP

Pol

IG

SNCdc45Pol

Mcm2-7

Topo I

Topo II

Pol

Pol

RPARPARPA

RPARPA

RPA

Pol

Pol

PolCdc45GI NS

Pol ε Cdc45

IG

SN

Pol

RPARPARPA

RPARPA

RPA

RPARPA RPA

RPARPA

RPARPA

RPA RPA

RPARPA

RPA

Pol

Cdc45GI NS

Pol

Cdc45GI NS

Pol

Pol

Cdc45GI NS

7Pol

Cdc45GI NS

7u

uu

K48

Topo II

A

B

C

D

E

F

G

Topo II

Figure 2. Gambus

uu

uCdc53

K48

Cdc48

Cdc45GI NS

7

Cdc45GI NS

7u

uu

K48

A B C

Dia2 uu

uCul2K48

p97

Cdc45GI NS

7

Cdc45GI NS

7u

uu

K48

Lrr1

Ufd1Npl4

Pol

Pol

IG

SNCdc45Pol

Mcm2-7

uCdc45G

I NS

Cdc45GI NS

u

uCdc45G

I NS

Cdc45GI NS

u

p97Ufd1

Npl4 Faf1

Pol

Pol

IG

SNCdc45Pol

Mcm2-7

Senp6/7

Pol

Pol

IG

SNCdc45Pol

Mcm2-7

uCdc45G

I NS

Cdc45GI NS

u

uCdc45G

I NS

Cdc45GI NS

uCdc45G

I NS

Cdc45GI NS

u

Figure 3. Gambus

cullinRING(E3)

substrateadaptor

N-terminus C-terminus

E2

substrate

UbUb

Ub

Ub

substratereceptor

cullin E3 ligase complexesin higher eukaryotes

Cdc53Hrt1Skp1

N-terminus C-terminus

Cdc34

Mcm7

UbUb

Ub

Ub

Dia2

S. cerevisiaeSCFDia2 complex

Cul2Rbx1

N-terminus C-terminus

E2

Mcm7

UbUb

Ub

Ub

Lrr1

metazoanCRL2 complexLrr1

A B C

EloB/C

Figure 4. Gambus