Single-strand annealing between inverted DNA repeats

29 Pages·2017·9.69 MB·English

RESEARCHARTICLE
Single-strand annealing between inverted
DNA repeats: Pathway choice, participating
proteins, and genome destabilizing
consequences
SreejithRamakrishnan1,2,3,ZacharyKockler1,RobertEvans2,BrandonD.Downing2¤,
AnnaMalkova1,2*
1 DepartmentofBiology,UniversityofIowa,IowaCity,IA,UnitedStatesofAmerica,2 IndianaUniversity
PurdueUniversityIndianapolis,Indianapolis,IN,UnitedStatesofAmerica,3 DepartmentofDevelopmental
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Biology,StanfordUniversitySchoolofMedicine,Stanford,CA,UnitedStatesofAmerica
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¤ Currentaddress:WashingtonUniversitySchoolofMedicine,St.Louis,MO,UnitedStatesofAmerica
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Abstract
DoublestrandDNAbreaks(DSBs)aredangerouseventsthatcanresultfromvarious
OPENACCESS
causesincludingenvironmentalassaultsorthecollapseofDNAreplication.Whiletheeffi-
Citation:RamakrishnanS,KocklerZ,EvansR,
cientandpreciserepairofDSBsisessentialforcellsurvival,faultyrepaircanleadtogenetic
DowningBD,MalkovaA(2018)Single-strand
instability,makingthechoiceofDSBrepairanimportantstep.Herewereportthatinverted
annealingbetweeninvertedDNArepeats:Pathway
choice,participatingproteins,andgenome DNArepeats(IRs)placednearaDSBcanchannelitsrepairfromanaccuratepathwaythat
destabilizingconsequences.PLoSGenet14(8): leadstogeneconversiontoinsteadabreak-inducedreplication(BIR)pathwaythatleadsto
e1007543.https://doi.org/10.1371/journal.
geneticinstabilities.TheeffectofIRsisexplainedbytheirabilitytoformunusualDNAstruc-
pgen.1007543
tureswhenpresentinssDNAthatisformedbyDSBresection.WedemonstratethatIRs
Editor:MitchMcVey,TuftsUniversity,UNITED
canformtwotypesofunusualDNAstructures,andthechoicebetweenthesestructures
STATES
dependsonthelengthofthespacerseparatingIRs.Inparticular,IRsseparatedbyalong
Received:January24,2018
(1-kb)spacerarepredominantlyinvolvedininter-molecularsingle-strandannealing(SSA)
Accepted:July6,2018 leadingtotheformationofinverteddimers;IRsseparatedbyashort(12-bp)spacerpartici-
Published:August9,2018 pateinintra-molecularSSA,leadingtotheformationoffold-back(FB)structures.Bothof
thesestructuresinterferewithanaccurateDSBrepairbygeneconversionandchannel
Copyright:©2018Ramakrishnanetal.Thisisan
openaccessarticledistributedunderthetermsof DSBrepairintoBIR,whichpromotesgenomicdestabilization.Wealsoreportthatdifferent
theCreativeCommonsAttributionLicense,which proteincomplexesparticipateintheprocessingofFBscontainingshort(12-bp)versuslong
permitsunrestricteduse,distribution,and
(1-kb)ssDNAloops.Specifically,FBswithshortloopsareprocessedbytheMRX-Sae2
reproductioninanymedium,providedtheoriginal
complex,whereastheRad1-Rad10complexisresponsiblefortheprocessingoflongloops.
authorandsourcearecredited.
Overall,ourstudiesuncoverthemechanismsofgenomicdestabilizationresultingfromre-
DataAvailabilityStatement:Allthedataare
routingDSBrepairintounusualpathwaysbyIRs.GiventhehighabundanceofIRsinthe
availablewithinthepaperandSupporting
Information. humangenome,ourfindingsmaycontributetotheunderstandingofIR-mediatedgenomic
destabilizationassociatedwithhumandisease.
Funding:Thisworkwassupportedbygrantsfrom
NationalInstitutesofHealthGM084242,
GM127006,andES029306(toAM),Thefunders
hadnoroleinstudydesign,datacollectionand
analysis,decisiontopublish,orpreparationofthe
manuscript.
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. Authorsummary
Efficientandaccuraterepairofdouble-strandDNAbreaks(DSBs),resultingfromthe
exposureofcellstoionizingradiationorvariouschemicals,iscrucialforcellsurvival.
Conversely,faultyDSBrepaircangenerategenomicinstabilitythatcanleadtobirth
defectsorcancerinhumans.HerewedemonstratethatinvertedDNArepeats(IRs)placed
inthevicinityofaDSB,interferewiththeaccuraterepairofDSBsandpromotegenomic
rearrangementsandchromosomeloss.Thisresultsfromannealingbetweeninverted
repeats,locatedeitherindifferentDNAmoleculesorinthesamemolecule.Inaddition,
wedescribeanewrolefortheRad1-Rad10proteincomplexinprocessingfold-back(FB)
structuresformedbyintra-molecularannealinginvolvingIRsseparatedbylongspacers.
Incontrast,FBswithshortspacersareprocessedbytheMre11-Rad50-Xrs2/-Sae2com-
plex.Overall,wedescribeseveralpathwaysofDSBpromotedinteractionbetweenIRsthat
canleadtogenomicinstability.GiventhelargenumberofIRsinthehumangenome,our
findingsarerelevanttothemechanismsdrivinggenomicdestabilizationinhumanscon-
tributingtothedevelopmentofcancerandotherdiseases.
Introduction
Doublestrandbreaks(DSBs)inDNAresultfromtheinteractionsofDNAwithenvironmental
agentsorcellularmetabolites,andareamajorsourceofgeneticinstability(reviewedin[1,2]).
TheefficientrepairofDSBsacquiredovertimeiscriticalforcellsurvival.SincesomeDSB
repairpathwaysresultinmutationsandchromosomalrearrangementsandothersdonot
(reviewedin[3–5]),thechoiceofsaferrepairpathwaysisimportanttomaintaingenomic
integrity.
ThetwomaintypesofDSBrepairpathways,non-homologousend-joining(NHEJ)and
homologousrecombination(HR),areconservedfromyeasttohumans[2,6].NHEJoccursby
re-ligationofbrokenDNAends(Fig1A).HRproceedsviaDNA5’-3’endresectionfollowed
byrepairusingahomologousDNAsequence(e.g.,asisterchromatidorahomologouschro-
mosome)forrepair(Fig1B).HRpathwaysincludesynthesis-dependentstrandannealing
(SDSA),double-Hollidayjunction(dHJ)repair,break-inducedreplication(BIR)andsingle-
strandannealing(SSA)[1,7].BothSDSAanddHJrepaircanleadtogeneconversion(GC)
(Fig1B-i,ii).dHJcanalsoleadtoGCassociatedwithcrossing-over(CO)anditisfrequently
usedfortherepairofmeioticDSBs[2].While,SDSApredominatesinmitoticcellsandrarely
leadstocrossing-over(reviewedin[1,2]),BIRisapathwayusedinsituationswhereonlyone
brokenDNAendcanfindhomologyforrepair(reviewedin[8–10]).BIRisinitiatedbyinva-
sionofa3’single-stranded-DNA(ssDNA)endintothehomologoussequence,followedbyan
unusualtypeofDNAsynthesiscopyinglarge,oftenchromosomalsizedDNAregions(Fig1B-
iii).Insteadofareplicationfork,BIRproceedsviaamigratingDNAbubble,whichcausesfre-
quentmutationsandlargechromosomalchangesincludingdeletions,duplicationsandtrans-
locations[10–14];(reviewedin[8]).Thus,BIRismoredeleteriousthanSDSA,whichvery
rarelyleadstogenomicinstability(reviewedin[1,2]).SSAproceedsviaannealingbetween
DNArepeatsthatbecomesingle-strandedfollowingresectionofbrokenDNAends.SSA
betweendirectDNArepeatscanleadtodeletions(Fig1B-iv),whileSSAbetweeninverted
repeatspromotestheformationofunusualDNAstructuresnamelyinverted,sometimes
dicentric,chromosomedimers(ID)andfold-backs(FB)(Fig1B-v)[15–18].Sincethepotential
forgenomicdestabilizationvariesbetweenvariousDSBrepairpathways,itisimportantto
understandhowthechoicebetweenthepathwaysismadebylivingcells.WhenDSBsare
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
Fig1.DSBrepairpathways.DSBscanberepairedbyeither(A)NonHomologousEndJoining(NHEJ)thatproceedsby
ligationofbrokenendsor(B)HomologousRecombination(HR)wheretheDSBendsundergo5’-3’resection,andrepair
proceedsviaoneofthefollowingsub-pathwaysofHR:(i).SDSA.3’endinvadesandpairswithahomologousdonortemplate
producingadisplacementloop(D-loop).The3’endoftheinvadingstrandisusedasaprimertoinitiatenewDNAsynthesis.
ThesecondendoftheDSBisresectedandpairswiththenewlycopiedstrandandinitiatesasecondroundofDNAsynthesis.
(ii)DoubleHollidayjunction(dHJ)pathway;analternativegeneconversionmechanismthatsynthesizesonlyashortDNA
patchandinvolvesstrandinvasionstepfollowedbystabilizationofaD-loopvia“capturing”ofthesecondbrokenDSBend.
TworoundsofsynthesisleadtotheformationofadHJthatcanberesolvedtoproducenon-crossoverorcrossoveroutcomes.
(iii)BIR;employedwhenonlyonebrokenendisavailableforstrandinvasion,thisinvasionleadstotheinitiationofDNA
synthesisthatproceedsviamigratingbubblewithasynchronoussynthesisofleadingandlaggingstrandsandleadsto
conservativeinheritanceofnewly-synthesizedDNA.(iv)SSAbetweendirectDNArepeats.5’-3’resectioncontinuesuntil
flankinghomologoussequencesareexposedandannealedtoeachother.Theprotrudingnon-homologous3’endsareclipped
offandthe3’endsareusedasprimerstofillinthegaps.(v)SSAbetweeninvertedDNArepeats.(v-a)Inter-molecularSSA
betweenIRs.DSBsoccurintwoDNAmolecules(e.g.sisterchromatids),followedby5’-3’strandresection,leadingtoexposure
oftheIRsasssDNA,followedbyannealingbetweenIRslocatedontwodifferentDNAmolecules.Afterclippingoffthe3’-non-
homologoustails,DNAsynthesisfillsinthegapsleadingtotheformationofinverteddimersthatcanalsobedicentric.(v-b)
Intra-molecularSSAbetweenIRs.5’-3’resectionexposestheIRsasssDNAwhereintra-molecularSSAbetweenIRsoccurs.
Thisisfollowedbyclippingoffofthenon-homologoustails,DNAsynthesisandligationthatfillsinthegapsultimatelyleading
totheformationoffold-back(hairpin)structures.
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initiatedinyeastcellsinsuchawaythatbothendspossessthehomologytothehomologous
template,repairpredominantlyproceeds(inmorethan90%ofthecells)bySDSAordHJlead-
ingtogeneconversion(GC),whilerepairbyBIRisrare[19,20].ThesuppressionofBIRlead-
ingtothepredominanceofGCoutcomeswasproposedtoresultfromarecombination
executioncheckpoint(REC)thatstimulatesthechoiceofhealthierDSBrepairmechanisms
[19].Inaddition,severalfactors,includingthetypeofDNAdamage,chromatinstructure,the
extentofDSBresection,andtheageofthecellscaninfluencethechoiceofDSBrepairpathway
[21,22–24],andalsoreviewedin[1,7].Forexample,olderyeastcellsuseBIRmorefrequently
ascomparedtoyoungercells[21].Also,multipleDNAbreakscausedbygammairradiationof
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
yeast,frequentlyresultingenomicrearrangementsviaBIR[25].Suchrearrangementscould
resultfrombreaksintroducedatthepositionofrepeatedelements,aswellasitisalsopossible
thatmultiplebreakssuppressREC,andthuspromotetherepairofindividualbreaksviaBIR.
Thepresenceofinvertedrepeats(IRs)inthevicinityofaDSBcanalsoaffectDSBrepair
[17,26,27].IRsarecharacterizedbyasymmetrythatallowsthemtoswitchbetweeninter-and
intra-strandbasepairing(reviewedin[28])toformsecondaryDNAstructures,suchascruci-
forms,hairpins,orfold-backswhichcanpromotegeneticinstabilities.Forexample,IRsare
implicatedintheformationofrearrangementsassociatedwithseveraldiseasesincludingcan-
cer,Emanuelsyndrome,andX-linkedcongenitalhypertrichosis[29–31].IRsinducegenomic
instabilitybyvariousmechanisms,includingimpedimentofDNAreplicationattheposition
ofnon-BDNAstructureformedbyIRsorbyaffectingtherepairofDNA.Forexample,we
demonstratedthatIRslocatedincistoHO-inducedDSBaffecteditsrepair[17].Inparticular,
weobservedthatDSBsthatwereintroducedbyHO-endonucleasepromotedSSAbetweenIRs
located30-kbcentromereproximaltothesebreaksontwodifferentsisterchromatids,andthis
ledtotheformationofinverteddicentricdimers(Inverteddimers,IDs)[17].Mitoticbreakage
ofthesedicentricchromosomesleadstochromosomerearrangementsincludingtransloca-
tions,deletions,andduplications.WeestablishedthatIR-mediatedinter-molecularSSA
requiresRad52,butisRad51-independent,similartoSSAinvolvingdirectDNArepeats[24].
Ourexperimentsalsosuggestedthatinter-molecularSSAcanpotentiallyre-routeDSBrepair
fromonepathwaytoanother.Specifically,weobservedthatinter-molecularSSAcansuccess-
fullycompetewithDSBrepairbyallelicBIRandre-routeitintoectopicBIR.Theproblem,
however,wasthatallelicBIRinourprevioussystemwasaveryslowandinefficientprocess.A
muchmoreinterestingquestioniswhetheranefficientDSBrepairpathway,allelicgenecon-
version,canalsobeoutcompetedbyIR-mediatedSSA.However,thisquestioncouldnotbe
answeredusingouroriginalexperimentalsystemwhereIRswerelocated30-kbawayfromthe
DSB-site[17],asittookmorethan7hourstocompleteSSAduetotheneedtoresectover30
kbtoinitiateSSA,whereasGCtakesonly2to3-hours[20].Inaddition,itremainedunclear
whichpropertiesoftheIRsplayedaroleintheabilityoftheIRstoundergoSSAandtopro-
moterearrangements.Forexample,ithasbeenpreviouslyshownthatduringDNAreplication,
IRscanformcovalentlyclosedhairpin-cappedDNAmoleculescalledfold-backs(FBs)[15,16,
28].Inyeast,theMre11-Rad50-Xrs2(MRX)/Sae2complexcancleaveFBswithshort(12-bp)
hairpinloopsgeneratingDSBsthatcanleadtochromosomalrearrangements[16].Therepli-
cationofunprocessedFBsleadstotheformationofinverteddicentricdimerspromoting
breakage-fusion-bridgecycles[15,16]leadingtochromosomalrearrangementsincluding
DNAamplifications,deletions,andtranslocations[15,18].WhiletheformationofFBsbyIRs
duringreplicationiswell-studied[18],theformationandprocessingofFBsfollowingDSB
inductionisnotwellinvestigated.ItremainsunknownwhetherformationofFBsdependson
thestructureofIRsandhowFBscontaininglonghairpinloopsareformedandprocessed.
Also,themolecularmechanismsofIR-mediatedgenomedestabilizationremainunclear.
Weherepresentanewexperimentalsystem,withIRslocatedclose(~3-kb)totheDSBsite.
Usingthissystem,wetestedwhetherDSBscanpromoteIR-mediatedSSA,andwhetheritcan
competewithpathwaysleadingtotheformationofGC.OurdatasuggeststhatIRscanpartici-
pateinbothinter-andintra-molecularSSAleadingtotheformationofinverteddicentric
chromosome(ID)andfold-back(FB)structures,respectively.Weshowthatthechoice
betweeninter-andintra-molecularSSAisdeterminedbythelengthofthespacerseparating
theIRs.InstrainswhereIRsareseparatedbyalong1-kbspacer,inter-molecularSSAismore
efficient,whileintra-molecularSSAdominatesinstrainscontainingIRsseparatedbyashort
12-bpspacer.Wealsodemonstratethatdifferentproteincomplexesareinvolvedinthepro-
cessingofFBscontaininglongandshorthairpinloops.Specifically,FBswithshort(12-bp)
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
hairpinloopsareprocessedbytheMRX-Sae2complex,whereasFBswithlong(1-kb)loopsare
processedbytheRad1-Rad10complex.Inaddition,wedemonstratethatthepresenceofIRs
inthevicinityofDSBspromotestherepairoftheseDSBsviaBIR,whiledecreasingthefre-
quencyofGC.Takentogether,ourresultsindicatehowinvertedDNArepeatsinfluencethe
choiceandoutcomeofDSBrepairandcanresultinchromosomalrearrangements.
Results
InvertedrepeatschannelrepairofanearbyDSBintoSSApathwaybetween
sisterchromatids
OurgoalwastoexaminetheeffectofinvertedDNArepeatsontherepairofnearbyDSBs.To
accomplishthis,wegeneratedahaploidstrain(IR-1000)withanIRconsistingoftwo2-kb-
longsequencesseparatedbya1-kb-longspacerandlocated3kbcentromereproximalto
MATa,onchromosomeIII(Fig2A-i).ThisIRwasshorterthantheTyelementsthatweprevi-
ouslyusedtodescribeIR-mediatedSSA[17],butwaswithinalengthrangethatwaspreviously
usedbyvariousgroupstostudySSAgeneticsandIR-mediatedGCRs[18,25,32–34].TheIR
inthisstudywascreatedbyinsertinga2-kblongcopyofPHO87geneinaninvertedorienta-
tionnexttotheendogenousPHO87geneoriginallylocatedatthischromosomalposition.
ADSBwasintroducedatMATabytheHO-endonucleaseexpressedunderthecontrolofa
galactose-induciblepromoter[35].TofollowthekineticsofDSBrepair,samplesfromyeast
liquidculturewerecollectedatvarioustime-pointsafterDSBinduction,andrepairwasana-
lyzedusingcontour-clampedhomogeneouselectricfield(CHEF)gelelectrophoresisfollowed
bySouthernblothybridizationusingADE1-specificsequenceasaprobe(Fig2A-i,ii,Fig2B).
A450-kbDSBrepairproductwasaccumulatedbetween2and5hoursfollowingDSBinduc-
tion(Fig2B,wt).Basedonthesizeofthisrepairproduct,wehypothesizedthatitrepresentsan
inverteddimer(ID)formedbysinglestrandannealing(SSA)betweenIRslocatedondifferent
sisterchromatids,similarlytopreviouslydescribed[17](Fig2A-ii,seealsoFig1B-v-a).This
wasconfirmedbydigestinggenomicDNAwithAvrIIrestrictionenzyme,followedbygelelec-
trophoresisandSouthernblothybridizationusingaprobespecifictoRBK1gene,asequence
locatednearandcentromereproximaltotheIRs(Fig2C,probeP-1,2D).Weobservedthat
the23-kboriginalfragment(OF),presentinchromosomeIIIbeforetheinducedDSBinthe
0-hrtime-point,disappearedaftertheinductionofaDSB(Fig2C–OF,2D-wt),whereasatthe
0.5-hrtime-pointa9-kbfragmentcorrespondingtotheDSBcutfragments(CF)appeared,
andfinallyatthe6-hrtimepointa7-kbfragmentappearedconsistentwiththemolecular-size
ofID(SSArepairproduct)fragment.TheamountofIDproductdetectedat6hoursafterDSB
inductionwas76%oftheamountofbrokenchromosome-III(cut-fragment,CF)inwtcells
(Fig2D-wt,S1AFig),indicativeofahighefficiencyofthisIDpathwayresultingfrominter-
molecularSSA.Thecut-fragmentband(0.5-hrtime-pointpostDSBinduction)wasusedto
calculatetheamountsofrepairproductsbecauseitrepresentedthetotalamountofthechro-
mosomethatwasbroken,followingDSBinduction.Inaddition,thehighmolecularweightof
theoriginal-chromosomefragment(OF)at0-hrtime-pointmadetheSouthern-transferofthis
fragmentinefficientandthereforeunreliableforcalculations.Overall,ourresultsindicated
thatinter-molecularSSAleadingtotheformationofIDisthepredominantpathwayofDSB
repairinthishaploidstrain.Similarresultswereobtainedinthesamestraininthepresenceof
nocodazole,whichpreventsmitoticdivisionsandexcludesthepossibilityofdicentricforma-
tionafterreplicationofafold-backstructureresultingfromintra-molecularannealing
betweeninvertedrepeats.Aspredicted,formationofIDsoccurredmuchfasterinthestrain
containingIRsthatarelocated3kbawayfromMATacomparedtowhatweobservedinour
previousexperimentalsystemwhereIRswerelocated>30-kbawayfromtheDSBsite[17].
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
Fig2.SSAbetweenIRsseparatedby1-kbspacer.(A)ExperimentalsystemtostudySSAbetweenIRs.(i)Two2-kb
longIRsseparatedby1-kblongspacer(IR-1000)wereinsertedintoChromosomeIII(ChrIII),centromereproximal
toMATawhereDSBwasinducedbygalactose-inducibleHOendonuclease.(ii)ThestructureofInverteddicentric
dimer(ID)resultingfromSSAbetweenIRslocatedonthetwodifferentsisterchromatids,asdemonstratedinFig1v-a.
BlueboxesunderthechromosomesindicatethelocationsoftheADE1-specificprobeonChrIII.(B)DSBrepair
analysisusingCHEFgelelectrophoresisofDNAisolatedfromwt(RAD1),rad51Δ,rad52Δ,andrad1Δstrainsbefore(0
hour(Hr)),aswellas0.5,2,4,and5hoursfollowingDSBinduction.Thefull-lengthChrIII,ID,cutfragment(CF)of
ChrIIIgeneratedbyHO-inducedDSB,andintactChrIweredetectedbyhybridizationwithADE1-specificprobe,and
DNAfragmentsizesareindicatedinparenthesis.Asterisksindicatetheshiftedcut-fragmentbandsfollowingDSB
resectionasdescribedin[17,86].(C)ThestructureoforiginalAvrIIrestrictionfragments(OF)ofchromosomeIIIin
IR-1000strain(2-kb-longIRsseparatedby1-kb-longspacer)anditsderivativesfollowingDSB:CF,ID,andFB.The
locationofhybridizationprobeP-1,specifictoRBK1sequenceisindicatedbyblackbox.(D)Southernblotanalysisof
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
DSBrepairinIR-1000wtstrainanditsrad1ΔderivativefollowinghybridizationtoprobeP-1.Thepositionsand
correspondingsizesofOF,CF,IDandFBfollowingAvrIIrestrictiondigestionofDNAisolatedbefore(0-hr),aswell
as0.5and6hoursfollowingDSBinductionareindicated.ThemedianefficiencyofFBformation(%)andtherangeof
themedian[inbrackets]areindicated(seeS1FigandMaterialsandMethodsfordetails).Everygelwasrunwith
appropriatemolecular-sizemarkersthatallowedtoestimatethesizesofeverybandshownineachfigure.
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TheformationofIDsrequiresRad52,butnotRad51(Fig2B),consistentwiththecurrent
modelsforSSA[24,36].WealsoaskedwhetherIDformationrequirestheflapendonuclease
Rad1knowntobeinvolvedinSSAbetweendirectDNArepeats[37].IntheabsenceofRad1,
thelevelofIDswasreducedmorethan10-foldascomparedtoRAD1(wt)strain(Fig2B-5-hr
wtvsrad1Δ).TheanalysisofgenomicDNAdigestedwithAvrIIendonuclease(Fig2C)also
showedthattheamountofIDalsowassignificantlydecreasedinrad1Δ(3%)comparedto
RAD1(wt,76%)(Fig2D;seealsoS1AFig).Overall,weconcludethatinter-molecularSSA
betweenIRsisapredominantpathwayofDSBrepairwhentheDSBoccursnearIRs.Surpris-
ingly,theanalysisofDSBrepairinrad1Δrevealedtheformationofanadditional3.5-kbband
at6-hrtime-point(Fig2D-rad1Δ).Wehypothesizedthatthisfragmentrepresentedafold-
backhairpinmoleculeformedbyannealingofIRswithinthesameresected3’-singlestranded
DNAwiththespacerbetweenIRsformingasinglestrandedloopatthetipofthehairpin(Fig
1B-v-b),similartothosereportedpreviouslywheninvertedrepeatswereincludedinasingle-
strandedDNAregion[15,16]althoughtheroleofRad1intheaccumulationofFBswasnot
reportedbefore.
TheroleofRad1intheprocessingoffold-backstructurescontaininglong
spacers
Toconfirmthatthe3.5-kbrepairproductdetectedinIR-1000-rad1Δstrain(Fig2D)hada
structureexpectedofaFBmolecule(Fig1B-v-b),weusedacombinationofnativeanddena-
turinggelelectrophoresisofgenomicDNAdigestedwithAvrIIandSphIenzymes(Fig3Aand
3B).ThetwoAvrIIsitesflankedtheIRregion,whiletheSphIsitewaslocatedinthespacer
DNAbetweentheIRsandwasexpectedtoberefractorytocuttingbySph1followingFBfor-
mationduetosingle-strandednatureoftheloopformedbythespacerDNAbetweenIRsin
theFBmolecule(Fig1B-v-b,Fig3A-FB).Duetothesereasons,suchanFB-fragmentis
expectedtoopenintoanopen-FBmoleculewithasizethatistwicethesizeunderdenaturing
conditionscomparedtothesizeoftheFBmoleculeundernativeelectrophoresisconditions.
Indeed,denaturinggelelectrophoresisshowsanew7-kbband,expectedfromthedenaturing
ofa3.5kbhairpinmolecule(Fig3Aand3B-denaturinggel).Moreover,the3.5-kbbandwas
consistentlydetectedinourexperimentsinIR-1000-rad1Δstrainat6-hrtime-pointfollowing
hybridizationtoeithertheRBK1-specificprobeP-1(Fig2Cand2D-rad1Δ)orthePHO87-spe-
cificprobeP-2(Fig3Aand3B-nativegel).Further,the3.5-kbbanddidnotshowhybridization
toaprobespecifictothe3’-flapsequence,includingtheregionlocatedbetweenIRsandMATa
(S1BFig,probeP-3),whichwasexpectedtoberemovedpriortoFBformation(S1CFig).
Theseresultssuggestthatthe3.5-kbrepairproductobservedinIR-1000-rad1Δstrainhasa
structurethatisconsistentwithFBhairpinmolecule.BasedonourdataweproposethatRad1
isresponsiblefortheprocessingofFBswith1-kbloops,andwhenRad1ismissing,FBsaccu-
mulateinthecells.
AnotherpossibilitywasthattheaccumulationofFBsintheabsenceofRad1inourIR-
1000-rad1Δstrainresultedfromthechannelingofinter-molecularSSAintermediatesthatare
incapableofformingIDsintheabsenceofRad1intointra-molecularSSAgeneratingFBs.
ThispossibilitywasaddressedbyrepeatingtheexperimentincellsarrestedattheG1stageof
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
Fig3.TheroleofRad1inprocessingoffold-backswith1-kbspacers.(A)TheschematicsofAvrII/SphIdigestof
chromosomeIIIinIR-1000(OF)anditsderivatives,including:CF,ID,FB,and“open”fold-backresultingfromdenaturation
ofFB.ThelocationofprobeP-2,specifictoPHO87sequence,isindicatedbybrownbox.(B)SouthernblotanalysisofDSB
repairinrad1ΔderivativeofIR-1000strainfollowingnativeordenaturinggelelectrophoresisafterAvrII/SphIdigestof
DNAisolatedbefore(0hr),or2,5,6hoursafterDSBinduction,followedbyhybridizationwithprobeP-2.(C)FBformation
inIR-1000arrestedatG1stageofthecellcycle.DNAextractedfromRAD1orrad1Δat0-hr(priortoDSBinduction)and5
or12-hrpost-DSB-induction,digestedwithAvrIIandSphIfollowedbynativegelelectrophoresisandhybridizationwith
probeP-2.(D)SchematicsofAvrIIdigestedChrIIIanditsderivatives;OF,CF,IDandFBinIR-1000.(E)AnalysisofFBand
IDformationinIR-1000(wt)anditsderivativesbyAvrIIdigest,nativegelelectrophoresis,andhybridizationwithprobeP-1.
ThemedianefficienciesofFBformation(%)andtherangeofthemedian[inbrackets]areindicated(seeS1FigandMaterials
andMethodsfordetails).
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Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
thecellcyclewheninter-sisterSSAcannotoccur.WeaskedwhetherFBscanbedetectedin
RAD1+(wt)cellsthatarearrestedinG1stagewherethereisnosisterchromatidtoformIDs.
TheexperimentwasperformedinRAD1+andrad1ΔstrainswithIRsseparatedbya1-kb
spacerthatwerealsobar1Δ(tofacilitatecellcyclearrestatG1byα-factorpheromone)[38].
ThecomplicationforourexperimentwasthatresectionofDSBendsrequiredfortheforma-
tionofFBsisnormallydefectiveincellsarrestedatG1.Toovercomethisproblem,wemade
thecellsku70Δsimilartodescribedin([39]),andthisrestoredresectiontoanextent.Follow-
ingtheexperimentinG1-arrestedcells,wedetectedaccumulationofFBsinrad1Δ,butnotin
RAD1cells(Fig3Aand3C).Ittookabout12-hoursfollowingDSBinductiontodetectFBsin
thisrad1Δstrain.ThisslowerrateofFBformationlikelyresultedfromslowerDSBresection
inG1-arrestedcells.ThekeyresultisthateveninG1-stageofthecellcyclewherenointer-
molecularSSAcouldoccur,westilldetectedanaccumulationofFBsinrad1Δ,butnotin
RAD1cells.TheseresultssuggestedthatFBrepresentsanindependentpathwayofitsown
ratherthantheresultofchannelingfromanunsuccessfulIDformationandsupportedour
hypothesisofdirectinvolvementofRad1inprocessingofFBstructurescontaininglong
spacers.
ToconfirmthatprocessingofFBsbyRad1isnotsequence-specific,wemodifiedthecon-
structbyreplacingitsinter-IRspacerwithDNAofbacteriophagelambdathatwas1-kbor
1.5-kblong(S2Fig).Inboththesestrains,FBsweredetectedintherad1Δbackground,butnot
intheisogenicRAD1+strains.ThisisconsistentwithRad1beinginvolvedintheprocessingof
FBswithlong((cid:21)1-kb)ssDNAloopsregardlessoftheirDNAsequence.
DifferentproteincomplexesprocessFBscontaininglongandshortssDNA
loops
ThenovelroleofRad1inprocessingofFBsthatweobserveinourIR-1000-rad1Δstrainis
similartotheknownfunctionoftheMRX-Sae2complexinFBprocessing[16].However,we
didnotobserveFBsinthesae2ΔderivativeofourIR-1000strain(Fig3Dand3E-sae2Δ).This
couldbeduetothedifferenceinthepropertiesofIRsinourstrainascomparedtoIRsusedin
previouslypublishedstrains[16].Inparticular,theroleofMRX-Sae2inFBprocessingwas
previouslydescribedinstrainswhereIRswereseparatedbyshort(~12-bp)spacers,whilethe
distancebetweenIRsinourstrainis1000bp.Wethereforereducedthelengthofthespacer
betweenIRsinourstrainfrom1000bpto12bpgeneratingtheIR-12strain.Consequently,we
observedarobustaccumulationofFBsinsae2Δderivative,butnotinrad1ΔderivativeofIR-
12strainat6-hoursfollowingDSBinduction(Fig4Aand4B-sae2Δ,4B-rad1Δ).Inparticular,
at6-hrafterDSBinduction,theintensityofa3-kbFBbandobservedinIR-12-sae2Δwas86%
oftheintensityofthecutfragmentmeasuredat0.5-hr(Fig4B-sae2Δ,S1DFig),demonstrating
thattheFBisthepredominantoutcomeforIR-12.TheFBstructureofthisproductwasalso
confirmedbyacombinationofnativeanddenaturinggelelectrophoresisofgenomicDNA
digestedwithBseYIrestrictionenzyme(Fig4Cand4D).Inparticular,theFBmoleculeformed
a2.4-kbbandfollowingthenativegelelectrophoresis,butondenaturinggelelectrophoresisa
4.8kbbandwasobserved.Thisisexpectedfromthedenaturingandopeningofa2.4-kbhair-
pinshapedFBmoleculetoform4.8-kbopen-FBmolecule(Fig4C).(Pleasenotethatsome
2.4-kbbandremainedpresentin6-hrsamplefollowingdenaturinggelelectrophoresisasthe
remainingOF,CF&IDalsogenerate2.4-kbband).
WealsoobservedtheaccumulationofFBstructuresinIR-12-rad1Δsae2Δstrain(Fig4B).
However,theamountofFBsaccumulatedinIR-12-rad1Δsae2Δwas3-foldlowerascompared
totheamountofFBsaccumulatedinIR-12-RAD1sae2Δ(Fig4B,S1DFig).Thefold-back
structureoftherepairproductsaccumulatedinrad1Δsae2Δwasalsoconfirmedbya
PLOSGenetics|https://doi.org/10.1371/journal.pgen.1007543 August9,2018 9/29
Mechanismofsingle-strandannealingbetweeninvertedDNArepeats
Fig4.Inverteddimersandfold-backsinIR-12.(A)SchematicsofAvrII-digestedChrIIIinIR-12(OF)anditsderivatives:CF,ID,FB(locationof
probeP-1isindicatedbyblackbox).(B)DSBrepairinIR-12(wt,rad1Δ,sae2Δandrad1Δsae2Δ)followingAvrIIdigestandhybridizationtoprobeP-
1.Therespectivepositionsof,CF,IDandFBareindicated.ThemedianefficienciesofFBformation(%)andtherangeofthemedian[inbrackets]are
PLOSGenetics|https://doi.org/10.1371/journal.pgen.1007543 August9,2018 10/29