TONSOKU prevents the formation of large tandem duplications and restrains ATR–WEE1 checkpoint activation

Domain: biology
Published: 2026-03-27
Editorial outcome: minor_revision

Abstract
Copynumbervariation(CNV)playsafundamentalroleinmodulatingplantagronomictraitsandtumorigenesisinanimals.Whilefrequentlylinkedtoreplicationstress,themechanismsgivingrisetoCNVsarenotfullyelucidated.HerewecharacterizethemutationalconsequencesassociatedwithlosingtheconservedTONSOKU(TSK/TONSL)pathway(CAF-1-H3.1-TSK),whichisrequiredtoresolveimpairedDNAreplicationforks.UsingArabidopsisthaliana,wedemonstratethattskmutantsrapidlyaccumulatelarge,heritabletandemduplicationswithintheirgenomesthatareconsistentwithDNA
Polymeraseθ(Polθ)activity.Theseduplicationsareassociatedwithlatereplicatingheterochromatinenrichedinsourcesofreplicationstress.Wealso
showthatstochasticdevelopmentalphenotypesintskplantsaretheresultoftheDNADamageResponse(DDR),withphenotypesuppressionoccurringwhenATR–WEE1checkpointsignalingisremoved.Wethusdescribeapreviouslyuncharacterizedsourceoflargetandemduplicationsthatarerelevanttounderstandinggenomestabilityindiverseeukaryotes,andindiseasecontexts.

Introduction
CNVs,wherelargesegmentsofthegenomeareeitherlostorduplicated,areafrequentlyobservedclassofstructuralvariants.Inmanycases,they underlie key agriculturaltraits selected for during plant domestication and, in humans, they occur somatically in many cancers1–5.Inthelattercase,CNVsareconsideredkeycomponentsofintratumorheterogeneity6–8,whichcangiverisetoresistancetospecificcancertreatments.TheclassificationofCNVsthereforehasimportantbiomedicalapplicationsincategorizingtumors,guidingappropriatecoursesoftreatmentandpredictingpatientprognosis4,8,9.

Tandem duplications are a common class of CNV. When such duplicationsarisefrequentlyacrossthegenomeofcanceroustissue,itisreferredtoasatandemduplicatorphenotype,whichisfurtherclassifiedbythesizeoftheduplications5,10.Forexample,cellsdeficientinthehomologousrecombination(HR)DNArepairproteinBRCA1formtandemduplicationsthatspan~11kb(Group1),whileaccumulationsoflargerduplications(~231kb,Group2;~1.7Mb,Group3)areassociatedwithothergeneticdriversfoundinspecificcancers5.Allthreegroupsoftandemduplicationsarelinkedtotheoccurrenceofstressatreplicationforks,leadingtoDNAbreaksandtheopportunityforduplicationstoarise11.

Centraltotheresolutionofstressoccurringatstalledorcollapsedreplication forks is the widely conserved protein TONSOKU-LIKE(TONSL), which directly interacts with unmethylated histones H3.1/H3.2andH4incorporatedintonewlysynthesizedDNAbyCAF-112–16.InconcertwithitspartnerMMS22L,TONSLparticipatesintheremovalofRPAatresectedssDNAandloadingoftheHRrecombinaseRAD5112,13,17.Loss of TONSL functionality is thus expected to induce genome instability, however,whether thisis the caseis unknown. Null tonsl mutations in vertebrates are lethal at the organism level18,19, thus makingitdifficulttoassessthemutationalprofileassociatedwithlackofTONSLactivity.

In Arabidopsis thaliana (Arabidopsis), the TONSL ortholog(TONSOKU/TSK)isnotrequiredforviabilityorfertility,althoughtskmutantsexhibitmanydevelopmentalphenotypes20–24.ArabidopsisisthusauniquesystemtostudythegenomicanddevelopmentalconsequencesthatoccurwhenTSKislost.TheTSKgenehaslongbeenassociated with genome stability in Arabidopsis, as tsk mutants are highlysensitivetogenotoxicstress21,23.ThissuggeststhattskplantsareunabletoadequatelyrepairDNAdamage,whichwehypothesizedmayleadtoelevatedlevelsofmutationsoverdevelopmentaltimeand/orgenerations.

Results
LargeCNVsariseintheabsenceofTSK
To investigate the genomic stability of tsk mutants, we performed whole-genome sequencing (WGS; Illumina) on three tsk-4 mutantplants(SALK_034207)thathadbeengrownformultiplegenerations(i.e.,Gen.Xplants).Acrossthegenomesoftheseplants,wedetectedbackground levels of single-nucleotide variants (SNVs) and small insertions/deletions(indels;<50bp)(SupplementaryFig.1A).Incontrast,asubstantialnumberoflargeCNVswerereadilyapparentinthegenomesoftsk-4mutants(Fig.1A).WhilewedidnotobserveCNVsconsistentwithfixeddeletionsevents(oneexceptionbeinga699bpdeletionpresentinChromosome4ofplants#2and#3),wefound42,31and44amplificationsinthethreetsk-4Gen.Xplants(Fig.1B;SupplementaryData1).Themeancoverageleveloftheseamplificationswas 1.85× over Col-0, consistent with duplications of these regions(Supplementary Fig. 1B). These duplications range in size from lessthan1kbtonearly1.5Mb,with44.6–74.4%beinggreaterthan100kb(Fig.1B).Together,theyadd13.5–18.8MbofDNAtothegenomesofthethreetsk-4Gen.Xplants,anincreaseof6.4–7.9%overthediploidgenome size of Arabidopsis(TAIR10) (Fig. 1C).Whilesomeduplicationsweresharedbetweenthetsk-4Gen.Xplants,amajoritywereonlyobservedinasingleplant.Thissuggeststhattheduplicationsaroseindependently.

CNVsintsk-4plantsareexpectedtointerferewithtranscriptionalactivity25,26,andlikelyaccountforthepreviouslyobservedlocalclusteringofupregulatedgenesintsk-4mutants24.Toassessthis,weperformedpaired WGS and RNA-seq on three plants grown without afunctionalTSKgeneforthreegenerations.Theseplantshad22,18and27duplications,respectively.RNA-seqanalysisshowedthattranscriptabundancewasincreasedacros，theduplicatedregions,withaverageincreases of 5.2, 3.3 and 9.7 fold in the three tsk-4 plants. Overall,38–46%oftranscriptswithinduplicatedregions,andafurther12–15%oftranscriptsoutsideofduplications,hadabundances>2foldgreaterthanCol-0(Fig.1D).

CNVsintskplantsandassociatedmutantscorrespondtotandemduplications
CNVs detected using short-read WGS can represent different structuralarrangementswithinthegenome.ExaminingalignmentsattheedgesofCNVregions(>100kb)intsk-4mutantsrevealednoveljunctionsbetweentheendandthestartofduplicatedsegmentsbasedonthepresenceofchimericreadspartiallymappingtobothendsoftheCNVs.Inthethreetsk-4Gen.Xplants,88%,93%and79%oftheCNVs
were associated with these chimeric reads, respectively. This resultsuggests that the CNVs are tandem duplications, with the chimericreadsrepresentingbreakpointjunctionsbetweentheoriginalsegmentanditsduplicatedcopyintegratedintothechromosomeinthesameorientation(Fig.2A).However,cellsexperiencinggenomicinstabilityhavealsobeencommonlyreportedtocontainextrachromosomalDNA(ecDNA), whichoftentimes formcircularstructures27. ItisthereforepossiblethattheCNVsintsk-4mutantscorrespondtoecDNA,withthechimericreadsrepresentingthepointatwhichthecirclesclose.

Because short-read WGS is unable to effectively distinguishbetween tandem duplications and circular ecDNA, we performedoptical genome mapping on the pooled progeny of a previouslysequencedtsk-4(thirdgeneration)plant.Comparisonoftheresultingmapped DNA molecules and 11 CNVs observed in the parent plantrevealedlongDNAmoleculescoveringbothcopiesoftheduplicationaswellasflankingsequencesforallCNVsupto178kbinsize(6CNVs)(Fig. 2B; Supplementary Data 2). While definitive results for the
remaininglarger5CNVswerelimitedbytechnicalconstraintsoftheassay,longDNAmoleculesconsistentwithtandemduplicationswerealsofoundfortheseremainingCNVs(SupplementaryData2,3).These
resultsdemonstratethattheCNVsdetectedintheabsenceofTSKaretandemduplicationsintegratedintotheArabidopsischromosomes.

TSK is also known as MGO3, as tsk-4 plants phenotypicallyresemblemgo1andmgo2singlemutants,whichhavedisruptedmeristem organization20,28. TSK/MGO3 has been proposed to act in thesame pathway as MGO120, which encodes a type 1B topoisomerase(TOP1α)29.WGSofsecond-generationmgo1mutants(SupplementaryFig. 1C) revealed tandem duplications (35.5kb and 74.7kb in size)(Fig.2C,SupplementaryFig.1D),consistentwithasharedmechanism
involvingbothTSKandTOP1α.Asmallnumberoftandemduplicationshave also been previously reported in plants deficient in the CAF-1chaperonecomplex(≥fourth-generationmutants)26,whichloadsthe
specific histone variant that TSK binds, H3.1, onto newly replicatedchromatin15,30,31. By re-analyzing published data, we also observedduplications in the genomes of H3.1 knockdown (H3.1kd) lines andother tsk-4 plants (Fig. 2D)32,33. These results point to a previouslyunrecognized molecularpathway (i.e., CAF-1, H3.1, TSK and TOP1α)required to maintain genome stability at replication forks by preventingtandemduplications.

TSKguardsagainstsomaticandgermlineduplicationsgeneratedviatheactivityofPolθ
Thedisparityinthenumberoftandemduplicationsobservedintsk-4Gen.Xplants(Fig.1A),andthoseofthepublisheddatasets(Fig.2D),
suggeststhattsk-4plantsaccumulateCNVsovertime.Wethustestediftheduplicationsareheritable.Fromapopulationofplantssegregatingforthetsk-4mutation,weidentifiedthreeplantswheretsk-4wasfixedfor the first time (first-generation mutants). We sequenced thoseplants, along with seven descendants each, acrosstwogenerations.Consistentwithgenerationalaccumulation,thenumberofobservedduplications increased from one generation to the next (Fig. 3A).
Intriguingly, the rate at which new duplications arise appeared toincreaseacrossgenerations(Fig.3B).

Oftheduplicationsthatcouldbetrackedacrossgenerations,69%(33/48)werefirstobservedtohavenormalizedreaddepths~50%largerthanWT,consistentwithheterozygouseventsinwhichoneallelewasduplicatedandinheritedviathegermline.Additionally,25%(13/51)ofinheritanceeventsledtoafurtherdoublingofthereaddepthinthenextgeneration,consistentwiththembecominghomozygous(Supplementary Fig. 1E). These results supportthatthe duplications areinheritedinaMendelianfashion.Whenthesizeoftheduplicationsiscomparedbetweengenerations,theinheritedduplicationsshowthesamedistributionasthosenotobservedtobeinherited(Fig.3C).

In light of TSK’s established role in DNA replication, we inferredthatduplicationsmayariseconcurrentlythroughouttheplant,asDNAreplicationoccursinalltissuetypesovertheplant’slifetime.Totestthis, samples weretaken from multipletissues (i.e.,flowers, caulineleaves,rosetteleaves,androots)ofthesamesecond-generationtsk-4plant(Fig.3D).Whilethemajorityofduplicationsinagivensampleweresharedamongstthedifferenttissues,tissue-specificeventswerealso observed in all sampled tissue types (Fig. 3E, F). These resultsindicatethatduplicationsarecontinuallybeinggeneratedthroughoutthelifeoftheplant.

Tandem duplications can arise from a DNA repair pathwayincorrectlyresolvingdamagedDNA2,11.SinceTSKparticipatesintheHRDNArepairpathway12,13,15,17,wereasonedthatanotherrepairpathwaymay be acting on the damaged DNA in tsk-4 mutants. The classicalnonhomologous end-joining pathway (c-NHEJ) is the dominantmechanism forrepairingdouble-strandedDNAbreaksineukaryoticcells,withtheKuheterodimer(Ku70/Ku80)beingakeyplayer34–36.Wegenerated both tsk-4 ku70-2 and tsk-4 ku80-7 double mutants, andobservedcomparablelevelsofduplicationsbetweentheseplantsandtsk-4plantsgrownalongside(Fig.3G).Thus,c-NHEJisnotresponsibleforthetandemduplicationsintsk-4mutants.

WenexttestedtheinvolvementofPolθ,thekeycomponentofthetheta-mediatedendjoining(TMEJ)repairpathway.PolθhaslongbeenassociatedwithCAF-1andTSKonaccountofsimilardevelopmental phenotypes when mutated37, and it plays a central role inmaintaining the meristem when DNA replication is impaired38. Wecrossedtsk-4toteb-5,amutantinwhichPolθactivityislost37.Strikingly,wedidnotrecovertsk-4teb-5doublemutantsfromthiscross,evenaftergenotypingtheprogenyoftsk-4+/−teb-5mutants(Supplementary Data 4). Looking at the siliques of tsk-4+/− teb-5 plants, wefoundasubsetofseedsaborted(Fig.3H),indicatingthatteb-5issyntheticlethalwithtsk-4.

TheTMEJpathwayhasbeenshowntocausecharacteristicsmall
deletions by joining two sequences that share microhomology, orsmalltemplatedinsertions,whichoftenmatchasequenceadjacenttotheDNAbreak39.ToassesswhetherTMEJcontributestotheformationof tandem duplications, we analyzed the sequence of the chimericreadsspanningthetandemduplicationbreakpointjunctions(Fig.2A).MutationalsignaturesconsistentwithTMEJwereidentifiedin85.3%ofthe204tandemduplicationsassessed(Fig.3I).Specifically,61.8%ofbreakpointjunctionshadsmalldeletionswithmicrohomologiesattheintersection, 13.2% had insertions from which an identical templatecouldbeidentifiedinadjacentsequences,and10.3%hadaninsertionforwhichnotemplatewasimmediatelyapparent.Theseresultssuggestthatthemajorityofduplicationsintsk-4mutantsarecreatedbytheactivityofTMEJ,whichactsasabackuprepairpathwaywhenTSKactivityislost.ThisconclusionissupportedbyarecentstudyinCaenorhabditiselegans,whichdemonstratesakeyroleforTMEJincreating tandem duplications in the absence of the TSK orthologueTONSL40.

Tandemduplicationspreferentiallyariseinrepetitive,late-replicatingregionsofthegenome
Tandemduplicationsintsk-4mutantsarewidelydistributedacrossthegenome(Fig.1A).Toinvestigatetheirassociationwithknowngenomicfeatures,weassembledalargesetofindependentduplications>10kbin size(n=481;medianspan199kb;84.4%>100kb;SupplementaryData5).Thelevelofintersectionamongseduplicationsmatchedrandom expectation(Fig. 4A, B), as did their overlap with differentchromatin states (Fig. 4C). However, a trend towards depletion ineuchromatin(statesE1-11)andenrichmentinheterochromaticstates(statesH1-6)waspresent(Fig.4C).

Examinationoftheduplicationbreakends(+/-50bpofjunctionswithunduplicatedgenome;Fig.4D)showedamodest1.5%increaseinmeanGCcontent(Fig.4E),thoughnoclearpatternwasseenacrossthem(SupplementaryFig.2A).Motifenrichmentanalysisfoundtwooverrepresentedmotifs,apoly(dA:dT)tract,andonewithaguanineevery 3bp (Supplementary Fig. 2B). These motifs, which rarely co-occurred (Supplementary Fig. 2C), may reflect distinct sources ofreplication stress. Short tandem repeats such as these motifs havebeendemonstratedtocausereplicationstressviaforkslippage41–43.Wefoundthatshorttandemrepeatsareenrichedatbreakends(SupplementaryFig.2D),andthisisattributabletothesetwomotifs(SupplementaryFig.2E).

Atrendtowardsconstitutiveheterochromatinwasagainobservedatbreakends(SupplementaryFig.2F)withenrichmentalsopresentforlate-replicating chromatin (Fig. 4F)44, and chromatin modificationsassociated with constitutive heterochromatin when analyzed on anindividualbasis(SupplementaryFig.2G–M)45–47.

In cancer cells, transcriptional-replication collisions are majorcauses of replication fork collapse and large CNVs4. In tsk-4, mostduplication breakends intersectwithtranscribedregionsinprotein-codinggenes(49.9%)ortransposons(TE)(24.7%)(Fig.4G).Wedonotinterpretthisasevidenceoftranscriptional-replicationcollisions,asthehighlevelofintersectionwithprotein-codinggenesisconsistentwiththeArabidopsisgenomebeinggenedense(approximatelyonegene/4–5kb)48. Furthermore, breakends intersecting protein-codinggenesshowednobiasonaveragetowardstranscriptionalstart(TSS)ortermination(TTS)sites(SupplementaryFig.2N),aswouldbeexpectedif they stemmed from collisions with paused RNA polymerase II.Moreover,noassociationwasdetectedwithgenesize(SupplementaryFig.2O),andgeneshighlyexpressedduringSphasewerefoundtobe
depletedatbreakends(Fig.4H).Together,thesefindingssuggestthatinthesenceofTSKactivity,transcriptional-replicationcollisionsareunlikelytobeacommoncauseoftheobservedtandemduplications.

ReplicationforkcollapsecanleadtolongresectiontractswhenHRisimpaired49.Wethusextendedouranalysistotheregionsbordering the duplication breakends (+/−20kb; Fig. 4I). We found thattheseborderregionsareenrichedforsingle-nucleotidevariants,indelvariants and tandem duplications present in diverse Arabidopsisaccessions (Fig. 4J,SupplementaryFig. 3A–C)50, consistent with elevatedreplicationstressinsomeregionsofthegenome.Theseregionsalsoshoweda67%increaseonaverageinshorttandemrepeattractlength (Fig. 4K, Supplementary Fig. 3D, E). TE coverage was alsoincreasedby22%intheseregions(Fig.4L,SupplementaryFig.3F).LTRretrotransposonsareparticularlyrelevant,astheycompriseapproximately one-third of replication origins in Arabidopsis51,52. Borderregions were enriched for originsofreplication,asdefinedbyshortnascentstrandsequencing(SupplementaryFig.3G),inaTE-dependentmanner (Supplementary Fig. 3H). LTR retrotransposons also commonlyfeatureG-quadruplexes53,whicharenon-canonicalDNAstructureslinkedtobothoriginsofreplication(SupplementaryFig.3I)54,55and replication stress56–59. Border regions are enriched forG-quadruplexes present in TEs (Supplementary Fig. 3J–M). Thesefindings suggest that replication origins and G-quadruplexes withinLTRsarehotspotsofreplicationstress,whichleadstotandemduplicationsintsk-4mutants.

Liketheorigins,sitesofreplicationterminationarealsostress-prone.Inmammaliansystems,terminationsitesalignwithtopologically associated domain boundaries, which often show elevatedmutation rates60–62. While Arabidopsis chromatin lacks these structures, transcription-linked chromatin boundaries are prevalent63–65.Theseboundariesalignwithreplicationtimingedges(SupplementaryFig. 3N), and the binding of the negative transcription elongationfactorBDR1(SupplementaryFig.3O).Thisindicatesanorganizationallinkbetweentranscription,chromatinstructure,andDNAreplication.

Consistentwiththeenrichmentofbreakendsinheterochromatin,theborder regions are also depleted of protein-coding genes (Supplementary Fig. 3P), have no association with R-loops (SupplementaryFig. 3Q)66, and are either depleted or unassociated with features ofchromatinstructure(Fig.4M,SupplementaryFig.3R–T).

Lastly, we observed that the border regions overlapped significantlywithT-DNAinsertionsitesinArabidopsis(Fig.4N)67,whichhavebeenhypothesizedtorelyonthepresenceofdouble-strandedDNA breaks68. Overall, while tandem duplications occur across thegenomeintheabsenceofTSK,thisanalysisshowsthatlate-replicatingregions,andheterochromaticfeatures,frequentlyco-occurwiththetandemduplications.

TheDNAdamageresponseinducesdevelopmentaldefectsintsk-4mutants
Arabidopsis tsk-4 mutants display many developmental phenotypesthat vary stochastically between individualplants (Fig. 5Ai),allconsistent with dysregulated meristematic tissue. These include nodeswithdoublesiliques(Fig.5Aiii),thickenedandsplitstems(Fig.5Aiv),fasciation(Fig.5Av),terminatedmeristems,increasedbranching,andsmallstature20–24.Wehypothesizedthattheremaybealinkbetweenthesedevelopmental phenotypes and the tandem duplicationsobservedintsk-4mutants.Toinvestigatethis,weperformedreciprocalcrossesbetweenfourth-generationtsk-4mutantsandCol-0.IntheF2generation,thetsk-4mutationandtandemduplicationsareexpectedtosegregateindependently.Asarepresentativetsk-4phenotype,wequantifiedthefrequencyofnodeswithdoublesiliques(Fig.5Aii-iii),and observed that this phenotype was mainly present in F2 plantshomozygousforthetsk-4mutation(Fig.5B).Thisresultsuggeststhatthedevelopmentalphenotypesoftsk-4plantsareassociatedwiththeabsenceofTSKratherthanthepresenceoftandemduplicationswithinthegenome.

TheinabilitytorepairdamagedDNAintheabsenceofTSKisa
sourceofcellularstress22,23.Thus,weconsideredwhetherthedevelopmental defects observed in tsk-4 mutants result from the DNAdamage response (DDR) pathway. This is a dose-dependent stressresponseprogressivelyactivatingDNArepair,cell-cyclearrestand,ifrequired,programmedcelldeath69.Cell-cycledisruptionandstemcelldeathhavepreviouslybeendescribedintsk-4mutants22,23.TheNACtranscription factor SOG1is a central componentofthe plantDDR,particularly in regulating the G2/M cell-cycle transition69–71. At thetranscriptional level, SOG1 regulates ~60% of the genes in the DDRpathway71.AnalysisofourRNA-seqdatasetsindicatedtranscriptionalmodulation of SOG1-dependent genes in tsk-4 plants, which is consistentwithDDRactivationthroughSOG1.ThisincludesupregulationofDNArepairgenes(groups1–3),anddownregulationofcell-cycleassociatedgenes(groups9–11)(Fig.5C).

TodirectlytesttheinvolvementoftheDDR,wefirstintroducedthesog1-1mutantalleleintothetsk-4backgroundandobservedthatsecond-generationdoublemutantscontinuedtogeneratetandemduplications(Fig.5D).However,thedevelopmentalphenotypesassociatedwithtsk-4mutantswerepartiallysuppressed.Specifically,thefasciation, thickened and split stems, and frequent double siliquenodeswereeliminated(Fig.5E,SupplementaryFig.4A).However,theirregular phyllotaxy, reduced bolt height, and increased branchingphenotypes were still in evidence within the populations (SupplementaryFig.4A–C).Acrossalllines,32%ofplantsresembledwildtype,and a further 26% only showed reduced stature (SupplementaryFig.4A–C).Inthesubsequentgenerationoftsk-4sog1-1mutants,themajorityofplantsexhibitedgrowthreductionandincreasedbranching(SupplementaryFig.4D).

Since TSK functions at nascent chromatin following DNAreplication12–15,wenextinterrogatedtheDDRcomponentsthatactivateasaresultofreplication-associatedgenomestress.ATRisakinasethatinitiatessignalinginresponsetopersistentsingle-strandedDNA,whichiscommonlyfoundatstalledreplicationforks69,72,73.ThistriggersCDKinhibitionthroughtheconservedcheckpointkinaseWEE1,which rapidly delays S phase progression74–76. This mechanism isreinforcedbyATRalsoactivatingtranscriptionfactors,suchasSOG1,which further increase WEE1expression77,78. Looking attsk-4doublemutants with either wee1-2 or atr-2, we observed near-complete
suppressionofalldevelopmentalphenotypespresentintsk-4singlemutants (Fig. 5F, G, SupplementaryFigs. 5,6). In these two doublemutantbackgrounds,amodestreductioningrowthwasobservedin
thethird generation (Supplementary Figs. 5E–G, 6E–G), and increasedbranching was present specifically in some lineages of tsk-4 wee1-2plants(SupplementaryFig.6E–G).However,thisphenotypewaspresenttoamuchlesserextentthaninthird-generationtsk-4sog1-1lines.
Inthethirdgenerationoftsk-4atr-2plants,71%ofplantsacrossalllinesresembledwildtype,andafurther22%showedonlyareducedstature;intsk-4wee1-2,theseproportionswere40%and40%,respectively.Inaddition,fasciationandsplitstemswerenotobservedintsk-4plantslackingWEE1orATR.Finally,weanalyzedtandemduplicationlevelsin
tsk-4 wee1-2 plants, and observed similar results as in tsk-4 sog1-1mutants,inthatinactivatingWEE1doesnotaffectthegenerationofduplications (Fig. 5D). Together,these results demonstrate thatthedevelopmentalphenotypesintsk-4plantsareaconsequenceoftheDDRpathway,andaparalleloutcomeoftheDNAdamagewhichleadstotandemduplications.

Methods
Plantmaterials
Arabidopsisseedswerestratifiedat4༠Cfor2-3daysandgerminatedon ½ MS agar plates before being transferred to soil (Pro-Mix BXMycorrhizaeGrowingMix;PremierHorticultureIncQuakertown,PA,USA). Plants were grown under cool-white fluorescent lights(~100μmolm−2s−1)inlong-dayconditions(16hlight/8hdark).Multiple SALK T-DNA lines were utilized in this work, from the Col-0background87; tsk-4 (aliases mgo3-4, bru1-4; At3g18730,SALK_034207)20, ku70-2 (At1g16970, SALK_123114)88, ku80-7(At1g48050, SALK_112921)89, teb-5 (At4g32700, SALK_018851)37, atr-2(At5g40820,SALK_032641)73andwee1-2(At1g02970,SALK_147968)74.Thesog1-1mutationisaG >R missensemutationatcodon155inGGA AGAtheLer-0background,whichhassincebeenintrogressedinCol-070,90.Themgo1CRISPRbackgroundwascreatedusingplasmids(describedbelow)transformedusingfloraldipwithAgrobacteriumtumefaciensGV3101(pMP90)91.

Plasmidconstruction
ThebinaryvectorcontainingCRISPR/Cas9reagentsusedtogeneratemgo1CRISPRwascreatedwiththemodularcloning(MoClo)toolkit92,93.AnnealedoligonucleotidescomprisingthespacersequencetargetingMGO1(At5g55300)(5‘-attgATCATCAGTTCCATCTTCTC-3‘,5‘-aaacGA-GAAGATGGAACTGATGAT-3‘)wereclonedusingtheEsp3Irestrictionenzyme into the previously described Level1_blank_sgRNAv2_Sp_AtU6_(Position 3) vector94. It was then combined into the Level 2backbone pAGM4723 with Level 1 vectors containing the followingcassettes: nos::BAR::nosT, RPS5a::zCas9i::rbcS-E995 and a pFAST-Rselectioncassette.

Plantphenotyping
Thenumberofnodeswithdoublesiliqueswasusedasarepresentativephenotypeoftskplants.Suchnodesweredefinedastwosiliquesbeing+/−2mm of each other. The fraction of such nodes per plant wasassessedin five-week-old plantsbycounting themon primaryboltsexcludingthosewithin1cmofthetopofthebolt.Stemlengthwasmeasuredincentimetersfromrosettetotip.
Thedegreeof“bushyness”intsk-4sog1-1plantswasaqualitativecategorization, with representative photos given in SupplementaryFig.4A.

Nucleicacidextractionandsequencing
GenomicDNAwasextractedfromleaftissue(unlessspecificallystatedotherwise)usingtheCTABprotocol96.DNAsequencinglibrarieswereprepared atthe Yale Center for Genome Analysis (YCGA). GenomicDNA was sonicated to an average fragment size of 350bp using aCovaris E220 instrument (Covaris, Woburn, MA) and libraries weregeneratedusingthexGenPrismlibraryprepkitforNGS(IntegratedDNATechnologies,Coralville,IA).Paired-end150bpsequencingwasperformedonanIlluminaNovaSeq6000usingtheS4XPworkflow(Illumina,SanDiego,CA).
RNA was extracted from three-week-old leaf tissue using theRNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RNA quality wasverifiedusingtheAgilent2100BioanalyzerNanoRNAAssay.Librarieswere prepared at the YCGA with 1μg of total RNA using Illumina’sTruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina). ThelibrarieswereamplifiedwitheightPCRcycles,validatedusingAgilentBioanalyzer2100HighsensitivityDNAassayandquantifiedusingtheKAPALibraryQuantificationKit(Illumina®Platforms).SequencingwasdoneonanIlluminaNovaSeq6000usingtheS4XPworkflow.

WGSanalysis
Analysis of all sequencing datasets utilized the TAIR10 genome97,98.DNAsequencingreadswereprocessedbyFastp(v0.23.2;-q20-l20)99and aligned using BWA-MEM (v0.7.17)100. Duplicate reads were
removedfromalignmentsusingSamtools(v1.16)101.AlignmentstatisticsofgenomicdatasetsproducedinthisstudyarelistedinSupplementaryData6.
LocalreadrealignmentwasthenperformedusingAbra2(v2.23)102.Analysis of single-nucleotide variants and small insertions/deletionswascarriedoutusingtheStrelka2(v2.9.10)germlinepipeline103.CNVsweredefinedusingsequencingdepthidentifiedusingGATK(v4.2.0.0/v4.4.0.0)104tosegmentthegenome.Thisfollowedtutorials#11682and#11683, omitting allele specific steps, a strategy which agreed withmanualinspections.ThispipelinealsousedPicard(v2.25.6)105toaddreadgroup(‘RG’)tags.GenomesegmentswithcoverageaboveCol-0weremerged(+/−100bp)intocontiguousamplifiedregions,whichweterm CNVs. The coverage of the segment with the highest level ofcoverage assigned as the CNV coverage. Thiswas achieved using R(v4.1.2)106, Bedtools (v2.30.0)107 and GNU Awk (v5.1.0)108. CNVs lessthan1.5xCol-0coveragewereremovedtoavoidfalsepositives.Thiswas augmented by manual inspection in the analysis of tissue-specificCNVs.
IdentificationofchimericreadsandcharacterizationofjunctionswasachievedbyfilteringSA-taggedreadsusingSamtools,andusingPysam(v0.19.0)101,109toanalyzealignments.Intheexperimentswhichtracked CNVs over generations, or between tissues, identical CNVsweredefinedasthosestartingandending+/−200bpofeachother,ThesewereidentifiedusingtheRfuzzyjoinpackage110.
TheDeeptools(v3.5.1)111andpyGenomeTracks(v3.8)112programs
wereusedtovisualizeCNVs.

Extractionofultra-highmolecularweight(uHMW)DNAandopticalmapping
Optical mapping was performed by The French Plant GenomicResources Center (CNRGV), INRAE. uHMW DNA was purified from0.8gofyoungfrozenpooledleaves(6individualplants,progenyoftsk_4_Gen3_Fam2_plant2) combining the Bionano Prep Plant TissueDNAIsolationBaseProtocol(30068;BionanoGenomics,SanDiego,CA) and a CTAB DNA purification protocol113. Briefly, frozen leavesweregroundinto2mmpiecesandthendisruptedwitharotorstatorinhomogenizationbuffercontainingspermine,spermidineand2-mercaptoethanol.Nucleiwerewashedinhomogenizationbuffer,TCbuffer(50mMTris-HClpH7.5,75mMNaCl,6mMMgCl ,0.1mMCaCl )2 2and finally resuspended in 2ml of TC. The nuclei suspension wasmixedwithanequalvolumeof2XCTABbuffer(1.4MNaCl,100mMTris-HClpH8.0,2%CTAB,20mMEDTA,0.5%(w/v)Na S O,2%(v/v)2-2 2 5mercaptoethanol) and incubated at 50°C for 40min under gentlehomogenization.Afterincubation,thenucleiwereisolatedwithcholoform:isoamylalcohol(24:1)andprecipitatedwith0.7volumeofisopropanol.Following70%ethanolwashes,theDNAmedusawasfinallyresuspendedinTEbuffer.Aftercompleteresuspension,theDNAwasquantifiedusingtheQubitdsDNABRAssay(Invitrogen,ThermoFisherScientific,Waltham,MA).The presenceof mega basesize DNAwasvisualizedbypulsedfieldgelelectrophoresis(PFGE).
Labeling and staining of the uHMW DNA were performedaccordingtotheBionanoPrepDirectLabelandStain(DLS)protocol(CG-30553-1;BionanoGenomics).Briefly,labelingwasperformedbyincubating750nggenomicDNAwithDLE-1enzyme(recognizingthesiteCTTAAG)inthepresenceofDL-Greendye.FollowingproteinaseKdigestionandDL-Greencleanupbymembraneadsorption,theDNAbackbone was stained by mixing the labeled DNA with DNA Stainsolutionandincubatedovernightatroomtemperature.TheDLSDNAconcentration was assessed with the Qubit dsDNA HS Assay(Invitrogen).
LabeledandstainedDNAwasloadedontheSaphyrchip.LoadingofthechipandrunningoftheBionanoGenomicsSaphyrSystemwereperformedaccordingtotheSaphyrSystemUserGuide(30247;BionanoGenomics).DataprocessingwasperformedusingtheBionanoAccess and Solve v.3.8 software (https://bionano.com/software-and-data-analysis-support/).

Opticalmappinganalysis
Apseudo-referencegenomewascreatedbyinsertingtandemduplications into the TAIR10 genome, based on 11 tandem duplicationslikely to be inherited from the tsk_4_Gen3_Fam2_plant2. A correspondingBEDfiledocumentingthecoordinatesofthetandemduplications was also created alongside. A reference CMAP file for thispseudo-reference genome was constructed using the OMTools(v.1.4a)114 FastaToOM (--enzymestring CTTAAG) command. TheOMToolsDataTools(--minsize100000--minsig9)commandwasusedtofiltertherawmappingdata,whichwasthenmappedtothepseudo-reference genome using OMTools OMBlastMapper (--filtermode 1--alignmentjoinmode 1 --minscore 0.3 --closeref 500000 --closefrag50000).Moleculeswithmultiplealignmentsegmentsthatwerenon-overlappingandcolinearonboththereferenceandthemoleculeweregroupedinto“chained”moleculesandanalyzedseparately.
Molecules with alignments intersecting one of the 11 inheritedtandemduplicationswereclassifiedintothreegroups;1)thosethatspanabreakpointjunction,2)thosethatspanabreakpointjunctionand one breakend, or 3) those thatspana breakpoint junction andboth breakends. All molecules were required to have ≥2 DLE siteseithersideofajunction.

AssociationoftskCNVswithannotatedgenomicfeatures
Simulation testing was used to assess the overlap between CNVsobservedintskplants.UniqueCNVs>10kbwerecompiledandbedtoolsintersect(-wao)wasusedwithindividualfeatures.SimulatedCNVsetswereimplementedusingbedtoolsshuffletorandomizetheposition of the CNVs before again using bedtools intersect (-wao) toascertainthelevelofoverlapcontrollingforCNVsize.Thiswasrun10,000times.TheresultingoutputwasthensummarizedusingR.ThesamestrategywasimplementedtolookattheoverlapbetweenCNVsandpreviouslyannotatedfeatures,orattheoverlapbetweenfeaturesthemselves.WhenlookingatCNVbreakends,anRscriptwasusedtocreatetheBEDfileofbreakendsforeachnewsetofrandomizedCNVs.Whensummarizingpreviouslyannotatedfeatures,countswereusedfor discrete features,and number of bp covered was used for continuousfeatures.
Wereporttheprobability(Pr)thatarandomizedmeanlevelofoverlap is less than whatis observed. Values>0.975are consideredenriched, while those less than 0.025 are considered depleted. GCoverlapwascalculatedbyintersectingabedgraphfileofGCcontentat10bpresolution.ShorttandemrepeatswereannotatedusingTandemRepeatsFinder115.Thepositionofotherfeatureswascarriedoutusingpublishedanalyses,withtheexceptionofSphasegenes116,replicationtimings44 and T-DNA insertions67 for which raw data were used toreplicatethepublishedanalysis.Wetermedthe6548geneswithnormalizedcounts≥1inSphaseshootscRNA-Seq(replicatesmerged)tobeSphasegenes.
ReanalysisofChIP-seqdataassayingchromatinmodifications45–47wasalsocarriedout.SequencingreadswereprocessedbyFastp(-q20-l 20) and aligned using BWA-MEM. Duplicate reads were removedfrom alignments using Samtools. MACS2 (v2.2.9)117 was used to callpeaks with the --broad parameter used for H3K27me1, H3K9me2,H3K27me3,H2AK121ub,andH3K36me3.Allpeakswerefilteredoutforq<0.05and,ifmultipledatasetswereavailable,thosepresentin≥50%ofthedatasets.
MotifenrichmentutilizedtheXSTREMEtool118.Alistofpreviouslypublisheddatasetsutilized44–47,50,52,63,66,67,97,116,119–123arelistedinSupplementaryData7.

RNA-seqanalysis
RNA sequencing reads were processed using Fastp (-q 20 -l 20).TranscriptabundanceswerequantifiedwithSalmon(v1.4.0)124usingthe Araport11 genome annotation97. Features were filtered to havecountsofatleast10.Globaltrendsintranscriptionalperturbationwerecalculatedbytakingtheratioofabundances,normalizedtotranscriptsper million, in tsk-4 plants relative to those of Col-0 sequencedalongside.

Statisticalanalysis
Testingtheassociationoftandemduplicationswithgenomicfeaturesmade useof simulationtesting outlined above. For comparisons ofplantphenotypes,thedifferencesinmeansweretestedusingone-wayANOVA followed by Tukey’s Honestly Significant Difference posthoctests.
Wasserstein distances were computed between empirical distributionsof log2 tandem duplication size within each plant, andgenotype effects were assessed by permutation (upper-tail)ofthePERMANOVApseudo-Fstatisticwithlabelsattheplantlevel.Thep-values wereadjusted across contrasts using the Bonferroni–Holmmethod.

Reportingsummary
Further information on research design is available in the NaturePortfolioReportingSummarylinkedtothisarticle.

Dataavailability
Source data is provided with this paper sufficient to recreate allpresentedresults.Sequencingdata(DNA-seqandRNA-seqdatasets)generated for this study are available from the NCBI SRA underBioProjectIDPRJNA1332561.IndividualaccessionnumbersarelistedinSupplementaryData6.OpticalmappingdatahasbeendepositedintheEMBL-EBIENAwiththeaccessionnumberPRJEB106903.Previously published data plotted in Fig. 2D has the SRA identifiersERR3852258 and BioProject ID PRJEB3583226, SRR5818181 and BioProject ID PRJNA39389233 and SRR24877660 and BioProject IDPRJNA97893832. For the purposes of testing the association oftandem duplications with genomic features in Fig. 4 and SupplementaryFigs.2,3severaldatasetswerereanalyzed.TheseareRepli-Seq data with the BioProject ID PRJNA33054744, shoot scRNA-Seqdata with the GSA ID: CRX125603 and CRX125604, BioProject IDPRJCA003094116andT-DNAintegrationsiteswiththeBioProjectIDPRJNA39361367. Previously published ChIP-seq data for assayingchromatinmodificationsplottedinSupplementaryFig.2G–Mweresourced for H3K27me1, H3K9me2, H3K4me3, H3K36me3 with theBioProject ID PRJNA96476345, H3K27me3, H2AK121ub with BioProject ID PRJNA35187047 and H3K9Ac with BioProject IDPRJNA35312146.Sourcedataareprovidedwiththispaper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript from the Jacob’s laboratory characterizes the tsk mutant in Arabidopsis thaliana and reveals the rapid accumulation of large Tandem Duplications (TD) in this mutant, provoking in few generations an increase of more than 6% of the genome size. They demonstrate that the TD arose independently, are inherited in a mendelian fashion and possess specific signatures consistent with TMEJ.\n\nImportantly, the original data presented, as well as the reanalyze of previously published data allowed the authors to confirm the existence of a molecular pathways required to maintain genome stability at replication forks by preventing tandem duplications via the DDR, and composed of several mutants confirm CAF-1, H3.1, TSK and TOP1α.\n\nThe data presented are well described, robust and the conclusions appropriate. Ultimately, the information revealed in this paper provides important insights into understanding the emergence of these tandem duplications. The fact that they are revealed in an in-planta system does not detract from their importance, especially in a context where studying this pathway is particularly complex in mammals due to the lethality induced by mutations in the actors involved in this pathway.\n\nI only have one main comment :\nThe authors analysed whether TD accumulation was linked to specific chromatic states in Figure 3C, but they did not specifically and individually analyse the different chromatic modifications and histone variants enriched in these regions. However, their systematic analyses could reveal the presence of a specific modification. Furthermore, previous observations of induced TD boundaries (reference #23 in this MS) corresponded to the boundaries of topologically associated domains (TADs). Given that more than 200 TDs were identified in tsk-4 lines, I am curious to see whether they are enriched within TAD boundaries or in the transition between compartments A and B.", "reviewer_2": "Thomson et al. examine the role of the TONSL (plant TSK) complex in the formation of genomic tandem duplications resulting from replication stress in Arabidopsis. TSK loss increases duplication occurrence and genetically interacts with DNA Pol theta (POLQ) deficiency. Presumed checkpoint activation in response to DNA damage in tsk mutant plants leads to developmental phenotypes.\n\nTandem duplications are an interesting and important subclass of genomic alterations in cancer, agriculture, and evolution and their mechanisms are poorly understood. The current work is highly valuable as the impact of TONSL loss of function on mammalian cell genomic instability has been harder to study due to lethality. The paper is well written and clear, and figures are well drawn and easy to understand, except for minor concerns below.\n\nThe specific and substantial induction of relatively large tandem duplications in plant genomes, and their accumulation across generations and within somatic tissues, is clear and striking from WGS data. The data from other plants with alterations in related genes is supportive. In total, the most important observation in the paper – increased tandem duplication occurrence resulting from TSK loss – is solid and the strength of the paper.\n\nThe limitation is that the study is largely phenomenological with few observations that clearly reveal mechanistic insight. In this regard, I am not especially compelled by the genome enrichment analyses that are likely underpowered, the checkpoint studies address the organism-level growth defect more than the genomic events and are not terribly surprising, and even the POLQ studies don’t fully arrive at establishing the unique role of TMEJ in duplication junction formation (although that is entirely plausible and most consistent with the data). However, nothing in the paper tells us much about why TONSL and its partners lead to the unique duplication phenotype, which is the striking finding of the study, as opposed to other outcomes, what role chromatin or recombination plays, etc.\n\nMajor additional comments:\n1) It is unclear why tsk teb (polq) double mutants are synthetic lethal. Even if the observed tandem duplications are POLQ-mediated, is the lesion burden high enough to lead to lethality, especially in the face of other DSB repair pathways? Cellular studies could help explore these issues by separating cell autonomous and organismal defects.\n2) Regardless of the reason, the synthetic lethality makes it tough to fully conclude that the tandem duplications are POLQ-mediated. I very much agree with the importance and interpretation of the junction properties, especially the templated insertions. But some conclusions around POLQ are likely overstated since the double mutant phenotype does not actually show a reduction, or change in the properties, of CNVs when POLQ is absent, it shows synthetic lethality for reasons that are not established by the data provided.\n3) When performing enrichment analysis by simulation, it is important that simulated events are only allowed to occur in locations that could have been detected in short sequencing reads that fell in that location; poorly mappable regions of the genome should be excluded. Otherwise, enrichment effects can be dominated by sequencing bias (more of a problem in the plant genome? I am not expert on that point). The methods do not indicate that such effects were considered, since simulated CNVs (unlike real CNVs) were allowed to fall anywhere in the genome as it is written. A secondary strategy can examine whether total aligned reads/read coverage show an enrichment bias relative to scored genomic spans.\n4) Regarding data sufficiency, there are a relatively small number of plants per group, but in most cases this seems sufficient since tsk mutant effects are large per individual, especially after multiple mutant generations. However, the number of plants sequenced of course correlates to the number of duplication events observed. Here, 481 events is decent but not overly powerful at detecting genomic feature enrichment, unless effects are large. The enrichment analysis in Figure 3 is hampered by relatively low total event numbers leading to suggestive trends that could be made more compelling. Given the large size of the duplications, it would seem plausible to sequence more plants at low coverage (and thus similar net cost). The stated coverage in Table S4 is quite high, often exceeding 100-fold (I assume “coverage” means “fold genome coverage”). Large duplications can be detected with a lot less read density than that, more than 10-fold less (the authors could explore for themselves how much coverage they would need per plant by downsampling current data). I would welcome the accumulation of many more events to power the genomic analysis. That said, I understand it may not be the best use of resources, since genomic location is probably not the thing that drives the most critical mechanistic questions in any case.\n\nA few minor comments about figures:\n5) I like the CNV size plots stratified by sequenced plant, but the plots can be hard to interpret since the number of plotted events can be difficult to discern. I suggest putting the event number on the plots. Also, on these plots, is it possible (was it done?) to isolate duplications that arose in that individual separately from those in the parental lineage? I understand that information may not always be available in the absence of multi-generational studies.\n6) It’s a small detail, but I don’t think the authors should use the same red and green coloring for the traces and the arrows in figures like Fig. 3F. I don’t think they intend for the arrow colors to match the trace colors.\n7) I especially like Fig. 5A, which does an excellent job placing the current observations in the context of tandem duplications in human cancer.", "reviewer_3": "In this manuscript, the authors reported that the tsk mutants rapidly accumulate large, heritable tandem duplications, which may be mediated by Pol θ. They also found that mutations in WEE1 and SOG1 partially rescued the developmental defects of tsk. The possible role of TSK in the formation of tandem duplications is very interesting. But the underlying mechanism is not well explored. More evidence is needed to further support their conclusions.\n\n1. The authors concluded that the tandem duplications in the tsk mutant were attributed to Pol θ, which was supported by the mutational signatures of the tandem duplications. Since this is one of the major findings of the manuscript, the genetic evidence is needed to further support it. As the tsk-4 teb-5 double mutant was lethal, the authors may consider to use the weak teb allele teb-3 (SALK_001669) or knock down TEB using RNAi. The lethality of tsk-4 teb-5 actually suggests that TSK and TEB can function independently. Given that the authors found that c-NHEJ was not responsible for the tandem duplications in tsk-4 mutants, it is possible that the HR pathway may be involved in this process. Therefore, it is worthwhile to cross tsk-4 with HR mutants such as brca1 and rad51 and test the phenotype of the double mutant.\n2. They performed reciprocal crosses between the tsk-4 mutant and Col-0 and concluded that the developmental phenotypes of tsk-4 plants are associated with the absence of TSK rather than the presence of tandem duplications within the genome. But the evidence to support this conclusion is not strong. Based on Figure 4B, compared with TSK +/+ plants, many more TSK +/- plants showed a high percentage of nodes with double siliques (>25%), especially in tsk-4 x Col-0. The statistical insignificance of the difference between TSK +/- and TSK -/- may be due to the small sample size. It is still possible that the tandem duplications cause the developmental phenotypes. Therefore, on the one hand, the authors need to increase the sample size. On the other hand, the authors need to examine the number of tandem duplications in TSK +/+, TSK +/-, and TSK -/- in the segregating population.\n3. It was very interesting that wee1 and sog1 partially suppressed the developmental phenotypes of tsk-4. The authors mentioned that“The inability to repair damaged DNA in the absence of TSK is a source of cellular stress”(line 270). Given that WEE1 and SOG1 positively regulate DNA repair, it is likely that tsk wee1 and tsk sog1 accumulate more DNA damage, which needs to be tested. Alternatively, the phenotypes of tsk-4 are not due to DNA damage per se but due to the activation of DDR, which is suppressed when WEE1 or SOG1 is mutated. WEE1 and SOG1 are known to function downstream of ATR. Given the difference of wee1 and sog1 in suppressing tsk-4, it will be interesting to test whether atr can suppress tsk-4 better than wee1 and sog1.\n4. In the title, the authors concluded that tandem duplications are induced by replication stress. Although TSK is proposed to regulate DNA replication, whether replication stress indeed induces tandem duplications is not clear. Some unknown factors may account for the enhanced tandem duplications in tsk-4. If the authors want to conclude that tandem duplications are induced by replication stress, they may examine tandem duplications in Col-0 and tsk-4 treated by replication stress-inducing chemicals such as hydroxyurea.\n5. Statistical analysis is needed for Figures 2B, 2C, 2G, and 4D to test whether the difference is significant or not.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We want to thank the reviewers for the comments provided on our initial manuscript. A point-by-point response to all comments is provided below.\n\nWe have also added new data in the revised manuscript that was not suggested by the reviewers, but that we believe is very important to our study based on questions we received when presenting this work. These experiments were initiated last summer and provided results after the initial submission of our manuscript to Nature Communications.\n\nIn the course of this study, we came to the conclusion that the copy number variations (CNVs) we were observing in tsk-4 mutant plants were tandem duplications. This was on account of chimeric short (150bp) reads being found linking the beginning and end of the CNV indicative of a breakpoint junction. Additionally, as shown in Figure S1E, these CNVs occurred at levels 1.5-2x the genomic DNA content of the wild type, suggesting a doubling of DNA.\n\nHowever, mammalian cells experiencing various forms of genome instability have been shown to produce circular extrachromosomal DNA (ecDNA), which are structures not integrated into chromosomes. Importantly, such structures are indistinguishable from tandem duplications in high-throughput short-read sequencing data. Therefore, there was a possibility that the CNVs identified in our study could be circular ecDNA.\n\nConfirming a tandem duplication requires the identification of long DNA molecules that span the amplification itself and both junctions between amplified and non-amplified genomic DNA. To do this, we have now performed Optical Genome Mapping of a sample of pooled tsk-4 sibling plants and compared the resulting DNA molecules to 11 CNVs observed in the parent plant. We observed DNA molecules covering both copies of the duplication as well as flanking sequences for all CNVs up to 178 kb in size (6 CNVs), thus demonstrating that they are tandem duplications. While definitive results for the remaining larger CNVs were limited by the technical constraints of the assay, long DNA molecules consistent with tandem duplications were also found for all the remaining CNVs. Together, this new data confirms the conclusion that we are observing tandem duplications integrated into the chromosome of tsk-4 plants and not ecDNA. We included this data in the revised manuscript in Fig. 2.\n\nReviewer #1\nThis manuscript from the Jacob’s laboratory characterizes the tsk mutant in Arabidopsis thaliana and reveals the rapid accumulation of large Tandem Duplications (TD) in this mutant, provoking in a few generations an increase of more than 6% of the genome size. They demonstrate that the TD arose independently, are inherited in a mendelian fashion and possess specific signatures consistent with TMEJ.\n\nImportantly, the original data presented, as well as the reanalyze of previously published data allowed the authors to confirm the existence of a molecular pathways required to maintain genome stability at replication forks by preventing tandem duplications via the DDR, and composed of several mutants confirm CAF-1, H3.1, TSK and TOP1α.\n\nThe data presented are well described, robust and the conclusions appropriate. Ultimately, the information revealed in this paper provides important insights into understanding the emergence of these tandem duplications. The fact that they are revealed in an in-planta system does not detract from their importance, especially in a context where studying this pathway is particularly complex in mammals due to the lethality induced by mutations in the actors involved in this pathway.\n\nThank you for the positive comments on our work.\n\n1. The authors analysed whether TD accumulation was linked to specific chromatic states in Figure 3C, but they did not specifically and individually analyse the different chromatic modifications and histone variants enriched in these regions. However, their systematic analyses could reveal the presence of a specific modification.\n\nThe chromatin states we used were defined by Jamge et al., (2023; PMID: 37467143) who used thirteen histone variants and twelve prominent chromatin modifications as input criteria. As shown in Figure 2A of that paper (reproduced below), histone variants and chromatin modifications cluster quite strongly within the Arabidopsis genome. Thus, the chromatin states reflect the co-occurrence of these features well.\n\nWe selected seven chromatin modifications and tested their association with both breakends and the wider border regions. Consistent with our analysis at the level of chromatin states (Fig. 4B and S2F), we saw strong enrichment in two known marks of constitutive heterochromatin, H3K27me1 and H3K9me2. Marks in more open regions of the genome on the whole had no association, and many were trending towards depletion. The most extreme example was the euchromatic H3K36me3 mark, which was depleted in the border regions.\n\nWe added these new results to the revised manuscript as Fig. S2G-M.\n\n2. Furthermore, previous observations of induced TD boundaries (reference #23 in this MS) corresponded to the boundaries of topologically associated domains (TADs). Given that more than 200 TDs were identified in tsk-4 lines, I am curious to see whether they are enriched within TAD boundaries or in the transition between compartments A and B.\n\nWe were also curious about the relationship between 3D chromatin structure and the tandem duplications. However, it is important to note that Arabidopsis chromatin lacks structures of the size as those usually referred to as TADs, although they are present in other plants. Why this is the case is not well understood but it has been proposed to be related to the small size and high gene density of the Arabidopsis genome. However, smaller transcription-linked chromatin boundaries are prevalent in Arabidopsis, which we showed to be depleted in the regions bordering breakends (Fig. 4M). In addition, a recent study has demonstrated that TAD-like structures emerge in cohesion cofactor pds5 quadruple mutants. However, as shown in Fig. S3S of our revised manuscript, there is no association between these structures and the regions bordering breakends.\n\nReviewer #2\nThomson et al. examine the role of the TONSL (plant TSK) complex in the formation of genomic tandem duplications resulting from replication stress in Arabidopsis. TSK loss increases duplication occurrence and genetically interacts with DNA Pol theta (POLQ) deficiency. Presumed checkpoint activation in response to DNA damage in tsk mutant plants leads to developmental phenotypes.\n\nTandem duplications are an interesting and important subclass of genomic alterations in cancer, agriculture, and evolution and their mechanisms are poorly understood. The current work is highly valuable as the impact of TONSL loss of function on mammalian cell genomic instability has been harder to study due to lethality. The paper is well written and clear, and figures are well drawn and easy to understand, except for minor concerns below.\n\nThe specific and substantial induction of relatively large tandem duplications in plant genomes, and their accumulation across generations and within somatic tissues, is clear and striking from WGS data. The data from other plants with alterations in related genes is supportive. In total, the most important observation in the paper – increased tandem duplication occurrence resulting from TSK loss – is solid and the strength of the paper.\n\nThe limitation is that the study is largely phenomenological with few observations that clearly reveal mechanistic insight. In this regard, I am not especially compelled by the genome enrichment analyses that are likely underpowered, the checkpoint studies address the organism-level growth defect more than the genomic events and are not terribly surprising, and even the POLQ studies don’t fully arrive at establishing the unique role of TMEJ in duplication junction formation (although that is entirely plausible and most consistent with the data). However, nothing in the paper tells us much about why TONSL and its partners lead to the unique duplication phenotype, which is the striking finding of the study that begs some explanation.\n\nThank you for the constructive comments on our study. We agree that a focus of our work has been on the genomic analyses and consequences of tandem duplications (e.g., RNA-seq data), and the genomic/epigenomic features that give rise to them in tsk mutants of Arabidopsis. However, we also believe that the discovery in our study of the role of the DDR checkpoints in regulating Arabidopsis development in the absence of TSK activity is an important outcome of our work, as noted by reviewer #3.\n\nIt may be important to provide a bit more context for this result. In Arabidopsis, a few previous studies (e.g., 2023; PMID: 37393621) have suggested a role for TSK in epigenetic inheritance that would be independent from its DNA repair/genome stability function, and could account for the developmental phenotypes observed in tsk mutants. This model was always tenuous, as mutations in the key epigenetic regulators identified as interacting with TSK (e.g., the H3K9 methyltransferases SUVH5 and SUVH6) does not recapitulate the developmental phenotypes observed in tsk mutants. In addition, our lab failed to replicate the published protein-protein interaction results (e.g., 2023; PMID: 37393621) substantiating this model. In our new study, we show that the DDR checkpoint regulators WEE1 and SOG1 (and also ATR in the revised manuscript) are suppressors of the developmental phenotypes observed in tsk mutants. These results provide an explanation for how the developmental phenotypes arise, linking them to replication-associated stress and/or DNA damage that induces DDR signaling, and not an independent role for TSK in epigenetic inheritance.\n\nMajor additional comments:\n1) It is unclear why tsk teb (polq) double mutants are synthetic lethal. Even if the observed tandem duplications are POLQ-mediated, is the lesion burden high enough to lead to lethality, especially in the face of other DSB repair pathways? Cellular studies could help explore these issues by separating cell autonomous and organismal defects.\n\nUnfortunately, cell culture is uncommon in plant systems and we do not have an expertise using them in our lab (very few labs do this due to the difficulty in using and maintaining these plant cell lines). We do not know for sure why tsk-4 teb-5 double mutants are synthetic lethal in Arabidopsis, but the most parsimonious explanation is that POLQ is required to patch the DNA damage which arises in the absence of TSK, and this is an essential activity. This conclusion is consistent with the mutational signatures at the breakpoint junctions of tandem duplications in tsk mutants. Supporting our conclusions are results from a companion study currently also under revision at Nature Communications (van Schendel et al., 2026; Marcel Tijsterman’s lab) that obtained the same findings as us using the roundworm Caenorhabditis elegans as a model system. In C. elegans, they observed the same POLQ/TMEJ signature at breakpoint junctions of duplications in tsk mutants, and synthetic lethality when tsk and polq mutations are combined. However, about 20% of the progeny of tsk polq double mutant worms survive, and these worms contain a strongly reduced set of duplications, and these residual duplications lack the signature of POLQ activity. The van Schendel et al manuscript concludes that their results are supportive of an exclusive role for POLQ/TMEJ in tandem duplication formation when TSK is missing. We learned of these results after we submitted our manuscript to Nature Communications.\n\nRegarding the point about the lesion burden being high enough to cause lethality, it is interesting to note that the tandem duplication frequency per generation in Arabidopsis tsk mutants appears to be 5-10 larger than in worm tsk mutants (M. Tijsterman, personal communication), a finding consistent with higher transposon levels in the Arabidopsis genome (A. thaliana Col ecotype; Genome size: 133 Mb, 21% transposons) than in worms (C. elegans N2; Genome size: 100 Mb, 15% transposons) which we have shown in our study to correlate with tandem duplication formation. This suggests the presence of a higher lesion burden in Arabidopsis compared to worms in the absence of TSK, which may explain why we could not recover any double mutant in our plants, while 20% of worms can still survive the absence of both TSK and POLQ. In addition, TSK has been shown to be essential in mice (2019; PMID: 30773278), zebrafish (2019; PMID; 30773277), and tomato plants (cv Micro-Tom; our unpublished data), which all have genomes that are more complex (e.g., larger and with a much higher transposon load) than A. thaliana Col or C. elegans N2. Taken together, this suggests that POLQ activity can only compensate for the loss of TSK and allow survival in organisms with smaller, less complex genomes, presumably because less lesions are created.\n\n2) Regardless of the reason, the synthetic lethality makes it tough to fully conclude that the tandem duplications are POLQ-mediated. I very much agree with the importance and interpretation of the junction properties, especially the templated insertions. But some conclusions around POLQ are likely overstated since the double mutant phenotype does not actually show a reduction, or change in the properties, of CNVs when POLQ is absent, it shows synthetic lethality for reasons that are not established by the data provided.\n\nTo better reflect the results obtained in our Arabidopsis study, we amended our conclusions to say that our results are “consistent” with PolQ activity in tandem duplication formation (instead of concluding that TMEJ is responsible). We also referenced the van Schendel et al. study as supportive evidence.\n\nAbstract:\n“...accumulate large, heritable tandem duplications within their genomes via DNA Polymerase θ (Pol θ).”\nwas changed to\n“...accumulate large, heritable tandem duplications within their genomes that are consistent with DNA Polymerase θ (Pol θ) activity.\"\n\nResults:\nThese results suggest that the majority of duplications in tsk-4 mutants are created by the activity of TMEJ, which acts as a backup repair pathway when TSK activity is lost. This conclusion is supported by a recent study in Caenorhabditis elegans, which indicates a key role for TMEJ in creating tandem duplications in the absence of the TSK orthologue TONSL41.\n\n3) When performing enrichment analysis by simulation, it is important that simulated events are only allowed to occur in locations that could have been detected in short sequencing reads that fell in that location; poorly mappable regions of the genome should be excluded. Otherwise, enrichment effects can be dominated by sequencing bias (more of a problem in the plant genome? I am not expert on that point). The methods do not indicate that such effects were considered, since simulated CNVs (unlike real CNVs) were allowed to fall anywhere in the genome as it is written. A secondary strategy can examine whether total aligned reads/read coverage show an enrichment bias relative to scored genomic spans.\n\nThis is an excellent point, thank you. We welcome suggestions to improve our methodology going forward. It is true that the placement of simulated CNVs was not constrained in our original manuscript, other than to occur within the nuclear genome. However, in this case, we do not believe that variation in the mappability of specific regions of the genome significantly biased the distribution of observed CNVs, which is based on average normalized coverage across large segments of the genome.\n\nIt is correct that many plant genomes are highly repetitive with large regions of poor mappability. However, this is not true of the Arabidopsis genome which has only 26.6% repetitive elements, and is 84% uniquely mappable (defined using genmap map -K 100 -E 2). Notably, 79% of the regions with poor mappability are less than 100 bp. Since our duplications span a median of 199 kb, they can usually span regions of poor mappability. In the uncommon event of an observed breakend coinciding with a region of poor mappability, it is probable that only a partial intersection occurs given our conservative definition of breakends (+/- 50bp) and border regions (+/- 20 kb) creating only negligible effects on simulation statistics.\n\nThe rationale for excluding regions of poor mappability in CNV calling is that the precision of breakend mapping may be impaired in the observed data relative to the simulated data.\nAs can be seen in the graph to the right (left panel), the intersection of breakends (+/- 50bp) with regions of poor mappability is within the bounds of what would be expected by chance, suggesting that the precision of breakend mapping is not impaired. Consistent with this, there are negligible effects on features both depleted (middle panels) and enriched in these locations (right panels).\n\nIf the precision of breakend mapping were to be impaired, we would expect a bias against the regions of poor mappability, which are usually heterochromatic. This is because observed breakends which occur in these regions would be incorrectly mapped and thus would not be included, while simulated ones would. In contrast, we observe the opposite. We detect an enrichment in heterochromatic regions and features. This suggests there is little effect of bias impairing our interpretation of the data. Excluding regions of poor mappability make marginal increases in the power of our tests, but does not change the conclusions qualitatively.\n\n4) Regarding data sufficiency, there are a relatively small number of plants per group, but in most cases this seems sufficient since tsk mutant effects are large per individual, especially after multiple mutant generations. However, the number of plants sequenced of course correlates to the number of duplication events observed. Here, 481 events is decent but not overly powerful at detecting genomic feature enrichment, unless effects are large. The enrichment analysis in Figure 3 is hampered by relatively low total event numbers leading to suggestive trends that could be made more compelling. Given the large size of the duplications, it would seem plausible to sequence more plants at low coverage (and thus similar net cost). The stated coverage in Table S4 is quite high, often exceeding 100-fold (I assume “coverage” means “fold genome coverage”). Large duplications can be detected with a lot less read density than that, more than 10-fold less (the authors could explore for themselves how much coverage they would need per plant by downsampling current data). I would welcome the accumulation of many more events to power the genomic analysis. That said, I understand it may not be the best use of resources, since genomic location is probably not the thing that drives the most critical mechanistic questions in any case.\n\nWe agree that more power would be obtained through the collection of a greater number of duplication events, and we recognize that we can obtain informative data at lower levels of coverage. We have learned a lot about genome sequencing and the depth needed to detect large duplications in the process of this multiyear project. We anticipate that as we, and others, continue to study TSK in Arabidopsis and other organisms, more data will accumulate such that higher powered analyses could be run in the future. However, collecting more data solely for this purpose is, as suggested, probably not the best use of our limited resources at the moment.\n\nA few minor comments about figures:\n5) I like the CNV size plots stratified by sequenced plant, but the plots can be hard to interpret since the number of plotted events can be difficult to discern. I suggest putting the event number on the plots. Also, on these plots, is it possible (was it done?) to isolate duplications that arose in that individual separately from those in the parental lineage? I understand that information may not always be available in the absence of multi-generational studies.\n\nWe have now added event numbers to all the CNV size plots in the manuscript, an example is shown below. Regarding the origin of the duplications within lineages, we depicted in our multi-generational study (Fig. 3A) the number of tandem duplications inherited from the parent (i.e., numbers in brackets), from which can be inferred the number that arose in that individual (or at least after parental sampling). In Fig. 3C, we show the distribution in size of these tandem duplications, separating the ones transmitted to the next generation (in yellow) from the ones not transmitted (in red).\n\n6) It’s a small detail, but I don’t think the authors should use the same red and green coloring for the traces and the arrows in figures like Fig. 3F. I don’t think they intend for the arrow colors to match the trace colors.\n\nWe have now changed the colors of the arrows.\n\n7) I especially like Fig. 5A, which does an excellent job placing the current observations in the context of tandem duplications in human cancer.\n\nThank you.\n\nReviewer #3\nIn this manuscript, the authors reported that the tsk mutants rapidly accumulate large, heritable tandem duplications, which may be mediated by Pol θ. They also found that mutations in WEE1 and SOG1 partially rescued the developmental defects of tsk. The possible role of TSK in the formation of tandem duplications is very interesting. But the underlying mechanism is not well explored. More evidence is needed to further support their conclusions.\n\nThank you for all the comments provided on our manuscript.\n\n1. The authors concluded that the tandem duplications in the tsk mutant were attributed to Pol θ, which was supported by the mutational signatures of the tandem duplications. Since this is one of the major findings of the manuscript, the genetic evidence is needed to further support it. As the tsk-4 teb-5 double mutant was lethal, the authors may consider to use the weak teb allele teb-3 (SALK_001669) or knock down TEB using RNAi. The lethality of tsk-4 teb-5 actually suggests that TSK and TEB can function independently. Given that the authors found that c-NHEJ was not responsible for the tandem duplications in tsk-4 mutants, it is possible that the HR pathway may be involved in this process. Therefore, it is worthwhile to cross tsk-4 with HR mutants such as brca1 and rad51 and test the phenotype of the double mutant.\n\nWe do not know for sure why tsk-4 teb-5 double mutants are synthetic lethal in Arabidopsis, but the most parsimonious explanation is that POLQ is required to patch the DNA damage which arises in the absence of TSK, and this is an essential activity. This conclusion is consistent with the mutational signatures at the breakpoint junctions of tandem duplications in tsk mutants.\n\nAdditional evidence on the key role of Pol θ in duplication formation in tsk mutants is provided by a companion (but independent) manuscript also currently under revision at Nature Communications (van Schendel et al., 2026; Marcel Tijsterman’s lab) that obtained the same findings as us using the roundworm Caenorhabditis elegans as a model system. In C. elegans, they observed the same TMEJ signature at breakpoint junctions of duplications in tsk mutants, and synthetic lethality when tsk and pol θ (polq) mutations are combined. However, about 20% of the progeny of tsk polq double mutant worms survive, and these worms contain a strongly reduced set of duplications, and these residual duplications lack the signature of Pol θ activity. The van Schendel et al manuscript concludes that their results are supportive of an exclusive role for TMEJ in tandem duplication formation when TSK is missing. We learned of these results after we submitted our manuscript to Nature Communications.\n\nWe are in the process of creating tsk-4 teb-3 double mutant lines, as well as tsk-4 brca1, but this will easily take over 6 months, especially if we include data generation and analysis, which in our study involves looking at second and/or third-generation mutants (tsk-4 rad51 will be sterile, making the analysis of duplications very difficult). In addition, it is not clear if combining the hypomorphic teb-3 allele with tsk-4 will allow us to recover viable Arabidopsis plants. The tandem duplication frequency in Arabidopsis tsk mutants appears to be 5-10 larger than in worm tsk mutants, a finding consistent with higher transposon levels in Arabidopsis than in worms, which we show in our study to correlate with tandem duplication formation. It is possible that partial Pol θ activity may not be sufficient to generate viable plants in the tsk mutant background.\n\nTo better reflect the results obtained in our Arabidopsis study, we amended our conclusions to say that our results are “consistent” with PolQ activity in tandem duplication formation (instead of concluding that TMEJ is responsible), and we referenced the van Schendel et al. study as supportive evidence.\n\nAbstract:\n“...accumulate large, heritable tandem duplications within their genomes via DNA Polymerase θ (Pol θ).”\nwas changed to\n“...accumulate large, heritable tandem duplications within their genomes that are consistent with DNA Polymerase θ (Pol θ) activity.\"\n\nResults:\nThese results suggest that the majority of duplications in tsk-4 mutants are created by the activity of TMEJ, which acts as a backup repair pathway when TSK activity is lost. This conclusion is supported by a recent study in Caenorhabditis elegans, which indicates a key role for TMEJ in creating tandem duplications in the absence of the TSK orthologue TONSL41.\n\n2. They performed reciprocal crosses between the tsk-4 mutant and Col-0 and concluded that the developmental phenotypes of tsk-4 plants are associated with the absence of TSK rather than the presence of tandem duplications within the genome. But the evidence to support this conclusion is not strong. Based on Figure 4B, compared with TSK +/+ plants, many more TSK +/- plants showed a high percentage of nodes with double siliques (>25%), especially in tsk-4 x Col-0. The statistical insignificance of the difference between TSK +/- and TSK -/- may be due to the small sample size. It is still possible that the tandem duplications cause the developmental phenotypes. Therefore, on the one hand, the authors need to increase the sample size. On the other hand, the authors need to examine the number of tandem duplications in TSK +/+, TSK +/-, and TSK -/- in the segregating population.\n\nWe would like to point out that Fig. 4B represents the phenotyping of 244 plants, which is a large sample size when the data is collected by hand. Increasing the sample size will increase the statistical power to detect smaller effect sizes. Given that there is a detectable effect at this sample size, it is unlikely to change as the sample size increases.\n\nWe could examine the number of tandem duplications in the TSK +/+, TSK +/-, and TSK -/- population. However, as we showed that the tandem duplications occur throughout the genome and that they can be inherited (Fig. 3A-C), we expect no difference in the complements of tandem duplications between TSK +/+, TSK +/-, and TSK -/- genotypes.\n\nMost importantly, consistent with the conclusion that we drew from this experiment is the work of Ohno et al. (2011, PMID: 21705754), and very recently Li et al. (2025, PMID: 41406205), which report that when tsk-4 plants were transformed with a pTSK::TSK-GFP or 35S::TSK-GFP transgene, the developmental phenotypes were fully complemented. The Li et al. study even performed the same double silique assay as our study. These results using an alternative method confirm our conclusion that lack of TSK activity causes the tsk-4 phenotypes, not the presence of tandem duplications.\n\nFinally, our results demonstrating that loss of ATR (see below) or WEE1 strongly rescues the developmental phenotypes of tsk-4 also support that it is the absence of TSK which is responsible for the phenotypes (Fig. 5F-G), as we showed that inactivating WEE1 activity in tsk-4 mutants does not eliminate tandem duplications (Fig. 5D).\n\n3. It was very interesting that wee1 and sog1 partially suppressed the developmental phenotypes of tsk-4. The authors mentioned that “The inability to repair damaged DNA in the absence of TSK is a source of cellular stress” (line 270). Given that WEE1 and SOG1 positively regulate DNA repair, it is likely that tsk wee1 and tsk sog1 accumulate more DNA damage, which needs to be tested. Alternatively, the phenotypes of tsk-4 are not due to DNA damage per se but due to the activation of DDR, which is suppressed when WEE1 or SOG1 is mutated. WEE1 and SOG1 are known to function downstream of ATR. Given the difference of wee1 and sog1 in suppressing tsk-4, it will be interesting to test whether atr can suppress tsk-4 better than wee1 and sog1.\n\nWEE1 and SOG1 both positively regulate DNA repair, so we agree that additional DNA damage may be observed in tsk-4 wee1 and tsk-4 sog1 double mutants. However, we consider it unlikely that increased DNA damage, if present, is responsible for the observed suppression of developmental phenotypes. Endogenous DNA damage levels in tsk-4 are already very high, frequently sufficient to trigger cell death (Suzuki et al, 2003, PMID: 15125773). If this existing DNA damage (i.e., tandem duplications) does not drive the developmental defects (see reply to point 2), further impairment of DNA repair would, if anything, be expected to exacerbate, rather than suppress, these phenotypes. The observed phenotypic suppression therefore argues against a simple DNA-damage–driven mechanism.\n\nWe favor the explanation that the tsk-4 developmental phenotypes primarily reflect activation of replication-associated DDR signaling. WEE1 and SOG1 function not only in DNA repair, but also as key cell-cycle checkpoint effectors, regulating cell-cycle arrest, differentiation, and stem-cell fate decisions in response to genotoxic stress (De Schutter et al., 2007; PMID: 17209125; Cools et al., 2011; PMID: 21498679; Ogita et al., 2018; PMID: 29430765). We therefore propose that loss of WEE1 or SOG1 suppresses tsk-4 phenotypes mainly by attenuating checkpoint-mediated developmental responses, rather than by altering the underlying level of DNA damage. Consistent with this interpretation, we find that tandem duplications—the primary genomic instability phenotype analyzed in this study—continue to arise in tsk-4 wee1 and tsk-4 sog1 double mutants at comparable frequency and size to those observed in tsk-4 single mutants. To clarify this point, we have added the number of tandem duplications alongside Fig. 5D in the revised manuscript.\n\nWhile interesting, we also believe that the manner in which the loss of WEE1 or SOG1 may alter the complex mutational profiles (i.e., indels, substitutions, structural variations, etc….) of the double mutants is outside the scope of this study for the practical reason of sample size (discussed in point 5 below).\n\nSince our initial submission, we finalized the generation and phenotypic analyses of tsk-4 atr double mutant lines. We found that atr mutation indeed does suppress the tsk-4 developmental phenotypes even better than wee1. These new data provide strong genetic support that replication-associated ATR-dependent DDR signaling is the primary driver of the tsk-4 developmental defects. The new results on the analysis of tsk-4 atr-2 double mutants are presented below, and in Fig. 5F and Supp. Fig. S5 in the revised manuscript.\n\n4. In the title, the authors concluded that tandem duplications are induced by replication stress. Although TSK is proposed to regulate DNA replication, whether replication stress indeed induces tandem duplications is not clear. Some unknown factors may account for the enhanced tandem duplications in tsk-4. If the authors want to conclude that tandem duplications are induced by replication stress, they may examine tandem duplications in Col-0 and tsk-4 treated by replication stress-inducing chemicals such as hydroxyurea.\n\nThis is a very good point, thank you. This is actually the project of a new graduate student in our lab, where they are trying to understand which types of replication stress increase duplications for the purpose of engineering these structural variations at high frequency in Arabidopsis and other plants. The companion study from the Tijsterman lab currently under revision at Nature Communications (van Schendel et al., 2026) indicates that exposing tsk mutant worms to irradiation can increase duplications by two fold (Marcel Tijsterman, personal communication). Because we have not yet identified the specific replication stress (or stresses) able to increase these duplications in plants, we changed the title of our manuscript to better reflect our main results.\n\n“TONSOKU prevents the formation of large tandem duplications and restrains ATR–WEE1 checkpoint activation”\n\n5. Statistical analysis is needed for Figures 2B, 2C, 2G, and 4D to test whether the difference is significant or not.\n\nFigure 3B (previously 2B). We have added statistical testing to this panel\n\nFigure 3C (previously 2C). We have performed statistical testing, and added it to the legend to retain clarity in interpreting the panel.\n\nFigures 3G and 5D (previously 2G and 4D). When assessing the incidence of tandem duplications in tsk-4 double mutants, we treat their presence or absence as the main genomic phenotype. This is because both the frequency and size of the tandem duplications within tsk-4 mutants themselves are quite variable between plants. A large shift in either the frequency, or the size of tandem duplications in a double mutant would therefore be required to be compelling evidence for altering the tandem duplicator phenotype (Fig. 3G, previously 2G), or for it to be a plausible explanation for the phenotypic suppression (Fig. 5D, previously 4D).\n\nFollowing the suggestion of reviewer 2, we are now listing in the revised manuscript the number of tandem duplications on the figure panel where similar numbers. We have also performed PERMANOVA on pairwise 1-Wasserstein distances between per-plant TD size distributions, finding no significant differences. We have added this information to the legends.\n\nReviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\n\nThank you for taking the time to review our manuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I would like to thank the authors for deepening their analyses by showing this link with repressive histone marks (i.e. H3K27me1 and H3K9me2), and for attempting to link the location of TD borders with the 3D organisation of the genome of A. thaliana. Moreover, the confirmation of TD presence using long molecules (via optical mapping here) is definitively a plus. At this stage, the authors have responded appropriately to my questions and I have no further concerns.", "reviewer_2": "Thomson et al. revise their manuscript addressing the role of TONSL (plant TSK) in the formation of genomic tandem duplications resulting from replication stress in Arabidopsis. As before, the association between TSK and suppression of this important mutation class is clear and of great interest.\n\nInterim improvements address some of my prior comments.\n\nThe importance of the checkpoint results in the context of TONSL/TSK literature was well defended and, perhaps obviously, was not well known to this reviewer. The context strengthens the value of those presented results, including the ATR addition.\n\nLikewise, the thoughtful response regarding lesion burden in interesting. It still begs the question why another DNA repair pathway, particularly NHEJ, cannot apparently repair the same ends, related to my major criticism below regarding mechanism.\n\nMy technical concerns regarding enrichment analysis were again well addressed and make me more confident in those conclusions.\n\nThe addition of optical genome mapping supporting that the events studied are true tandem duplications and not extrachromosomal circles (ecDNA) is nice and certainly adds good information, but the outcome is not surprising since ecDNA typically has multiple copies unlike the monotonous single-copy increase seen in the current results. Were they extrachromosomal, the results would have raised more questions than they answered, among other things, the apparently reliable heritability.\n\nHowever, the main limitation remains that the study is largely phenomenological with few observations that clearly reveal mechanism. As before, nothing in the paper tells us much about why TONSL and its partners lead to the unique duplication phenotype, which is the striking finding of the study that begs some explanation.", "reviewer_3": "The manuscript was improved through revision. I have no other comments.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter