DNA replication stress triggers Rapid DNA Replication Fork Breakage by Artemis and XPF

16 Pages·2017·3.61 MB·English

RESEARCHARTICLE
DNA replication stress triggers rapid DNA
replication fork breakage by Artemis and XPF
Re´myBe´tous1☯*,The´oGoulletdeRugy1☯,AlessandraLuizaPelegrini1,2,SophieQueille1,
Jean-PierredeVillartay3,Jean-Se´bastienHoffmann1*
1 CRCT,Universite´deToulouse,Inserm,CNRS,UPS;Equipelabellise´eLigueContreleCancer,Laboratoire
d’excellenceToulouseCancer,Toulouse,France,2 DepartmentofMicrobiology,InstituteofBiomedical
Sciences,UniversityofSãoPaulo,SãoPaulo,Brazil,3 Laboratory“GenomeDynamicsintheImmune
System”,INSERMUMR1163,Universite´ParisDescartesSorbonneParisCite´,InstitutImagine,Paris,France
a1111111111 ☯Theseauthorscontributedequallytothiswork.
a1111111111 *[email protected](RB);[email protected](JSH)
a1111111111
a1111111111
a1111111111 Abstract
DNAreplicationstress(DRS)leadstotheaccumulationofstalledDNAreplicationforks
leavingafractionofgenomiclociincompletelyreplicated,asourceofchromosomalrear-
rangementsduringtheirpartitioninmitosis.MUS81isknowntolimittheoccurrenceofchro-
OPENACCESS
mosomalinstabilitybyprocessingtheseunresolvedlociduringmitosis.Here,weunveilthat
Citation:Be´tousR,GoulletdeRugyT,PelegriniAL,
theendonucleasesARTEMISandXPF-ERCC1canalsoinducestalledDNAreplication
QueilleS,deVillartayJ-P,HoffmannJ-S(2018)
DNAreplicationstresstriggersrapidDNA forkscleavagethroughnon-epistaticpathwaysallalongSandG2phasesofthecellcycle.
replicationforkbreakagebyArtemisandXPF. Wealsoshowedthatbothnucleasesarerecruitedtochromatintopromotereplicationfork
PLoSGenet14(7):e1007541.https://doi.org/
restart.Finally,wefoundthatrapidchromosomalbreakagecontrolledbyARTEMISand
10.1371/journal.pgen.1007541
XPFisimportanttopreventmitoticsegregationdefects.Collectively,theseresultsreveal
Editor:JulianE.Sale,MRCLaboratoryofMolecular
thatRapidReplicationForkBreakage(RRFB)mediatedbyARTEMISandXPFinresponse
Biology,UNITEDKINGDOM
toDRScontributestoDNAreplicationefficiencyandlimitchromosomalinstability.
Received:December29,2017
Accepted:July4,2018
Published:July30,2018
Authorsummary
Copyright:©2018Be´tousetal.Thisisanopen
accessarticledistributedunderthetermsofthe
DNAreplicationisanessentialprocessthatneedstobeabsolutelyaccuratetopreventfix-
CreativeCommonsAttributionLicense,which
ationofmutationswhichcouldimpaircellularessentialfunctionsandpromotediseases
permitsunrestricteduse,distribution,and
suchascancers.DuringS-phaseDNAreplicationforksencountermanyobstaclesthat
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. blockthereplicativeDNApolymerasesandinduceforkstalling.Accumulationofstalled
forksorexcessiveforkslowingisreferredtoasDNAreplicationstresswhichpromotea
DataAvailabilityStatement:Allrelevantdataare
DNAdamageresponseelicitedbyATRfromthestalledforkstopreservegenomestability.
withinthepaperanditsSupportingInformation
files. However,howcellsdealwithpersistentlystalledreplicationforksisnotfullyunderstood.
IthasbeenshownthattheendonucleaseMUS81-EME1cancleavethestalledforksafter
Funding:RB’ssalarywasprovidedbythe
24hoursofreplicationstress.HowevernormalS-phaselength,iscommonlyofabout8
FondationdeFrance,#201400051605(https://
www.fondationdefrance.org/fr)ALP’ssalarywas hours.Thusweaskedwhatcouldhappenifforksstallmoretransiently.Weuncovered
providedbyFundac¸ãodeAmparoàPesquisado thatstalledDNAreplicationforkscanbreakrapidlyafterinductionofreplicationstress.
EstadodeSãoPaulo–FAPESP(http://www. WeshowthatthisRapidReplicationForkBreakage(RRFB)isachievedbytwoendonucle-
fapesp.br/).WorkinJSH’slaboratoryissupported
ases,ARTEMISandXPF-ERCC1,whichworkindependentlyofeachothertoresume
byfundingfromtheInstitutNationalduCancer
(PLBIO2016)(http://www.e-cancer.fr/),the
PLOSGenetics|https://doi.org/10.1371/journal.pgen.1007541 July30,2018 1/16
ArtemisandXPFmediateforkbreakage
AgenceNationaledelaRecherche(ANRPRC
2016)(http://www.agence-nationale-recherche.fr/), DNAreplicationfromthestalledforksandtopreventmitoticsegregationdefects.Hence,
andLaLiguecontreleCancer(Equipelabellise´e weidentifiednewpathwayspreservinggenomestabilityduringreplicationstress.
2017)(https://www.ligue-cancer.net/).Thefunders
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe
manuscript.
Introduction
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. Genomestabilityisaffectednotonlybyexogenousaggressionssuchaschemicalcarcinogens
andionizingradiationbutalsobyendogenouslyinducedDNAdamagegeneratedduringthe
processofDNAreplicationwhentheDNAreplicationforksaresloweddownorstalledby
diversenaturalreplicationbarriers,aphenomenonreferredasDNAreplicationstress(DRS)
[1].Thishasbeenwelldocumentedduringcancerprogression,inwhichoncogene-drivencell
proliferationinducesahighlevelofDRS,resultinginthegenomeinstabilitythatisahallmark
ofcancercells[2].DRSgeneratesstalledreplicationforkscontaininglargeamountsofsingle-
strandedDNAcoatedwiththeproteinRPA.TheseactivatethereplicationstresskinaseATR,
whichphosphorylateshundredsofsubstratesinordertostabilizeandrestartthestalledDNA
replicationforks[3].Ifthisreplicationstressresponsefails,DNAdouble-strandbreak(DSB)s
arecreatedatthestalledreplicationforks[4],aphenomenonknownasreplicationforkbreak-
age.Currently,themechanismsmediatingreplicationforkbreakagearepoorlyunderstood
andhaveonlybeenreportedtooccurinresponsetoextensiveDRSwhereitrequiresMUS81
[5,6],thecatalyticsubunitofastructure-specificendonucleasewhichformsacomplexwith
EME1orEME2[7].
DSBsareusuallyseenasthreatstogenomeintegrityandcellviability,soitisunclearwhy
eukaryoteshaveevolvedmechanismsthatinduceDSBsduringDNAreplication.Arecent
bodyofevidence,however,suggestsananswertothisconundrum.Twostudieshavefound
thatDRScanleadtounder-replicatedregionsthatpersistintolateG2andMphaseofthecell
cycle[8,9].Iftheseregionsremainedunresolvedinmitosis,thesecanleadtochromosomes
segregationdefects,whichcanresultinuncontrolledchromosomebreaksthataretransmitted
todaughtercellsuponcelldivision,thusaffectingthegenomicstabilityofthenextcellgenera-
tion.Importantly,Mus81wasdemonstratedtopreventthesedetrimentalconsequencesof
DRS[10,11].
Initially,MUS81dependentforkbreakagehasbeenobservedafterprolongedexposureto
DRS(18–24hours)andhasbeenproposedtooccurinS-phasecells[5].However,eventhough
MUS81exhibitsbasalactivitythroughoutthecellcycle,itsmainbiologicalfunctionseemsto
berestrictedtomitosistopromotetheresolutionofunder-replicatedchromosomes,holliday
junctionsorotherrecombinationintermediates[7]andmistargetingofMUS81toreplication
factoriesduringS-phaseinduceschromosomepulverization[12].Therefore,whetherstalled
replicationforksareactuallysubjectedtoendonucleolyticcleavageduringS-phaseremains
unclear.Interestingly,activationoftheDNAdamageresponseandrecruitmentofDSBrepair
proteinstostalledreplicationforksoccurasearlyas2hoursafterinductionofDRS[13]sug-
gestingthatatleastsomestalledreplicationforksarebeingcleavedwithinthistimeframe.
Thus,weinvestigatedifDNAreplicationforkscouldactuallybreakearlierthanpreviously
reported,whenS-phaseischallengedbyreplicationinhibitors.
Inthisstudy,weprovideevidencethatReplicationforkscanbreakquicklyinS-phaseupon
DRSinductionbyanendonucleolyticmechanismindependentofMUS81.Wedemonstrate
thattwonucleasesARTEMISandXPF-ERCC1areresponsibleforthisRapid-ReplicationFork
Breakage(RRFB)whichtakesplaceduringSandG2phasesofthecellcycle.Weproposethat
thisphysiologically-controlledDSBinductionisimportanttopreventgeneticinstabilityunder
DRS.
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ArtemisandXPFmediateforkbreakage
Results
Replicativestressinducesrapidreplicationforkbreakage
Toinvestigatethekineticsofreplicationforkbreakage,weusedthesensitiveneutralcomet
assaytomonitortheoccurrenceofDSBsatvariouslevelsofreplicativestress.Wetreatedthe
humancoloncarcinomacelllineRKOwith0.5,2and8mMhydroxyurea(HU),aninhibitor
ofdNTPbiogenesis,for1–24hours.ThisDRSinducedDSBsdetectableasearlyas1hourafter
HUtreatmentatthestrongestdoseofHU(Fig1A).DuringthefirstfourhoursofDRS,the
amountofDSBsincreasedlinearlyandreachedaplateaubeforeincreasingagainfrom16–24
hours.DSBsobservedduringtheearliesttimepointsoftheassayappearedconcomitantwith
activationofDNA-PK,whichisknowntobeactivatedbyDSBs,andtothephosphorylationof
oneofitssubstrates,RPA,onSer4and8(S1AFig).
TodeterminewhethertheobservedDSBswereinducedinS-phasecells,wecarriedoutquanti-
tativeimage-basedcytometry(QIBC),ahigh-throughputmicroscopymethoddescribedinFig1B
[14].QIBCallowedustoquantifyintheindividualcellsofapopulationtheamountsofseveral
DNAdamagemarkersandtocorrelatethiswiththestageofthecellcycle.After4hoursofDRS
inducedbyHU,weobserved,aspreviouslydescribed[14],astrong,dose-dependentaccumula-
tionofγH2AX,thephosphorylatedformofthehistonevariantH2AX,whichisphosphorylated
bythekinasesATR,ATMandDNA-PKinresponsetoDSBsbutalsotostalledforks(Fig1C).To
quantifymorepreciselyDSB,weusedadirectmarkerofDSB,thephosphorylatedformof53BP1
atSer1778.Indeed,phosphorylationofthissitehasbeenshowntocorrelatewiththeamountof
DSB[15].Wefoundthat53BP1isalreadysignificantlyphosphorylatedatmilddoseofHU(Fig
1C)andaccumulatesinfociinreplicatingcellsonly30minutesafterinductionofDRS(S1Band
S1CFig).Importantly,becauseweextractedsolubleproteinsbeforecellfixation,weonlyquanti-
fiedproteinsonchromatinandthereforeDSBboundp53BP1.Asimilarrapidaccumulationof
p53BP1onchromatinofS-phasecellswasfoundinresponsetotreatmentwithaphidicolin,an
inhibitorofthereplicativeDNApolymerases(S1DFig).Thisphosphorylationof53BP1andits
recruitmenttochromatinwasstronglyinhibitedbyachemicalinhibitorofATM(KU55933),
indicatingthatitisdependentuponDSBs(Fig1D).Together,thesefindingssuggestthatatleast
somestalledDNAreplicationforksarerapidlyconvertedintoDSBsduringSphaseinresponseto
DRS,aphenomenonthatwerefertoasrapidreplicationforkbreakage(RRFB).
RapidreplicationforkbreakageoccursinaMus81-independentmanner
ItwaspreviouslyreportedthattheformationofDSBsuponprolongedexposure(18–24hours)
toreplicationinhibitorsrequiresMUS81[5].Thus,weaddressedifitsroleinRRFBcould
havebeenoverlookedforexperimentalsensitivityreasons.DespitestrongdepletionofMUS81
byRNAinterference(Fig2A),wesawnodifferenceintheamountofγH2AXorp53BP1inS-
phasecellsafterDRS,asdeterminedbyQIBC(Fig2Band2C).Toconfirmthisfinding,we
assayedthegenerationofDSBsuponHUtreatmentdirectlybyneutralcometassay.Again,
DSBsweredetectedatearlytimepointsbothincellsdepletedofMUS81andincontrolcells
(Fig2D).After24hofDRS,however,Mus81depletionhadacleareffectontheamountof
DSBs,confirmingpreviouslypublisheddata[5].Together,thesedatademonstratethatRRFB
doesnotrelyontheendonucleaseMUS81.
ARTEMISandXPF-ERCC1areinvolvedinRRFBthroughtwodistinct
pathways
ToinvestigatewhichotherendonucleasesmightbeinvolvedinRRFB,weconductedneutral
cometassayinRKOcellsdepletedofanyoneofseveralcandidateendonucleasesthatmight
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ArtemisandXPFmediateforkbreakage
Fig1.Briefreplicationstressresultsinrapidreplicationforkbreakage.(A)QuantificationbyneutralcometassayofDSBsinduced,in
responsetotreatmentwithvariousdosesofHUforupto24hours.Representativeimagesofcellstreatedfor4hoursareshown.Eachdatapoint
correspondstothemeantailmoment(%DNAintailxtaillength)anderrorbarsrepresentstandarddeviationsfrom3independentexperiments
inwhichatleast150cometswerescored.(B)Schematicrepresentationofquantitativeimage-basedcytometry(QIBC).(C)Quantificationof
nuclearγH2AX(left)orp53BP1(right)fluorescenceintensitybyQIBCin>2500S-phasecellstreatedwiththeindicateddosesofHUfor4hours.
(cid:3)(cid:3)(cid:3)p<0.0001(Mann-Whitneytest).(D)Quantificationofnuclearp53BP1fluorescenceintensitybyQIBCin>2000S-phasecellseither
untreated(NT)orpretreatedfor1hourwithanATMinhibitor(ATMi;KU5593310μM)andthentreatedwiththeindicatedconcentrationsof
HUfor4hoursinthepresenceorabsenceoftheATMinhibitor.(cid:3)(cid:3)(cid:3)p<0.0001(Mann-Whitneytest).
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ArtemisandXPFmediateforkbreakage
Fig2.Rapidreplicationforkbreakage(RRFB)isindependentofMUS81.(A)WesternblotofwholecellextractsofRKOcellsshowingdepletionof
Mus81bysiMus81comparedtoacontrol,siLUC.Loading:nonspecificband.(B)RepresentativefieldsofQIBCimagesafterMUS81depletionand
treatmentwith4mMHUfor4hours.(C)QuantificationofnuclearγH2AX(left)orp53BP1(right)fluorescenceintensitybyQIBCin>1500S-phase
cellsdepletedofMUS81(siMUS81)ordepletedwithacontrolsiRNA(siLUC)andeithertreatedornottreatedwith4mMfor4hours.ns:notsignificant
(Mann–Whitneytest).(D)QuantificationofDSBsincellsdepletedofMUS81(siMUS81;blue)ordepletedwithacontrolsiRNA(siLUC;gray)by
neutralcometassay.Barscorrespondtothemediantailmoment(%DNAintailxtaillength).Errorbarsrepresentstandarddeviationsfrom3
independentexperimentsinwhichatleast150cometswerescored.ns:notsignificant,(cid:3)(cid:3)(cid:3)p<0.001(Student’sTtest).
https://doi.org/10.1371/journal.pgen.1007541.g002
functionduringSphase(S2AFig).Whereasdepletionofmostofthemhadnoeffectonthe
amountofDSBs,depletionofeitherARTEMISorXPFsignificantlyinhibitedDSBformation
after4hourstreatmentwithHU(Fig3A).Similarly,incellsdepletedofeitherArtemisorXPF
(Fig3B),wesawlessp53BP1onchromatinofSphasecellsbyQIBCwheneitherARTEMISor
XPFwasdepletedthanwesawincontrolcells4hours(Fig3C)butalsoonly1hourafterHU
addition(S2BFig)orafteraphidicolintreatment(S2CFig).Consistentwithourfindingswith
thedepletedRKOcells,primaryfibroblastsisolatedfrompatientssufferingfromRS-SCIDand
deficientforARTEMIS(Guetelcells)[16]orfromasevereformofXerodermaPigmentosum
anddeficientforXPF[17]whentreatedwithHUhadlessp53BP1inductionthanhadwild-
typeprimaryfibroblastsisolatedfromhealthydonor(Fig3D).Importantly,wefoundthesame
effectsofARTEMISorXPFdepletioninnon-transformedRPEcells(S2DFig).Interestingly,
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Fig3.ARTEMISandXPFareinvolvedinRRFB.(A)QuantificationbyneutralcometassayofDSBsinducedinresponsetotreatmentwith4mMHUfor
4hoursincontrolcells(siLUC)andincellsdepletedofoneofvariousendonucleases.Theplotshowsmeantailmoment(%DNAintailxtaillength).
ErrorbarsrepresentSEMs.Atleast150cometswerescored.(cid:3)p<0.05,(cid:3)(cid:3)(cid:3)p<0.0001(Mann–Whitneytest).(B)Westernblotsofwholecellextractsof
RKOcellsshowingdepletionofArtemis(siARTEMIS),XPF(siXPF)andboth(siXPF+ARTEMIS).(C)Quantificationofnuclearp53BP1fluorescence
intensitybyQIBCin>1500S-phaseRKOcellsdepletedofXPFand/orARTEMISandeitheruntreated(NT)ortreatedwith4mMofHUfor4hours,as
indicated.(cid:3)(cid:3)(cid:3)p<0.0001(Mann–Whitneytest).(D)Quantificationofnuclearp53BP1fluorescenceintensitybyQIBCinArtemis-deficientRS-SCID
patientcells(Guetelcells)andinXPF-deficientXPpatientcells(XPfibroblastsgroupF)comparedtoWTprimaryfibroblasts.Meanfoldchangecompared
tountreatedcellsisshown.Errorbarsrepresentstandarddeviationsfrom3independentexperiments.(cid:3)p<0.05(Student’sTtest).(E)Quantificationby
neutralcometassayofDSBsinuntreatedRKOcells(NT)andincellstreatedwith4mMHUfor4hoursafterdepletionofXPFand/orARTEMIS,as
indicated.Theplotshowsmeantailmoment(%DNAintailxtaillength).ErrorbarsrepresentSEMs.Atleast150cometswerescored.(cid:3)(cid:3)(cid:3)p<0.0001
(Mann–Whitneytest).
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weobservedcumulativeeffectonDSBandchromatinboundp53BP1whenbothendonucle-
asesweredepleted(Fig3Cand3E),stronglysuggestingthatARTEMISandXPFfunction
independentlytoproduceDSBunderDRS.
TodeterminewhetherARTEMISandXPFactdirectlyatstalledDNAreplicationforks,we
performediPOND,amethodologywhichallowsthepurificationoffork-associatedproteins.
HereweusediPONDinnativeconditionwhichhasshowntoimprovecaptureefficiencyand
thereforesensitivity[18].Aspreviouslyreported[13],weobservedlessPCNAatreplication
forksofcellstreatedwithHUcomparetountreatedcells,presumablybecausePCNAis
unloadedfromOkazakifragmentsduringHUtreatment.ConverselymoreRad51wasfound
atthestalledreplicationforks(Fig4A).Moreover,wefoundmoreXPFinsamplesfromcells
treatedwithHUthanincontrolcells,suggestingthatXPFisrecruitedtostalledforks.We
wereunabletodetectARTEMISbythenative-iPONDmethodprobablybecausetheantibody
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ArtemisandXPFmediateforkbreakage
Fig4.RegulationofARTEMISandXPFdependentRRFB.(A)Purificationoffork-associatedproteinsbynative-
iPONDinresponsetoHU(4mM–4hours).NoClick,negativecontrol;Chase,chromatinbehindtheforks.(B)
WesternblotsofthesolubleandinsolublefractionsofRKOcellseitheruntreated(-)ortreatedwith4mMHUfor4
hours(+).BargraphshowsImage-JquantificationsofARTEMISandRAD51bandsnormalizedtoORC2intensity.
(C)QuantificationbyneutralcometassayofDSBsincontrolcells(siLUC)andincellsdepletedofARTEMIS
(siARTEMIS)andtreatedornotfor4hourswith4mMofHUand/or2.5μMofDNA-PKinhibitor(NU7441).Theplot
showsmeantailmoment(%DNAintailxtaillength).ErrorbarsrepresentSEMs.Atleast250cometswerescored.
(cid:3)(cid:3)(cid:3)p<0.0001(Mann–Whitneytest).(D)Quantificationofnuclearp53BP1fluorescenceintensitybyQIBCin>1500
S-phaseRKOcellsdepletedofXPF,SLX4and/orERCC1andtreatedwith4mMofHUfor4hours.(cid:3)(cid:3)(cid:3)p<0.0001
(Mann–Whitneytest).
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wasoftoolowaffinityandiPONDcapturesverylowamountofproteinseveninnativecondi-
tion.However,wewereabletodetectitafterfractionationoflargeamountofcells(Fig4B).
WefoundthatARTEMISaccumulatesonchromatin(insolublefraction)intheHU-treated
cells,consistentwiththeideathat,likeXPF,ARTEMISmayalsoberecruitedtostalledDNA
replicationforks.Takentogether,thesedatasuggestthatArtemisandXPFarerecruitedto
stalledforksuponDRStoperformRRFB.IthasbeenshownthatDNA-PKactivityisrequired
foratleastsomeARTEMISfunctionssuchasVDJrecombination[19].Therefore,wetestedby
neutralcometassayifinhibitionofDNA-PKcouldphenocopyARTEMISdepletionafterHU
treatment.WefoundthatcellstreatedwithDNA-PKinhibitoraccumulateDSBevenin
absenceofreplicationstress(Fig4C).Moreover,weobserveasharpinductionofDSBupon
DNA-PKinhibitionandHUtreatment.However,ARTEMISdepletionwasstillabletoinhibit
DSBformationinthiscondition.Thus,weconcludedthatARTEMISfunctioninRRFBdoes
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ArtemisandXPFmediateforkbreakage
notrelyonDNA-PKactivity.Moreover,XPFassociatestoERCC1toformanactiveholoen-
zymeandinteractswiththescaffoldingproteinSLX4whichhasbeenshowntoregulate
XPF-ERCC1duringreplicationstress[20].Thus,weassessedthecontributionofERCC1and
SLX4toXPFdependentRRFBbyQIBC(Fig4D).WefoundthatERCC1orSLX4depleted
RKOcellsexhibitlesschromatinboundp53BP1afterHUtreatmentthancontrolcells.More-
over,doubledepletionofXPFandERCC1orXPFandSLX4didnotyieldtofurtherreduction
ofchromatinboundp53BP1comparedtosinglesiRNAtransfectedcellsindicatingthatXPF,
ERCC1andSLX4areallworkinginthesameRRFBpathway.
ARTEMIS—AndXPF-ERCC1-dependentRRFBsupportsreplicationfork
restartandpreventsanaphasesegregationdefects
ToinvestigatewhetherRRFBinducedbyDRSaffectsreplicationresumptionuponrelease
fromHUtreatment,weusedDNAfiberanalysistomonitorforkrestart.ARTEMIS—orXPF-
depletedcellsandcontrolcellswereincubatedwithIdUfor20minutestolabelongoingrepli-
cationforksandweresubsequentlyexposedfor4hoursto4mMHU.Thecellswerethen
incubatedwithoutHUinmediumcontainingCldUtolabeltherestartedforksfor40minutes,
whichwassufficienttoobserverestartof65%oftheforksincontrolcells(Fig5A).Incells
depletedofARTEMISorXPF,however,replicationforkrestartwassignificantlyimpaired
and,conversely,theproportionofforksthatremainstalledwashigherthaninthecontrols
(Fig5A).Thisobservationsuggeststhatsomebrokenforkscouldberapidlyrepairedtosup-
portforkrestart.Therefore,wefollowedbothbyQIBCandcometassaythebehaviorofXPF
andARTEMISdependentDSBsuponreleasefromaHUblock(Fig5BandS3AFig).Consis-
tentwithourreplicationrestartdataincontrolcells,weobservedthatmostoftheDRS
inducedDSBswererapidlyrepairedwithin30min.However,someDSBsremainedunrepaired
foratleast2hourswhichmightexplainwhynotallthestalledforksareabletorestartevenin
controlcells,asobservedbyDNAcombing.
NextweusedQIBCtosortcontrolandARTEMIS—orXPF-depletedcellsstainedwith
EdUintoearlySphase,middleSphase,andlateSphaseaccordingtotheirDNAcontent.
Then,weanalyzedtheamountofp53BP1onchromatininthevariousSphasepopulations
afterHUtreatment(Fig5C).Weobservedincreasingp53BP1onchromatinfromearlytolate
Sphasecells,suggestingthatthenumberofbrokenforksincreasedascellsprogressedthrough
Sphase.IncellsdepletedofARTEMISorXPF,weobservedoveralllessp53BP1ateachphase
thaninthecontrolcells,suggestingthatARTEMISorXPFdependentforkcleavageisnot
restrictedtoearly,midorlateSphase.Interestingly,wesawstrongp53BP1fluorescenceinten-
sityalsointheG2phasecellsafterHUtreatment,showingthatDRSinducesDNAdamage
evenwhenmostofthegenomeisalreadyduplicated.Moreover,thep53BP1fluorescenceinten-
sitywassignificantlylessinG2phasecellswhenARTEMISorXPFweredepleted(Fig5C),sug-
gestingthatARTEMIS—andXPF-dependentRRFBalsooccursinG2phase.
AlthoughG2phasecellshaveessentiallycompletedDNAreplicationandsoshouldsynthe-
sizerelativelylittleDNA,RRFBoccurstoalevelcomparabletoearlyandmiddleS-phase.This
observationsuggeststhatDNAreplicationforksaremorepronetobreakagewhencells
approachmitosis.WehypothesizedthatefficientRRFBduringG2couldsupportthecomple-
tionofDNAreplicationbeforethecellsentermitosisandthereforepreventtheaccumulation
ofunresolvedstructuresduringchromosomesegregation.Totestthishypothesis,wetreated
EdUlabeledRKOcellswith2mMHUfor2hours,adoseandtimethatinducesasignificant
numberofbrokenforks(Fig1A).WethenremovedtheHUfor6hourstoallowSphasecells
toreachmitosiswherewequantifiedtheproportionoflaggingchromosomesandDNAbrid-
ges(henceforthreferredassegregationdefects)inEdU-positivecells(Fig5D).Wefoundthat
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Fig5.ARTEMISandXPFpromoteefficientreplicationrestartandpreventchromosomesegregationdefects.(A)Schematicrepresentationof
theDNAfiberassaywithimagesoftypicalrestartedorstalledforks(top).Meansandstandarddeviationsofthepercentagesofrestartedorstalled
forkswerederivedfromthreeindependentexperiments.(cid:3)p<0.05,(cid:3)(cid:3)p<0.01(Student’sTtest).(B)RKOcellstreatedfor4hourswith4mMHU
andreleasedintofreshmediafortheindicatedtimesweresubjectedtocometassaytoquantifyDSBs.Theplotshowsmeantailmoment(%DNA
intailxtaillength).ErrorbarsrepresentSEMs.Atleast250cometswerescored.(cid:3)(cid:3)(cid:3)p<0.0001(Mann–Whitneytest).(C)Quantificationof
nuclearp53BP1fluorescenceintensitybyQIBCin>7500cellsdepletedofArtemis(siARTEMISs)orXPF(siXPF)ortreatedwithacontrolsiRNA
(siLUC)andtreatedwithwith4mMHUfor4hours.ThecellsweresortedintothefiveindicatedphasesofthecellcyclebasedontheirEdUand
DAPIcontent.ns:notsignificant,(cid:3)(cid:3)(cid:3)p<0.0001(Mann–Whitneytest).(D)Schematicrepresentationoftheexperimentperformedtoquantify
aberrantanaphasesincellstreatedwithHUandtypicalimagesofDNAbridgesandlaggingchromosomesinanaphasecells.(E)Quantificationof
aberrantanaphasesinuntreated(NT)orHU-treatedcellsdepletedofARTEMISorXPF.Errorbarsrepresentstandarddeviationsfrom3
independentexperiments.(cid:3)p<0.05,(cid:3)(cid:3)p<0.01(Student’sTtest).
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ArtemisandXPFmediateforkbreakage
thisshortDRSresultedinasubstantialincreaseofsegregationdefectsinthesecells.Impor-
tantly,priordepletionofeitherARTEMISorXPFfromthecellsaggravatedtheincidenceof
thesedefects(Fig5E).ToevaluatetheincidenceofRRFBoncellviabilitywetreatedRKOcells
depletedofARTEMISorXPFfor4hourswith4mMHUandassessedcellularviabilityby
MTSassay3dayslater.BecauseHUcouldonlyimpacttheviabilityofcellsthatareinS-phase,
wealsoevaluatedtheircellcycledistributionbyFACS.Interestingly,whilemostoftherepli-
catingcontrolcellswerekilledbythetreatment,wefoundthatARTEMISorXPFdepletion
conferresistancetoHU(S3BFig)Overall,thesefindingsdemonstratethatRRFBisimportant
tosupportefficientDNAreplicationinSandG2phaseandtopreventchromosomalinstabil-
ityduringmitosis.HoweveriftheamountofreplicationstressistoohighRRFBcouldtrigger
celldeath.
Discussion
Inthisstudy,wedescribeaphenomenoninwhichDNAreplicationforksthatstallinresponse
toDRSarerapidlyconvertedintoDSBs,whichwerefertoasrapidreplicationforkbreakage
(RRFB).Weidentifytwoendonucleases,ARTEMISandXPF-ERCC1thatpromoteRRFBat
stalledreplicationforksduringSandG2phasesinresponsetoDRStoensureefficientDNA
replicationandpreventmitoticsegregationdefects.
Interestingly,apreviousstudyfoundthatDSBsareinducedpoorlyinArtemis-deficient
cellsbyprolongedHUtreatment[21].Thismightbeexplainedbytheincapacityofthesecells
tocleavestalledreplicationforksearlyon.Similarly,anotherstudyfoundthatdepletionofthe
endonucleasescaffoldingproteinSLX4,whichinteractswithXPF-ERCC1,preventstheforma-
tionofreplication-associatedDSBsafterlongHUtreatment(24hours)[20];theauthorssug-
gestedthatthisfunctionofSLX4partiallyreliesonXPF-ERCC1butalsoSLX1endonucleases.
Here,earlierafterDRSinductionweclearlyshowthatforkbreakageisdependenton
XPF-ERCC1andSLX4butnotSLX1.
WhywouldthecelldevelopamechanismofforkrescuethatinvolveDSBisunclear.Our
datademonstratethatRRFBisusedtoresumeDNAreplicationatpersistentstalledforksto
preventmitoticsegregationdefects.WeshowthatRRFBalsooccursathighfrequencyinG2
phasecellstreatedwithHU.Thus,wespeculatethatRRFBmightbeusedbythecellasthe
finalattempttorescuestalledforks.Indeed,severalmechanismshavebeendescribedthathelp
stalledforksresumeDNAreplication[22]andextensiveforkremodelingsuchasforkreversal
hasbeenshowntooccuruponDRS[23].Interestingly,arecentstudyhasshownthat
SAMHD1promotesdegradationofnascentDNAatstalledreplicationforksbystimulating
theexonucleaseactivityofMRE11whichfacilitatesforkrestart.Whetherendo-andexo-
nucleolyticactivitiesworkinconcertatstalledreplicationforksneedfurtherattention.Impor-
tantly,theauthorsshowedthatexo-nucleolyticdegradationofnascentDNAatstalledforks
inducesexpressionofpro-inflammatorytypeIinterferons.AsweobservedthatRRFBcould
triggercelldeathwhenreplicationstressisexcessive,bothendoandexo-nucleolyticactivities
couldrepresentnewmechanismstoclearproblematiccellswhichexperiencehighreplication
stressfromthetissuesinordertopreventaccumulationofmutationsandtherefore
tumorigenesis.
HowRRFBfacilitatescompletionofDNAreplicationunderDRSisstillunclear.Arecent
studysuggestedthatreplicationforkbreakagemightbemanagedbybreak-inducedreplication
(BIR)duringSphaseinyeast[24].Thistemplateswitch-mediatedrepairmightcouplerepair
ofthesingle-endedDSBinducedbyRRFBtocompletionofreplication.Itwouldalsoprovide
arationaleforthecoexistenceofMus81andRRFBnucleases.Indeed,MUS81couldresolve
theD-loopinducedbyBIRinordertolimitthemutagenicsynthesisofPOLD3[24].Recent
PLOSGenetics|https://doi.org/10.1371/journal.pgen.1007541 July30,2018 10/16