The Smc5/6 complex regulates the yeast Mph1 helicase at RNA-DNA hybrid-mediated DNA damage

25 Pages·2017·6.25 MB·English

RESEARCHARTICLE
The Smc5/6 complex regulates the yeast
Mph1 helicase at RNA-DNA hybrid-mediated
DNA damage
JuanLafuente-Barquero1☯,SarahLuke-Glaser2,3☯,MarcoGraf2☯,SoniaSilva1,4,
Bele´nGo´mez-Gonza´lez1,AriannaLockhart2,MichaelLisby4,Andre´sAguilera1*,
BrianLuke2,3,5*
1 AndalusianCenterforMolecularBiologyandRegenerativeMedicine-CABIMER,UniversidaddeSevilla-
CSIC-UniversidadPablodeOlavide,Avda.AmericoVespucio24,Seville,Spain,2 InstituteofMolecular
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Biology(IMB),Mainz,Germany,3 Zentrumfu¨rMolekulareBiologiederUniversita¨tHeidelberg(ZMBH),
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DKFZ-ZMBHAlliance,Heidelberg,Germany,4 DepartmentofBiology,UniversityofCopenhagen,Ole
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Maaloeesvej5,CopenhagenN,Denmark,5 InstituteofNeurobiologyandDevelopmentalBiology,JGU
a1111111111 Mainz,Mainz,Germany
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☯Theseauthorscontributedequallytothiswork.
*[email protected](BL);[email protected](AA)
Abstract
OPENACCESS
Citation:Lafuente-BarqueroJ,Luke-GlaserS,Graf
RNA-DNAhybridsarenaturallyoccurringobstaclesthatmustbeovercomebytheDNArep-
M,SilvaS,Go´mez-Gonza´lezB,LockhartA,etal.
(2017)TheSmc5/6complexregulatestheyeast licationmachinery.IntheabsenceofRNaseHenzymes,RNA-DNAhybridsaccumulate,
Mph1helicaseatRNA-DNAhybrid-mediatedDNA resultinginreplicationstress,DNAdamageandcompromisedgenomicintegrity.Wedem-
damage.PLoSGenet13(12):e1007136.https://
onstratethatMph1,theyeasthomologofFanconianemiaproteinM(FANCM),isrequired
doi.org/10.1371/journal.pgen.1007136
forcellviabilityintheabsenceofRNaseHenzymes.TheintegrityoftheMph1helicase
Editor:FrancescaStorici,GeorgiaInstituteof
domainiscrucialtopreventtheaccumulationofRNA-DNAhybridsandRNA-DNAhybrid-
Technology,UNITEDSTATES
dependentDNAdamage,asdeterminedbyRad52foci.Mph1formsfociwhenRNA-DNA
Received:April5,2017
hybridsaccumulate,e.g.inRNaseHorTHO-complexmutantsandatshorttelomeres.
Accepted:November28,2017 Mph1,howeverisadouble-edgedsword,whoseactionathybridsmustberegulatedbythe
Published:December27,2017 Smc5/6complex.Thisisunderlinedbytheobservationthatsimultaneousinactivationof
RNaseH2andSmc5/6resultsinMph1-dependentsyntheticlethality,whichislikelydueto
Copyright:©2017Lafuente-Barqueroetal.Thisis
anopenaccessarticledistributedundertheterms anaccumulationoftoxicrecombinationintermediates.Thedatapresentedheresupporta
oftheCreativeCommonsAttributionLicense, model,whereMph1’shelicaseactivityplaysacrucialroleinrespondingtopersistentRNA-
whichpermitsunrestricteduse,distribution,and
DNAhybrids.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation
Authorsummary
files.
Funding:Thisworkwassupportedbygrantsfrom DNAdamagecaneitheroccurexogenouslythroughDNAdamagingagentssuchasUV
theSpanishMinistryofEconomyand lightandexposuretochemotherapeutics,orendogenouslyviametabolic,cellularpro-
Competitiveness(BFU2013-42918-P&BFU2016- cesses.TheRNAproductoftranscription,forexample,canengageintheformationof
75058-P),http://www.mineco.gob.es/portal/site/
RNA-DNAhybrids.SuchRNA-DNAhybridscanimpedereplicationforkprogression
mineco;JuntadeAndaluc´ıa(BIO1238),http://
andcausegenomicinstability,ahallmarkofcancer.ThemisregulationofRNA-DNA
www.juntadeandalucia.es/index.html;theEuropean
ResearchCouncil(ERC2014AdG669898 hybridshasalsobeenimplicatedinseveralneurologicaldisorders.Recently,ithasbecome
TARLOOP),https://erc.europa.eu;theDanish
PLOSGenetics|https://doi.org/10.1371/journal.pgen.1007136 December27,2017 1/25
TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
CouncilforIndependentResearch,http://ufm.dk/
en/research-and-innovation/councils-and- evidentthatRNA-DNAhybridsmayalsohavebeneficialrolesandtherefore,thesestruc-
commissions/the-danish-council-for-independent- tureshavetobetightlycontrolled.WefoundthatMph1(mutatorphenotype1),thebud-
research;theVillumFoundation,http://
dingyeasthomologofFanconiAnemiaproteinM,counteractstheaccumulationofRNA-
veluxfoundations.dk/da;EuropeanResearch
DNAhybrids.TheinactivationofMPH1resultsinaseveregrowthdefectwhencombined
Council(ERCStG,no.242905);BMBF-GerontoSys
withmutationsinthewell-characterizedRNaseHenzymes,thatdegradetheRNAmoiety
IInetworkAGENET(FKZ0315898),https://www.
bmbf.de;CancerTelSys(01ZX1302)intheE:med ofanRNA-DNAhybrid.Basedonthedatapresentedhere,weproposeamodel,where
programoftheGermanFederalMinistryof Mph1itselfhastobekeptincheckbytheSMC(structuralmaintenanceofchromosome)
EducationandResearch(BMBF),http://www.sys- 5/6complexatreplicationforksstalledbyRNA-DNAhybrids.Mph1actsasadouble-
med.de/de/;InstitutoCarlosIII(SpanishMinistryof
edgedsword,asbothitsdeletionandtheinabilitytocontrolitshelicaseactivitycause
Health)toJLB;ScientificFoundationoftheSpanish
DNAdamageandgrowtharrestwhenRNA-DNAhybridsaccumulate.
AssociationAgainstCancertoBGG;Fundac¸ãopara
aCiênciaeaTecnologiatoSS;EMBO/MarieCurie
co-fundfellowshiptoSLG(ALTF9-2010),http://
www.embo.org/funding-awards/fellowships;
EuropeanUnion(FEDER).Thefundershadnorole Introduction
instudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthe RNA-DNAhybridscanformduringtranscriptionwhenthenascentRNAbasepairswiththe
manuscript. templatestrandoftheDNA.Thisresultsina3-strandedstructure,referredtoasanR-loop
[1,2].R-loopsleadtoDNAreplicationstressandhenceincreasedratesofDNAbreakage,
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. unscheduledrecombination,andgenomerearrangements[3,4].ItisunclearexactlyhowR-
loopscausereplicationstress,butitmaybelinkedtotheirabilitytopromotelocalchromatin
compaction[5].R-loopscanberemovedbytheRNaseHenzymes(RNaseH1andRNaseH2),
whichcandegradetheRNAmoietyofanRNA-DNAhybridmolecule.Inyeast,RNaseH1is
encodedbytheRNH1gene,whiletheRNaseH2enzymeisatrimericcomplexmadeupofthe
geneproductsofRNH201(thecatalyticsubunit),RNH202,andRNH203[6,7].Mutationsin
allthreesubunitsofRNaseH2havebeenlinkedtotheneurologicalauto-immunedisorder,
Aicardi-Goutièressyndrome(AGS)[8].
InadditiontoRNaseH-mediatedhybriddegradation,theTHOcomplexcounteracts
theaccumulationofRNA-DNAhybrids.TheTHOcomplexisatranscription-coupledRNA
processingcomplexthatlimitstheformationofRNA-DNAhybridsinaco-transcriptional
manner[9]by“capturing”thenascentRNAandensuringthatitgetsefficientlyexported.Fur-
thermore,multiplehelicaseshavebeenimplicatedinRNA-DNAhybridremovalviadisplace-
mentoftheRNAstrand,includingyeastPif1[10]andSen1[11],humanSETX(theequivalent
ofyeastSen1)[12],AQR[13],DDX19[14],DDX23[15],DDX1[16]andDDX21[17].When
R-loopremovalfactorsareinactivated,manyDNArepairgenes(namelythoseinvolvedin
homologousrecombination)becomeessential,underliningthepropensityofR-loopstopro-
moteDNAdamageasaresultofreplicationstress[1,2].RNA-DNAhybridsexistattelomeres
inyeastandhumancellsandpromotehomology-directedrepair[18–20]demonstratingtheir
involvementintelomeremaintenance.Astelomeresshorten,RNA-DNAhybridsaccumulate
togetherwithincreasedlevelsofthelongnon-codingRNA,telomericrepeat-containingRNA
(TERRA)[21–25],suggestingthattelomericRNA-DNAhybridsaremadeupofTERRA.
TherecentobservationsthattheFanconiAnemiarepairpathway,whichisinvolvedinthe
repairofDNAinter-strandcrosslinksandobstaclesthatimpedereplicationforkprogression,
iscrucialtodissolveR-loopsthatmayblockreplicationforksinhumancells[26,27]suggesta
relevantroleofthisDNArepairpathwayinR-looppreventionandremoval.HumanFANCM
hastheabilitytobranchmigratereplicationstructures,resolveRNA-DNAhybridsinvitroas
wellastopreventRNA-DNAhybridaccumulationinvivoinhumanandchickencells[27].
AlthoughbuddingyeastcellslackacanonicalFanconiAnemiapathway,yeastMph1helicase
standsoutasahomologoftheFANCMprotein[28].Invitro,Mph1isabletodissociateD-
loopsandpromotereplicationforkreversal,similarlytoFANCM[29].Boththedeletionand
PLOSGenetics|https://doi.org/10.1371/journal.pgen.1007136 December27,2017 2/25
TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
overexpressionofyeastMph1areassociatedwithincreasedreplicationstressandgenome
instability[30,31],respectivelyindicatingthatitsactivityisimportantwhenfacingreplication
stress,butmustbetightlyregulatedinordertopreventtoxicity.Interestingly,thelossofMph1
hasbeenreportedtoleadtoasyntheticgrowthdefectintheabsenceofRNH203[32],pointing
toapossibleroleforMph1inR-loopregulation.
SeveralstudieshavereportedthattheSmc5/6complexisanegativeregulatorofMph1[33–
35].TwoSMCsubunits(Smc5andSmc6)and6non-SMCelementsformthishighlycon-
servedcomplex,whichstructurallyresemblesthecohesin,condensinandtheMRNcomplexes
[36].ThedeletionofMPH1isabletorescuethetemperaturesensitivegrowthdefectofsmc5/6
mutantallelesaswellastheaccumulationofX-shapedrecombinationintermediatesafter
treatmentwithDNAdamagingagents[33–35].Interestingly,theinactivationoftheSmc5/6
complex[37,38],aswellasoverexpressionofMph1[39],havebeendemonstratedtodrive
yeastintoprematurereplicativesenescence.
Giventheobservationsoutlinedabove,wesetouttoinvestigatetheroleofMph1andits
establishednegativeregulator,Smc5/6atRNA-DNAhybridsinvivointhebuddingyeastSac-
charomycescerevisiae.Inthisstudy,weshowthatMph1formsnuclearfociwhenRNA-DNA
hybridsaccumulate,i.e.inrnh1rnh201mutantsandatshorttelomeres.Wedemonstratethat
Mph1,andinparticularitshelicasefunction,suppressestheaccumulationofRNA-DNA
hybrids,atRNAPII-transcribedgenesaswellasattelomeres,andthatintheabsenceofMph1,
cellsaccumulaterecombinogenicDNAdamageinanRNA-DNAhybrid-dependentmanner.
Accordingly,Mph1becomesessentialwhenRNA-DNAhybridremovalisstronglyimpaired,
asseenbytheseveregrowthdefectofthernh1rnh201mph1triplemutants.However,when
thefunctionalityoftheSmc5/6complexiscompromised,Mph1’shelicaseactivitybecomes
deleteriousinsituationswhenRNA-DNAhybridsaccumulate.WeproposethatMph1plays
animportantroleinRNA-DNAhybridmetabolismandthatitsactivityhastobetightlycon-
trolledbytheSmc5/6complex.
Results
Mph1preventstheaccumulationofhybrid-inducedRad52foci
GiventheinvolvementofthehumanFanconiAnemiaMproteininpreventingR-loop-medi-
ateddamageandRNA-DNAhybridaccumulationinvivo,wedecidedtoaddresswhether
yeastcellsdeletedfortheFANCMhomologMPH1orothercandidatehelicasesimplicatedin
DNArepairarealsoinvolvedinR-loopmetabolism.DeletionmutantsofSGS1,SRS2,MPH1,
RRM3wereassayedfortheaccumulationofRad52foci,amarkerforDNArepairatsitesof
damage.GenotypeswereconsideredasspecificallyaccumulatingRNA-DNAhybrid-induced
damagewhentheincreasednumberofRad52fociwasabolisheduponRNaseH1overexpres-
sion.ThedeletionoftheRNaseHenzymes(rnh1rnh201)andHPR1,amemberoftheTHO
complex,whereR-loopaccumulationhasbeendemonstratedtoaccountforthemajorityof
DNAdamage[9],servedaspositivecontrols.ItisworthnoticingthatRad52fociwerenot
fullysuppressedinrnh1rnh2cells,whichsuggeststhathybridsnoteasilyaccessibletoRNase
H1mayaccumulateinthesedoublemutants.Althoughallfoursinglegenemutantstestedled
toasignificantincreaseinRad52focicomparedtowildtype,onlyMPH1deletedcellsshowed
anRNaseH1-sensitiveincrease(Fig1A).Therefore,wefocusedonachievingamorecomplete
understandingofMph1anditsroleatRNA-DNAhybrids.Toruleoutyeastgeneticback-
groundspecificeffects,weconfirmedtheaccumulationofRad52fociinmph1mutantsinthe
W303geneticbackground(Fig1B).Weperformedawesternblotonanendogenouslytagged
Rad52strain(Rad52-TAP)withandwithoutRNaseH1overexpressiontoruleoutaneffecton
Rad52proteinlevels(S1AFig).ThisindicatesthatMph1hasaroleinthepreventionof
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
Fig1.Mph1helicasedomainpreventstheaccumulationofRad52foci,andisrequiredintheabsenceofRNaseH1and
RNaseH2.A.QuantificationofspontaneousRad52-YFPfociformationinWTandcandidatemutanthelicasestrainsas
wellasthedoublemutantrnh1rnh201(ySLG428)andtriplemutantrnh1rnh201mph1(ySLG611)(BY4741background).
CellsweretransformedwithpWJ1314,thatcarriestheRad52-YFPfusion,andtheemptyplasmidpCM189(-RNH)or
pCM189::RNH1(+RNH).AverageandSDofthreetosixindependentexperimentsareshown.B.Rad52-GFPfoci
accumulationinWT(W303.1A)andmph1(WMPH.2B)mutantintheW303background,withandwithoutover-
expressionofRNH.(cid:3)P<0.05;(cid:3)(cid:3)P<0.01;(cid:3)(cid:3)(cid:3)P<0.001;(cid:3)(cid:3)(cid:3)(cid:3)P<0.0001,(Student’st-test).Blueasterisksindicatesignificant
differencescomparedtoWTandredasterisksindicatestatisticalsignificancebetween-/+RNHconditionsforeachmutant.
AverageandSDofthreeindependentexperimentsareshown.C.Leftpanel:RepresentativeimageofMph1-GFPfoci
accumulatinginrnh1rnh201mutants.Arrowheadsindicatefoci.Scalebar,3μm.RightPanel:Mph1-GFPfociwere
quantifiedinexponentiallygrowingwildtype(ySLG636),rnh1(ySLG639),rnh201(ySLG640),andrnh201rnh1(ySLG642)
cells(OD =0.3)at25˚C.CellsweredividedintobuddedcellsintheSorG2phaseofthecellcycleorintoun-budded,
600
G1-cells.Tworeplicatesof200–600cellswereexamined.Errorbarsindicate95%confidenceintervals.Significancerelative
tothewildtypedeterminedbyFisher’sexacttest((cid:3)(cid:3)P<0.05).D.Theheterozygousdiploidstrain(ySLG605,BY4741
background)wassporulatedandmicromanipulatedontoYPDagarplatestogeneratehaploidoffspring.Sporeswere
incubatedfor3daysat30˚Cbeforeimaging(leftpanel).E.yAL296wasarrestedinG1withα-factorandreleasedintoYPD
mediaat25˚C.Proteinextractswerepreparedattheindicatedtimepoints(minutespostG1release)andsubjecttowestern
blotting.F.yAL404wassporulatedanddissectedontoYPDmediaandimageswereacquiredfollowing3daysofincubation
at30˚C.StrainsweregenotypedbyselectablemarkersandPCRtechniques.G.ThestatusofRad53phosphorylation(up-
shift)wasanalyzedbywesternblottingwithanti-Rad53specificantibodies.Rnr3proteinlevelswereassessedbyprobing
withananti-Rnr3antibody.Ponceaustainingservedasaloadingcontrol.H.Wildtype(WT)(top)andmph1rnh1rnh201
cells(bottom)weretransformedwithanMph1wildtypeexpressingplasmidorwithhelicase-defectiveMph1variants
(E210QandQ603D).Serialdilutionswerespottedontoselectivemedia.Picturesweretakenaftergrowingcellsfor3daysat
30˚C.
https://doi.org/10.1371/journal.pgen.1007136.g001
RNA-DNAhybridformationorinminimizingthedamageinducedbyRNA-DNAhybrids
alreadyformed.
InordertogetanindicationofwhetherMph1maybeactingdirectlyatRNA-DNAhybrids,
wetestedwhetheritformsfociinmutantsthatareknowntoaccumulatehybrids.Weobserved
anincreasedfrequencyofendogenouslyexpressedMph1-GFPfociinrnh1rnh201double
mutantcells,wherehybridsaccumulatetohighlevels(Fig1C).Mph1-GFPfocispecifically
accumulatedinrnh1rnh201doublemutantcellsintheS-orG2phaseofthecellcycleandnot
inG1(Fig1C),suggestingthatMph1playsaroleatRNA-DNAhybridsduringDNAreplica-
tiontopreventtheaccumulationofDNAdamage.
MPH1isessentialintheabsenceofRNaseH1andRNaseH2
AsMph1formsfociwhenRNA-DNAhybridsaccumulate,wenextinvestigatedwhetherits
functionbecomesessentialwhenknownRNA-DNAhybridregulatingfactorsareimpaired.
Indeed,ithasbeenreportedthatthelossofMPH1leadstogrowthimpairmentintheabsence
ofRNH203intheS288Cbackground[32].IntheBY4741andW303geneticbackgrounds,the
mph1rnh1rnh201triplemutantswereeitherseverelygrowthcompromisedorcompletely
inviable,respectively(Fig1DandS1BFig,fordetailedgrowthcurvesandpopulationdoubling
timesseeS1CFig).InordertotestifthisessentialfunctionofMph1occursduringSphase,we
createdanalleleofRNH1(S-RNH1)thatisspecificallyexpressedinSphase,byplacing
RNH1-TAPundertheClb6promoterandfusingittoaClb6degronsequence[40](Fig1E,
S1DFig).Comparedtothernh1rnh201mph1triplemutant,theexpressionofRnh1inSphase
didnothaveagrowthdefectinrnh201mph1mutants(Fig1F).ConsistentwithMph1prevent-
ingR-loop-induceddamage,theslowgrowthofthernh1rnh201mph1mutantswasassociated
withaslightlyincreasedphosphorylationofRad53aswellasinducedexpressionofRNR3(Fig
1G),bothbeingestablishedmarkersforactivationoftheDNAdamagecheckpoint.Totest
whetherMph1’shelicaseactivityisessentialtorestorethegrowthofrnh1rnh210mph1triple
mutants,wereintroducedhelicase-compromisedMph1mutants,mph1-E210Qandmph1-
Q603D[34],onaplasmidbyexpressingthemfromtheendogenousMPH1promoter.
WhereaswildtypeMph1fullysuppressedthegrowthdefectandcheckpointactivation,neither
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
ofthehelicasemutantswasabletorescuethesyntheticinteraction(Fig1HandS1EFig),
despitebeingexpressedatsimilarlevels(S1FFig).
AsRNaseH2activityiscapableofremovingsingleribonucleotidesthathavebeenmisin-
corporatedintoDNAhelixbythereplicatingDNApolymerase[41,42],aswellasconsecutive
RNA-DNAhybrids(i.e.thoseformedinanR-loop),wewantedtodetermineatwhichtypeof
RNA-DNAhybridMph1wasfunctioning.Therefore,weemployedtheRNH201-P45D-Y219A
mutant,whichisspecificallydefectiveinmono-ribonucleotideexcisionrepair(RER),butis
proficientinremovinglongerhybridstretches(hereaftercalledRNH201-REDforribonucleo-
tideexcisiondefective)[43,44].Wetransformedthisplasmidintothernh1rnh201mph1cells
andobservedthatitsexpressioncomplementsthesyntheticgrowthdefecttothesameextent
aswildtypeRNH201(S1GFig).Consistently,thecheckpointactivation,asmonitoredbyRnr3
expression,wasalsoalleviateduponexpressionofbothwildtypeRNH201andRNH201-RED
(S1HFig).WeverifiedthattheRNH201-REDalleleisRER-defectiveinvivobydemonstrating
thattheexpressionofthismutantcouldnotsuppressthehydroxyurea(HU)sensitivityof
pol2M644Grnh201doublemutants,whichharborshighlevelsofmisincorporatedribonucleo-
tides[45],whereaswildtypeRNH201wasabletocomplement(S1IFig).Insummary,these
dataindicatethatMph1’shelicaseactivitybecomesessentialduringDNAreplicationwhen
consecutiveRNA-DNAhybrids(suchasthosepresentinR-loops)accumulateandmaynotbe
requiredatmisincorporatedribonucleotideinsertions.
AsMph1’sactivityisrequiredinrnh1rnh201doublemutantswetestedwhetherMph1is
alsoessentialinothermutantsthathavebeenreportedtoaccumulateRNA-DNAhybrids.
UnlikethelossofbothRNaseH1andH2functions,wefoundthatdeletingMPH1doesnot
resultingrowthimpairmentwheneitheroftheTHOcomponents,THP2(S2AFig)or
HPR1,aredeleted(S2BFig),orincombinationwiththetemperature-sensitivesen1-1allele
(S2CFig).Consistently,wealsodidnotseeevidenceofDNAdamagecheckpointactivation
asshownbybothlackofdetectableRad53phosphorylationandRNR3induction,inmph1
hpr1doublemutantsintheBY4741background(S2DFig).Nonetheless,Mph1fociwere
observedinhpr1mutantsandthesefociweresuppressedbyRNaseH1overexpression(S2E
Fig),consistentwiththenotionthatMph1fociareenrichedatR-loops.Theseresultssug-
gestthatMph1mayonlybecomeessentialwhenRNA-DNAhybridsreachveryhighlevels
asinRNaseH-deficientdoublemutantcells(seeFig2Aright).Alternatively,Mph1maybe
requiredtoactonasubsetofRNA-DNAhybridsthatisdistinctfromthoseaffectedbythe
THOandSen1proteins.
Mph1preventstheaccumulationofRNA-DNAhybrids
Thecombinedresultsofthesyntheticgrowthdefectbetweenmph1andrnh1rnh201mutants
togetherwiththefactthatRad52fociaccumulateinmph1cellsinanRNaseH1-sensitiveman-
nerledustospeculatethatRNA-DNAhybridsmayaccumulateinmph1mutants.SinceR-
loopsfrequentlyleadtohyper-recombination,wesoughttostudyrecombinationinthe
absenceofMph1.Forthat,weusedpreviouslydeviseddirect-repeatrecombinationassays
basedontwotruncatedcopiesoftheLEU2gene[46].UnlikemutantsoftheTHOcomplexor
sen1-1mutants,whereR-loopsaccumulate,deletionofMPH1resultsinonlyconservative
changes,ornochangesatall,inrecombination,dependingontheassayused(S3AFig,S3B
Fig).Thesedata,togetherwiththeRNaseH-sensitiveincreaseofRad52foci(Fig1A)suggest
thatintheabsenceoftheMph1helicaseactivity,thedamagecausedbyRNA-DNAhybridsis
notefficientlyresolvedintodetectablerecombinationproductsandhenceresultsintheaccu-
mulationofRad52foci.
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
Fig2.AccumulationofRNA-DNAhybridsinmph1cellsdependonMph1’shelicasedomain.A.S9.6focidetectedbyindirectimmunofluorescenceon
yeastchromosomespreadsinWT(W303.1A),mph1(WMPH1-2B),hpr1(HPBAR1-R)andrnh1rnh201(RNH-R)strainscarryingtheemptyplasmid
pCM189(-RNH)orpCM189::RNH1(+RNH).Representativespreadsareshown,DNAisstainedinblue(DAPI),redfocirepresentRNA-DNAhybrids
detectedbytheS9.6antibody.Scalebar=5μm(leftpanel).QuantificationofnucleicontainingS9.6fociisshown.DatarepresentmeanandSDofthreeto
fourindependentexperimentsinwhich150–200nucleiwerecountedforeachreplicate.Statisticalanalysiswasperformedwiththetotalnumberofsignals
foreachmutantwithrespecttoWTvalue(rightpanel).B.DRIPwithS9.6antibodyinWT(W303-1A),mph1(WMPH1-2B),mph1-Q603D(T597-1)and
mph1-E210Q(T617)strainsinasynchronousculturestreated(+)ornot(-)invitrowithRNaseHintheGCN4andPDC1genes(leftandrightpanel
respectively).DatarepresentmeanandSDoffourindependentexperiments.(cid:3)P<0.05;(cid:3)(cid:3)P<0.01;(cid:3)(cid:3)(cid:3)P<0.001(two-tailedStudent’st-test).Blackbar
depictstheapproximateprimerbindingregionforamplificationoftherespectivelocus.
https://doi.org/10.1371/journal.pgen.1007136.g002
ToassayforthepresenceofRNA-DNAhybrids,weperformedindirectimmunofluores-
cenceonchromosomespreads.WedetectedanincreaseinthenumbercellswithS9.6fociin
mph1mutantcells,indicatingincreasedlevelsofRNA-DNAhybrids(Fig2A).Cellscontaining
morethan3foci,wereespeciallyprevalentwhenMPH1wasdeleted.hpr1andrnh1rnh201
cellsservedaspositivecontrols,wherehybridsareknowntoaccumulate(Fig2A,rightpanel).
ToconfirmtheaccumulationofRNA-DNAhybridsinmph1mutantsweusedDNA-RNA
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
Fig3.AccumulationofRNA-DNAhybridsinthetriplemutantmph1rnh1rnh201.DRIPwiththeS9.6antibodyinWT(yBL7),mph1(ySLG2),rnh1rnh201
(ySLG428)andmph1rnh1rnh201(ySLG611)strainsinasynchronousculturestreated(+)ornot(-)invitrowithRNaseHintheGCN4andPDC1genes(top,left
andrightpanelsrespectively)andTEL06RandrDNA18Sloci(bottom,leftandrightpanelsrespectively).DatarepresentmeanandSDoffourindependent
experiments.(cid:3)P<0.05;(cid:3)(cid:3)P<0.01;(cid:3)(cid:3)(cid:3)P<0.001(two-tailedStudent’st-test).Blackbardepictstheapproximateprimerbindingregionforamplificationofthe
respectiveloci.
https://doi.org/10.1371/journal.pgen.1007136.g003
immunoprecipitation(DRIP)topulldownRNA-DNAhybridsaspreviouslydescribed[26,
47].Inagreementwiththeimmunofluorescencedata(Fig2A),RNA-DNAhybridsaccumu-
latedsignificantlywithintheproteincodinggenes,GCN4(Fig2B,leftpanel)andPDC1(right
panel)inmph1mutants.TheRNA-DNAhybridssignalcouldbestronglyreduceduponin
vitroRNaseH1treatment,therebydemonstratingthespecificityoftheDRIPsignal.Toinvesti-
gatewhetherMph1’shelicaseactivityisneededtopreventtheaccumulationofRNA-DNA
hybrids,wealsoperformedDRIPonhelicase-deadMph1mutants(mph1-E210Qandmph1-
Q603D)[36],whichaccumulatedRNA-DNAhybridsattheGCN4(Fig2B,left)andthePDC1
(right)locisimilarlytothecompletedeletion.
TobetterunderstandhowMph1isinvolvedinRNA-DNAhybridregulation,weexpanded
ourDRIPanalysistotelomeres(telomere6R)andtherDNAloci(Fig3).Whereaslossof
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
Mph1functiondidnotaffecthybridlevelsatthe18srDNAlocus,itledtoanincreaseattelo-
mere6R,suggestingthatMph1mayactpreferentiallyatRNApolymeraseIItranscribedloci.
WhenDRIPwasperformedinthemph1rnh1rnh2mutants(onlyviableinBY4741back-
ground,Fig1),wedidnotobserveanyfurtherincreaseinRNA-DNAhybridswithrespectto
thesinglemph1ordoublernh1rnh2mutants(Fig3).Asimilarepistasiswasobservedfor
Rad52foci(Fig1A),suggestingthatMph1mayremoveRNA-DNAhybridsviaRNaseH.
Mph1accumulatesatshorttelomeresinresponsetoincreasedTERRAand
R-loops
TheoverexpressionofMph1drasticallyincreasestherateofreplicativesenescenceincellshar-
boringshorttelomeresfollowingthelossoftelomeraseactivity[39],implyingthatitmaybe
functionallyrelevantattelomeres.Levelsofthenon-codingtelomericrepeat-containingRNA
(TERRA)increaseastelomeresshorteninyeastandhumancells[22,23,48].Moreover,
RNA-DNAhybrids(presumablyinvolvingTERRA)canbedetectedattelomeresandaccumu-
lateintheabsenceoftheRNaseHenzymes[18–20,49].Weallowedwildtypeandtelomerase
negative(tlc1)cellstoundergoapproximately60populationdoublingsand,asexpected,
observedanincreaseinTERRAlevelsatalltestedtelomeres(Fig4A).UponperformingDRIP
onwildtypeandtelomerasenegativecellstomonitorhybridlevels,weobservedanincreasein
telomerichybridsdespitethefactthattheywereshorter(Fig4B).Importantly,inanindepen-
dentexperiment,weobservedthattheoverexpressionofRNaseH1abolishedtheincreased
DRIPsignalintlc1cells(S4AFig),demonstratingthespecificityoftheRNA-DNAhybridsig-
nalshowninFig4B.ToverifythatMph1wasactingatshorttelomeresinanRNA-DNA
hybriddependentmanner,wemonitoredMph1-YFPfociformationastelomeresshortenin
telomerasenegativecells(est2).Importantly,weobservedthatMph1-YFPfociaccumulateas
telomeresshorten(Fig4Cand4D).Atthepeakofsenescencemorethan50%ofMph1focico-
localizewithCdc13(whichrepresentdysfunctionaltelomeres)andRad52foci,therebyindi-
catingthattheyaccumulateatcriticallyshortanddysfunctionaltelomeres(Fig4C–4E)[50].
Importantly,Mph1’sabilitytoformfociwasgreatlyreducedwhenRNaseH1wasoverex-
pressed(Fig4Cand4D).Strikingly,thenumberofCdc13fociwasalsodecreaseduponRNase
Hoverexpression,suggestingthathybridsmaybeasourceofDNAdamageattelomeresupon
shortening.ByChIPanalysiswecouldseethatlossoftheC-terminaldomainofMph1,which
hasbeenshowntointeractwithRfa1andMte1[30,51],preventedMph1fromlocalizingto
telomeresintelomerasepositivecells(S4BFig)indicatingthatMph1locationattelomeres
maydependonitsinteractionwithreplicationproteinA(RPA).Takentogether,weconclude
thatMph1accumulatesatshorttelomeres,inanRNA-DNAhybrid-dependentmanner.
TheSmc5/6complexnegativelyregulatesMph1atRNA-DNAhybrids
PreviousdataindicatesthattheSmc5/6complexisimportanttolimittheaccumulationof
toxicrecombinationintermediatesinthepresenceofMMS(methylmethanesulfonate),andat
naturalpausesites[34,35,52–54].Anegativegeneticinteractionbetweenthesmc6mutants
andrnh201aswellasrnh202waspreviouslydescribedinahigh-throughputSGAscreen[55].
Furthermore,thereisampleevidencethattheSmc5/6complexisimportantfortheaccurate
replicationoftherDNAlocus,arepetitivegenomicregionrichinRNA-DNAhybrids[56,57].
Basedontheseobservations,wehypothesizedthattheSmc5/6complexmightberequiredfor
theaccurateprocessingofDNAreplicationintermediatesthatarisewhenRNA-DNAhybrids
areencounteredbythereplisome.Totestthisnotion,weintroducedthesmc6-9andsmc5-6
temperaturesensitiveallelesintostrainsdefectiveforeitherRNaseH1(rnh1)orRNaseH2
(rnh201)activity,aswellasinrnh1rnh201doublemutants.TheabsenceofRNH201,butnot
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TheroleofMph1inconcertwithSmc5/6atRNA-DNAhybrids
Fig4.Mph1formsfociattelomeresduringsenescence,whenRNA-DNAhybridsaccumulate.A.Bothwildtypeandtlc1cellswerederivedfromtheTLC1/tlc1
heterozygousdiploidyAL95andgrownforapproximately60populationdoublings(PD)beforeRNAwasextractedfromexponentiallygrowingcells.Followingreverse
transcriptionwithatelomericsequence,TERRAlevelswereanalysedattheindicatedtelomeresviaqPCRwithsubtelomericspecificprimerpairs(seemethodsandS1
Tablefordetails).Threebiologicalreplicateswereusedforeachgenotype.Errorbarsindicate95%confidenceintervals.(cid:3)representssignificancerelativetowildtype
determinedbyStudent’st-test(P<0.05).B.Bothwildtypeandtlc1cellsweregrownforapproximately60populationdoublings.ChIPwiththeS9.6antibodythat
specificallyrecognizesRNA-DNAhybridsfollowedbyqPCR.Errorbarsrepresent95%confidenceintervals,(cid:3),significancerelativetowildtypeasdeterminedbya
Student’st-test(P<0.05).C.Representativeimagesforsenescingcellsshowingco-localizationbetweenMph1-YFP,Rad52-yEmRFPandCdc13-CFP.Scalebar,3μm.
QuantificationofMph1(D.)andCdc13-foci(E.).RNaseH1wasexpressedfromaplasmid(pBB39).
https://doi.org/10.1371/journal.pgen.1007136.g004
RNH1,resultedinaseveregrowthdefect,wheneitherSMC6(Fig5A)orSMC5(S5AFig)func-
tionwasreduced.
Next,wetestedwhetherthedeletionofMPH1wouldrescuetheabovedescribedsynthetic
growthdefectbetweensmc5/6mutantsandrnh201.Weobservedthatgrowthdefectsofboth
smc6-9rnh201andsmc5-6rnh201cellswerealleviatedintheabsenceofMPH1(Fig5B,and
S5BFig).Inaccordancewiththesyntheticgrowthdefectoccurringasaresultofreplication
stress,weobservedthatsmc6-9rnh201culturesaccumulatedcellswitha2NDNAcontentin
comparisontobothwildtypeandtherespectivesinglemutants(S5CFig).Importantly,the
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