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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
11
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
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(C).
Figure4.ModelofcullinligasesubiquitylatingMcm7duringtermination
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ModelofSCFDia2ubiquitylatingMcm7inS.cerevisiae(B).ModelofCRL2Lrr1
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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