Advertisement

Replication stress at microsatellites causes DNA double-strand breaks and break-induced replication

Open AccessPublished:September 01, 2020DOI:https://doi.org/10.1074/jbc.RA120.013495
      Short tandemly repeated DNA sequences, termed microsatellites, are abundant in the human genome. These microsatellites exhibit length instability and susceptibility to DNA double-strand breaks (DSBs) due to their tendency to form stable non-B DNA structures. Replication-dependent microsatellite DSBs are linked to genome instability signatures in human developmental diseases and cancers. To probe the causes and consequences of microsatellite DSBs, we designed a dual-fluorescence reporter system to detect DSBs at expanded (CTG/CAG)n and polypurine/polypyrimidine (Pu/Py) mirror repeat structures alongside the c-myc replication origin integrated at a single ectopic chromosomal site. Restriction cleavage near the (CTG/CAG)100 microsatellite leads to homology-directed single-strand annealing between flanking AluY elements and reporter gene deletion that can be detected by flow cytometry. However, in the absence of restriction cleavage, endogenous and exogenous replication stressors induce DSBs at the (CTG/CAG)100 and Pu/Py microsatellites. DSBs map to a narrow region at the downstream edge of the (CTG)100 lagging-strand template. (CTG/CAG)n chromosome fragility is repeat length–dependent, whereas instability at the (Pu/Py) microsatellites depends on replication polarity. Strikingly, restriction-generated DSBs and replication-dependent DSBs are not repaired by the same mechanism. Knockdown of DNA damage response proteins increases (Rad18, polymerase (Pol) η, Pol κ) or decreases (Mus81) the sensitivity of the (CTG/CAG)100 microsatellites to replication stress. Replication stress and DSBs at the ectopic (CTG/CAG)100 microsatellite lead to break-induced replication and high-frequency mutagenesis at a flanking thymidine kinase gene. Our results show that non-B structure–prone microsatellites are susceptible to replication-dependent DSBs that cause genome instability.
      Approximately 3% of the human genome comprises microsatellites or short sequence repeats of 1–9 base pairs (
      • Lander E.S.
      • Linton L.M.
      • Birren B.
      • Nusbaum C.
      • Zody M.C.
      • Baldwin J.
      • Devon K.
      • Dewar K.
      • Doyle M.
      • FitzHugh W.
      • Funke R.
      • Gage D.
      • Harris K.
      • Heaford A.
      • Howland J.
      • et al.
      Initial sequencing and analysis of the human genome.
      ,
      • Subramanian S.
      • Mishra R.K.
      • Singh L.
      Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions.
      ). The structure of these ubiquitous microsatellites is dynamic and susceptible to expansions, contractions, and DNA double-strand breaks (DSBs) (
      • Subramanian S.
      • Mishra R.K.
      • Singh L.
      Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions.
      ,
      • Neil A.J.
      • Kim J.C.
      • Mirkin S.M.
      Precarious maintenance of simple DNA repeats in eukaryotes.
      ,
      • Kim J.C.
      • Mirkin S.M.
      The balancing act of DNA repeat expansions.
      ,
      • López Castel A.
      • Cleary J.D.
      • Pearson C.E.
      Repeat instability as the basis for human diseases and as a potential target for therapy.
      ). The tendency of repetitive DNAs to form a variety of non-B DNA structures (hairpin, slipped strand, G quadruplex (G4), and triplex H-DNA) has been linked to interference with DNA replication and repair and to DSBs (
      • Mirkin S.M.
      DNA structures, repeat expansions and human hereditary disorders.
      ,
      • Mirkin S.M.
      Expandable DNA repeats and human disease.
      ,
      • Voineagu I.
      • Freudenreich C.H.
      • Mirkin S.M.
      Checkpoint responses to unusual structures formed by DNA repeats.
      ,
      • Wang G.
      • Vasquez K.M.
      Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability.
      ,
      • Shastri N.
      • Tsai Y.C.
      • Hile S.
      • Jordan D.
      • Powell B.
      • Chen J.
      • Maloney D.
      • Dose M.
      • Lo Y.
      • Anastassiadis T.
      • Rivera O.
      • Kim T.
      • Shah S.
      • Borole P.
      • Asija K.
      • et al.
      Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition.
      ).
      In humans, a growing cohort of neurodegenerative diseases has been attributed to DNA instability at microsatellite DNA noncanonical structures (
      • Neil A.J.
      • Kim J.C.
      • Mirkin S.M.
      Precarious maintenance of simple DNA repeats in eukaryotes.
      ,
      • Kim J.C.
      • Mirkin S.M.
      The balancing act of DNA repeat expansions.
      ,
      • Mirkin S.M.
      Expandable DNA repeats and human disease.
      ,
      • Schmidt M.H.
      • Pearson C.E.
      Disease-associated repeat instability and mismatch repair.
      ,
      • Kumari D.
      • Hayward B.
      • Nakamura A.J.
      • Bonner W.M.
      • Usdin K.
      Evidence for chromosome fragility at the frataxin locus in Friedreich ataxia.
      ,
      • Usdin K.
      Bending the rules: unusual nucleic acid structures and disease pathology in the repeat expansion diseases.
      ,
      • Usdin K.
      • Grabczyk E.
      DNA repeat expansions and human disease.
      ,
      Genetic Modifiers of Huntington's Disease (GeM-HD) Consortium
      CAG repeat not polyglutamine length determines timing of Huntington's disease onset.
      ,
      • Zhao X.N.
      • Lokanga R.
      • Allette K.
      • Gazy I.
      • Wu D.
      • Usdin K.
      A MutSβ-dependent contribution of MutSα to repeat expansions in fragile X premutation mice?.
      ,
      • Kumari D.
      • Usdin K.
      Sustained expression of FMR1 mRNA from reactivated fragile X syndrome alleles after treatment with small molecules that prevent trimethylation of H3K27.
      ,
      • Gerhardt J.
      • Bhalla A.D.
      • Butler J.S.
      • Puckett J.W.
      • Dervan P.B.
      • Rosenwaks Z.
      • Napierala M.
      Stalled DNA replication forks at the endogenous GAA repeats drive repeat expansion in Friedreich's ataxia cells.
      ). Thus, replication fork barriers are believed to provoke fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR) (
      • Lee J.A.
      • Carvalho C.M.
      • Lupski J.R.
      A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders.
      ,
      • Cocquempot O.
      • Brault V.
      • Babinet C.
      • Herault Y.
      Fork stalling and template switching as a mechanism for polyalanine tract expansion affecting the DYC mutant of HOXD13, a new murine model of synpolydactyly.
      ,
      • Zhang F.
      • Khajavi M.
      • Connolly A.M.
      • Towne C.F.
      • Batish S.D.
      • Lupski J.R.
      The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans.
      ,
      • Koumbaris G.
      • Hatzisevastou-Loukidou H.
      • Alexandrou A.
      • Ioannides M.
      • Christodoulou C.
      • Fitzgerald T.
      • Rajan D.
      • Clayton S.
      • Kitsiou-Tzeli S.
      • Vermeesch J.R.
      • Skordis N.
      • Antoniou P.
      • Kurg A.
      • Georgiou I.
      • Carter N.P.
      • et al.
      FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.
      ,
      • Verdin H.
      • D'Haene B.
      • Beysen D.
      • Novikova Y.
      • Menten B.
      • Sante T.
      • Lapunzina P.
      • Nevado J.
      • Carvalho C.M.
      • Lupski J.R.
      • De Baere E.
      Microhomology-mediated mechanisms underlie non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory domain.
      ,
      • Hsiao M.C.
      • Piotrowski A.
      • Callens T.
      • Fu C.
      • Wimmer K.
      • Claes K.B.
      • Messiaen L.
      Decoding NF1 intragenic copy-number variations.
      ,
      • Vogt J.
      • Wernstedt A.
      • Ripperger T.
      • Pabst B.
      • Zschocke J.
      • Kratz C.
      • Wimmer K.
      PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.
      ,
      • Deshpande M.
      • Gerhardt J.
      Break-induced replication sparks CGG-repeat instability.
      ). The induction of gross chromosomal rearrangements (GCRs) due to FoSTeS/MMBIR has been implicated in the etiology of several developmental disorders, including blepharophimosis syndrome (MIM no. 110100) (
      • Verdin H.
      • D'Haene B.
      • Beysen D.
      • Novikova Y.
      • Menten B.
      • Sante T.
      • Lapunzina P.
      • Nevado J.
      • Carvalho C.M.
      • Lupski J.R.
      • De Baere E.
      Microhomology-mediated mechanisms underlie non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory domain.
      ), CHARGE syndrome (MIM no. 214800) (
      • Vatta M.
      • Niu Z.
      • Lupski J.R.
      • Putnam P.
      • Spoonamore K.G.
      • Fang P.
      • Eng C.M.
      • Willis A.S.
      Evidence for replicative mechanism in a CHD7 rearrangement in a patient with CHARGE syndrome.
      ), and Pelizaeus–Merzbacher disease (MIM no. 312080) (
      • Beck C.R.
      • Carvalho C.M.
      • Banser L.
      • Gambin T.
      • Stubbolo D.
      • Yuan B.
      • Sperle K.
      • McCahan S.M.
      • Henneke M.
      • Seeman P.
      • Garbern J.Y.
      • Hobson G.M.
      • Lupski J.R.
      Complex genomic rearrangements at the PLP1 locus include triplication and quadruplication.
      ).
      In yeast, microsatellite DNAs promote chromosome breakage (
      • Sundararajan R.
      • Gellon L.
      • Zunder R.M.
      • Freudenreich C.H.
      Double-strand break repair pathways protect against CAG/CTG repeat expansions, contractions and repeat-mediated chromosomal fragility in Saccharomyces cerevisiae.
      ,
      • Zhang H.
      • Freudenreich C.H.
      An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S. cerevisiae.
      ,
      • Freudenreich C.H.
      • Lahiri M.
      Structure-forming CAG/CTG repeat sequences are sensitive to breakage in the absence of Mrc1 checkpoint function and S-phase checkpoint signaling: implications for trinucleotide repeat expansion diseases.
      ,
      • Balakumaran B.S.
      • Freudenreich C.H.
      • Zakian V.A.
      CGG/CCG repeats exhibit orientation-dependent instability and orientation-independent fragility in Saccharomyces cerevisiae.
      ,
      • Freudenreich C.H.
      • Kantrow S.M.
      • Zakian V.A.
      Expansion and length-dependent fragility of CTG repeats in yeast.
      ,
      • Kim H.M.
      • Narayanan V.
      • Mieczkowski P.A.
      • Petes T.D.
      • Krasilnikova M.M.
      • Mirkin S.M.
      • Lobachev K.S.
      Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair.
      ,
      • Voineagu I.
      • Narayanan V.
      • Lobachev K.S.
      • Mirkin S.M.
      Replication stalling at unstable inverted repeats: interplay between DNA hairpins and fork stabilizing proteins.
      ). In this model system, break-induced replication (BIR) has been shown to generate large-scale repeat expansions and mutations at a distance, as seen in human tumors (
      • Kim J.C.
      • Harris S.T.
      • Dinter T.
      • Shah K.A.
      • Mirkin S.M.
      The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats.
      ,
      • Shah K.A.
      • Mirkin S.M.
      The hidden side of unstable DNA repeats: mutagenesis at a distance.
      ,
      • Anand R.P.
      • Tsaponina O.
      • Greenwell P.W.
      • Lee C.S.
      • Du W.
      • Petes T.D.
      • Haber J.E.
      Chromosome rearrangements via template switching between diverged repeated sequences.
      ). BIR is also a consequence of replication stress in human cells (
      • Sotiriou S.K.
      • Kamileri I.
      • Lugli N.
      • Evangelou K.
      • Da-Re C.
      • Huber F.
      • Padayachy L.
      • Tardy S.
      • Nicati N.L.
      • Barriot S.
      • Ochs F.
      • Lukas C.
      • Lukas J.
      • Gorgoulis V.G.
      • Scapozza L.
      • et al.
      Mammalian RAD52 functions in break-induced replication repair of collapsed DNA replication forks.
      ,
      • Sakofsky C.J.
      • Malkova A.
      Break induced replication in eukaryotes: mechanisms, functions, and consequences.
      ,
      • Leffak M.
      Break-induced replication links microsatellite expansion to complex genome rearrangements.
      ,
      • Carvalho C.M.B.
      • Pfundt R.
      • King D.A.
      • Lindsay S.J.
      • Zuccherato L.W.
      • Macville M.V.E.
      • Liu P.
      • Johnson D.
      • Stankiewicz P.
      • Brown C.W.
      • Shaw C.A.
      • Hurles M.E.
      • Ira G.
      • Hastings P.J.
      • Brunner H.G.
      • et al.
      Absence of heterozygosity due to template switching during replicative rearrangements.
      ,
      • Sakofsky C.J.
      • Roberts S.A.
      • Malc E.
      • Mieczkowski P.A.
      • Resnick M.A.
      • Gordenin D.A.
      • Malkova A.
      Break-induced replication is a source of mutation clusters underlying kataegis.
      ,
      • Costantino L.
      • Sotiriou S.K.
      • Rantala J.K.
      • Magin S.
      • Mladenov E.
      • Helleday T.
      • Haber J.E.
      • Iliakis G.
      • Kallioniemi O.P.
      • Halazonetis T.D.
      Break-induced replication repair of damaged forks induces genomic duplications in human cells.
      ). Indeed in humans, replication-dependent single-ended DSBs (seDSBs) leading to BIR have been proposed to be responsible for copy number variation, several forms of GCR (
      • Lee J.A.
      • Carvalho C.M.
      • Lupski J.R.
      A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders.
      ,
      • Sakofsky C.J.
      • Roberts S.A.
      • Malc E.
      • Mieczkowski P.A.
      • Resnick M.A.
      • Gordenin D.A.
      • Malkova A.
      Break-induced replication is a source of mutation clusters underlying kataegis.
      ,
      • Kramara J.
      • Osia B.
      • Malkova A.
      Break-induced replication: an unhealthy choice for stress relief?.
      ), oncogenesis (
      • Kumari D.
      • Hayward B.
      • Nakamura A.J.
      • Bonner W.M.
      • Usdin K.
      Evidence for chromosome fragility at the frataxin locus in Friedreich ataxia.
      ,
      • Usdin K.
      Bending the rules: unusual nucleic acid structures and disease pathology in the repeat expansion diseases.
      ,
      • Usdin K.
      • Grabczyk E.
      DNA repeat expansions and human disease.
      ,
      • Carvalho C.M.B.
      • Pfundt R.
      • King D.A.
      • Lindsay S.J.
      • Zuccherato L.W.
      • Macville M.V.E.
      • Liu P.
      • Johnson D.
      • Stankiewicz P.
      • Brown C.W.
      • Shaw C.A.
      • Hurles M.E.
      • Ira G.
      • Hastings P.J.
      • Brunner H.G.
      • et al.
      Absence of heterozygosity due to template switching during replicative rearrangements.
      ,
      • Zhang F.
      • Carvalho C.M.
      • Lupski J.R.
      Complex human chromosomal and genomic rearrangements.
      ,
      • Holland A.J.
      • Cleveland D.W.
      Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements.
      ,
      • Nambiar M.
      • Goldsmith G.
      • Moorthy B.T.
      • Lieber M.R.
      • Joshi M.V.
      • Choudhary B.
      • Hosur R.V.
      • Raghavan S.C.
      Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma.
      ,
      • Nambiar M.
      • Kari V.
      • Raghavan S.C.
      Chromosomal translocations in cancer.
      ), and multiple developmental disorders (
      • Vatta M.
      • Niu Z.
      • Lupski J.R.
      • Putnam P.
      • Spoonamore K.G.
      • Fang P.
      • Eng C.M.
      • Willis A.S.
      Evidence for replicative mechanism in a CHD7 rearrangement in a patient with CHARGE syndrome.
      ,
      • Beck C.R.
      • Carvalho C.M.
      • Banser L.
      • Gambin T.
      • Stubbolo D.
      • Yuan B.
      • Sperle K.
      • McCahan S.M.
      • Henneke M.
      • Seeman P.
      • Garbern J.Y.
      • Hobson G.M.
      • Lupski J.R.
      Complex genomic rearrangements at the PLP1 locus include triplication and quadruplication.
      ,
      • Holland A.J.
      • Cleveland D.W.
      Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements.
      ,
      • Nagamani S.C.
      • Zhang F.
      • Shchelochkov O.A.
      • Bi W.
      • Ou Z.
      • Scaglia F.
      • Probst F.J.
      • Shinawi M.
      • Eng C.
      • Hunter J.V.
      • Sparagana S.
      • Lagoe E.
      • Fong C.T.
      • Pearson M.
      • Doco-Fenzy M.
      • et al.
      Microdeletions including YWHAE in the Miller-Dieker syndrome region on chromosome 17p13.3 result in facial dysmorphisms, growth restriction, and cognitive impairment.
      ,
      • Pehlivan D.
      • Hullings M.
      • Carvalho C.M.
      • Gonzaga-Jauregui C.G.
      • Loy E.
      • Jackson L.G.
      • Krantz I.D.
      • Deardorff M.A.
      • Lupski J.R.
      NIPBL rearrangements in Cornelia de Lange syndrome: evidence for replicative mechanism and genotype-phenotype correlation.
      ,
      • Lupski J.R.
      Structural variation mutagenesis of the human genome: Impact on disease and evolution.
      ,
      • Carvalho C.M.
      • Lupski J.R.
      Mechanisms underlying structural variant formation in genomic disorders.
      ).
      Here, we focus on replication-dependent DSBs that occur at two types of microsatellite elements, an expanded (CTG/CAG)100 trinucleotide repeat from the 3′-UTR of the human DMPK locus and the 88-bp asymmetric polypurine/polypyrimidine (Pu/Py)88 mirror repeat from the PKD1 IVS21 locus. Much work has concentrated on (CTG/CAG) expansions in the DMPK gene, inasmuch as expansions of this microsatellite beyond ∼40 repeats promote further enlargement of the tract and genetic anticipation leading to myotonic dystrophy type 1 (DM1, chr19q13.32, MIM no. 160900) (for a recent review, see Ref.
      • Pettersson O.J.
      • Aagaard L.
      • Jensen T.G.
      • Damgaard C.K.
      Molecular mechanisms in DM1—a focus on foci.
      ). Previous work showed that expanded (CTG/CAG) tracts can form hairpin structures in vivo (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ,
      • Liu G.
      • Chen X.
      • Leffak M.
      Oligodeoxynucleotide Binding to (CTG)·(CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability.
      ), and several reports have also shown that (CTG/CAG)n microsatellite repeats contribute to DNA DSBs in bacterial, yeast, and human model systems (
      • Sundararajan R.
      • Gellon L.
      • Zunder R.M.
      • Freudenreich C.H.
      Double-strand break repair pathways protect against CAG/CTG repeat expansions, contractions and repeat-mediated chromosomal fragility in Saccharomyces cerevisiae.
      ,
      • Freudenreich C.H.
      • Kantrow S.M.
      • Zakian V.A.
      Expansion and length-dependent fragility of CTG repeats in yeast.
      ,
      • Blackwood J.K.
      • Okely E.A.
      • Zahra R.
      • Eykelenboom J.K.
      • Leach D.R.
      DNA tandem repeat instability in the Escherichia coli chromosome is stimulated by mismatch repair at an adjacent CAG·CTG trinucleotide repeat.
      ,
      • Frizzell A.
      • Nguyen J.H.
      • Petalcorin M.I.
      • Turner K.D.
      • Boulton S.J.
      • Freudenreich C.H.
      • Lahue R.S.
      RTEL1 inhibits trinucleotide repeat expansions and fragility.
      ).
      The PKD1 (Pu/Py)88 asymmetric mirror repeats have the potential to form triplex H-DNA and G quadruplex DNA. In vitro, DNA triplex structures are visible in this sequence by atomic force microscopy (
      • Tiner Sr., W.J.
      • Potaman V.N.
      • Sinden R.R.
      • Lyubchenko Y.L.
      The structure of intramolecular triplex DNA: atomic force microscopy study.
      ). Mutations in the PKD1 gene are associated with at least 85% of the cases of autosomal dominant polycystic kidney disease (ADPKD) (chr16p13.3, MIM no. 173900). In more than 100 unrelated patients with ADPKD, mutations were at least twice as frequent in the exons flanking the PKD1 (Pu/Py)88 microsatellite as in 5′ exons 1–8 (
      • Rossetti S.
      • Strmecki L.
      • Gamble V.
      • Burton S.
      • Sneddon V.
      • Peral B.
      • Roy S.
      • Bakkaloglu A.
      • Komel R.
      • Winearls C.G.
      • Harris P.C.
      Mutation analysis of the entire PKD1 gene: genetic and diagnostic implications.
      ). Surprisingly, the (Pu/Py)88 microsatellite is not detectable as a hotspot for mutation in blood samples of ADPKD patients (
      • Kozlowski P.
      • Roberts P.
      • Dabora S.
      • Franz D.
      • Bissler J.
      • Northrup H.
      • Au K.S.
      • Lazarus R.
      • Domanska-Pakiela D.
      • Kotulska K.
      • Jozwiak S.
      • Kwiatkowski D.J.
      Identification of 54 large deletions/duplications in TSC1 and TSC2 using MLPA, and genotype-phenotype correlations.
      ,
      • Kozlowski P.
      • Bissler J.
      • Pei Y.
      • Kwiatkowski D.J.
      Analysis of PKD1 for genomic deletion by multiplex ligation-dependent probe assay: absence of hot spots.
      ).
      We have shown that the PKD1 IVS21 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the (Pu/Py)88 tract elicited a polar replication fork barrier. When the repeat was in the fork-stalling orientation, the binding of replication checkpoint proteins Rad9, RPA, and ATR near the repeat and the sensitivity of cells to Chk1 inhibition suggested that the DNA damage response is activated by replication fork stalling at this microsatellite (
      • Liu G.
      • Myers S.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
      ).
      In the present work, we describe a novel system to analyze replication-dependent DNA double-strand breaks in human cells, using fluorescent marker protein genes flanking the (CTG/CAG)100 or (Pu/Py)88 microsatellites. We find that the expanded (CTG/CAG)100 tract is sensitive to breakage following exposure to multiple forms of replication stress. Under nonperturbed conditions as well as after treatment with low-dose hydroxyurea (HU), DSBs occur in a narrow region near the downstream end of the (CTG/CAG) repeats. Moreover, these breaks are not repaired by the same mechanism as a restriction enzyme–generated DSB. Replication-dependent DSBs at the ectopic (CTG/CAG)100 microsatellite result in BIR and a greatly elevated frequency of mutagenesis of the neighboring thymidine kinase gene. The (Pu/Py)88 microsatellite is also sensitive to DSBs under unperturbed conditions and is highly vulnerable to DSBs when the purine-rich strand is the lagging-strand template for replication in cells treated with a G4-stabilizing drug.
      Our results show that diverse forms of replication stress cause DSBs at microsatellite repeats prone to forming non-B DNA structures. The frequency of DSBs depends on the structure-selective Mus81 endonuclease and translesion polymerases. Invasion of the sister chromatid by the broken DNA results in complex rearrangements and a high rate of base substitutions during break-induced replication.

      Results

      A dual-fluorescence reporter system for analysis of DNA DSBs in vivo

      Double-strand breaks are the most dangerous of DNA lesions because of the potential for error-prone repair, gross chromosomal rearrangement, and loss of heterozygosity. To identify factors affecting microsatellite DSBs and repair, we developed a system in which a DSB between two chromosomal reporter genes could be detected by microscopy or flow cytometry (Fig. 1). In this system, (CTG/CAG)100 or (Pu/Py)88 microsatellites were individually integrated at a single-copy FLP recombinase target (FRT) site at chromosome 18p11.22 in HeLa cells (
      • Liu G.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Unstable spinocerebellar ataxia type 10 (ATTCT)*(AGAAT) repeats are associated with aberrant replication at the ATX10 locus and replication origin-dependent expansion at an ectopic site in human cells.
      ), bordered by the c-myc replication origin core (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ,
      • Liu G.
      • Malott M.
      • Leffak M.
      Multiple functional elements comprise a mammalian chromosomal replicator.
      ,
      • Chen X.
      • Liu G.
      • Leffak M.
      Activation of a human chromosomal replication origin by protein tethering.
      ), an I-Sce1 site, and two fluorescent protein marker genes (dTomato, eGFP) flanked by three identical AluYa5 elements (Fig. 1A) (
      • Lewis T.W.
      • Barthelemy J.R.
      • Virts E.L.
      • Kennedy F.M.
      • Gadgil R.Y.
      • Wiek C.
      • Linka R.M.
      • Zhang F.
      • Andreassen P.R.
      • Hanenberg H.
      • Leffak M.
      Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.
      ). Control cell lines were also constructed that contain the same starting construct except that the dual-fluorescence (DF)/myc cell line is missing the microsatellite sequences (Fig. 1B), and the DF cell line is additionally missing the c-myc origin core (Fig. 1C). The (CTG/CAG)23 and (CTG/CAG)100 sequences are pure (CTG/CAG) repeats (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ). The sequence of the PKD1 (Pu/Py)88 microsatellite is shown in Fig. 1D. The cell lines are named to indicate the DNA sequence of the lagging-strand template when replicated from the c-myc origin (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ,
      • Liu G.
      • Myers S.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
      ).
      Figure thumbnail gr1
      Figure 1Maps of the ectopically integrated DF cell line constructs. A, DF/myc/(non-B DNA) cells contain the 2.4-kb c-myc core replication origin and individual microsatellites prone to forming non-B DNA. (CTG)23 and (CTG)100 are 23 repeats or 100 repeats of the CTG trinucleotide, respectively, in the lagging-strand template when replicated from the c-myc origin. (Pu)88 and (Py)88 refer to the 88-bp PKD1 microsatellite with the purine-rich strand or pyrimidine-rich strand, respectively, in the lagging-strand template when replicated from the c-myc origin. dTomato and eGFP genes are flanked by three identical AluYa5 repeats. B, DF/myc cells contain the same construct as in A but are missing the microsatellite sequences. C, DF cells contain the same construct as in A but are missing the ectopic c-myc origin and the microsatellite sequences. Hyg, hygromycin phosphotransferase (Hygr) gene; Neo, neomycin phosphotransferase (Neor) gene; TK, HSV thymidine kinase gene; FRT, S. cerevisiae FLP recombinase target, allowing site-directed integration. D, the PKD1 microsatellite sequence, showing two regions of mirror repeat symmetry; E, DF/myc(CTG)100 cells untreated; F, DF/myc(CTG)100 cells treated with I-Sce1.
      The DF/myc(CTG)100 cell line (hereafter referred to as (CTG)100) expresses both the dTomato and eGFP reporter genes and fluoresces yellow (Fig. 1E). Transfection of an I-Sce1 expression vector results in double-strand DNA cleavage 25 bp downstream of the (CTG/CAG)100 microsatellite. This leads to intrachromosomal homology-directed recombination (single-strand annealing) between the second and third Alu elements (
      • Lewis T.W.
      • Barthelemy J.R.
      • Virts E.L.
      • Kennedy F.M.
      • Gadgil R.Y.
      • Wiek C.
      • Linka R.M.
      • Zhang F.
      • Andreassen P.R.
      • Hanenberg H.
      • Leffak M.
      Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.
      ), which eliminates the eGFP reporter. The half-lives of eGFP and dTomato reporter proteins are ∼24 h (
      • Li X.
      • Zhao X.
      • Fang Y.
      • Jiang X.
      • Duong T.
      • Fan C.
      • Huang C.C.
      • Kain S.R.
      Generation of destabilized green fluorescent protein as a transcription reporter.
      ,
      • Snapp E.L.
      Fluorescent proteins: a cell biologist's user guide.
      ); therefore, I-Sce1 digestion resulted in cells that appear red after allowing 4–8 days for turnover of the reporter proteins present before digestion (Fig. 1F).

      Replication-dependent DSBs are not repaired in the same way as I-Sce1 DSBs

      To quantitate these observations over the entire cell population, (CTG)100 cells were analyzed by flow cytometry. The (CTG)100 cells initially expressed both dTomato (red) and eGFP proteins and appeared in the upper right quadrant (yellow, double-positive) (Fig. 2, A and B). A small percentage of cells (<2%) had spontaneously lost either the green reporter (upper left quadrant, red cells), the red reporter (lower right quadrant, green cells), or both reporters (lower left quadrant, double-negative cells). When these cells were transfected with the I-Sce1 expression vector, more than 40% of the cells lost the green reporter (generating red cells) or both reporters (resulting in double-negative cells) by 4 days after treatment (Fig. 2C). The loss of the eGFP reporter gene is the result of intrachromosomal single-strand annealing between the second and third Alu elements, whereas the double-negative cells result from single-strand annealing between the first and third Alu elements (
      • Lewis T.W.
      • Barthelemy J.R.
      • Virts E.L.
      • Kennedy F.M.
      • Gadgil R.Y.
      • Wiek C.
      • Linka R.M.
      • Zhang F.
      • Andreassen P.R.
      • Hanenberg H.
      • Leffak M.
      Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.
      ).
      Figure thumbnail gr2
      Figure 2I-Sce1 DSBs are repaired by homologous recombination, but replication-dependent DSBs are refractory to homology-directed repair. A, key to flow cytometry results; B, untreated DF/myc(CTG)100 cells; C, DF/myc(CTG)100 cells transfected with an I-Sce1 expression plasmid; D, DF/myc(CTG)100 cells treated with low-dose hydroxyurea (0.2 mm HU). Similar results were observed after treatment of DF/myc(CTG)100 cells with aphidicolin () or hydrogen peroxide ().
      In striking contrast, approximately half of the (CTG)100 cells exposed to low-dose hydroxyurea (0.2 mm) for 96 h had lost the dTomato marker after 4 days of recovery (Fig. 2D). This HU treatment quickly arrests replication forks in S phase and induces a low level of the phosphorylated replication stress proteins γH2AX and pChk1345 (Fig. S1), consistent with previous reports that γH2AX marks stalled forks before DSBs are detectable (
      • Petermann E.
      • Orta M.L.
      • Issaeva N.
      • Schultz N.
      • Helleday T.
      Hydroxyurea-stalled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair.
      ).
      DSBs were also induced in (CTG)100 cells by treatment with a low dose of the replication inhibitor aphidicolin (Fig. S2), or by using H2O2 as a source of ROS and replication stress (Fig. S3). In contrast, (CTG)23 cells did not exhibit these effects. We conclude that replication stress–dependent DSBs occur between the Tomato and eGFP marker genes near the ectopic (CTG)100 site. Based on the difference in flow cytometry patterns between cells treated with I-Sce1 and HU, we conclude that replication-dependent DSBs in the ectopic (CTG)100 locus are not repaired in the same way as “clean” restriction enzyme–generated DSBs and that replication-dependent DSBs caused by the (CTG/CAG)100 repeat are refractory to the most common repair pathways of homology-directed repair and nonhomologous end joining.

      (CTG/CAG)n repeat length–dependent DSBs

      To confirm that the HU-induced DSBs were dependent on the (CTG/CAG)100 repeat, several control cell clones were constructed and tested with HU, namely DF cells missing the c-myc origin and the (CTG/CAG)100 repeat (Fig. 3, A and B), DF/myc cells missing only the (CTG/CAG)100 repeat (Fig. 3, C and D), and DF/myc cells containing a shorter (CTG/CAG)23 repeat (Fig. 3, E and F). None of the control cell populations showed a significant difference in HU-induced DSBs (p > 0.999). Thus, the ectopic site displays replication-dependent DSBs contingent on the length of the (CTG/CAG)n microsatellite.
      Figure thumbnail gr3
      Figure 3Chromosome fragility due to (CTG)100 repeats and replication stress. A, DF cells, untreated; B, DF cells treated with HU (0.2 mm hydroxyurea); C, DF/myc cells, untreated; D, DF/myc cells treated with HU; E, DF/myc(CTG)23 cells, untreated; F, DF/myc(CTG)23 cells treated with HU; G, DF/myc(CTG)100 cells treated with HU (48 h) and allowed to recover (H–J) for 4–10 days.
      In previous work in which the position of the I-Sce1 site was changed, we showed that homology-directed repair that removes either the ectopic dTomato gene or the eGFP gene is not inherently deleterious to cells (
      • Lewis T.W.
      • Barthelemy J.R.
      • Virts E.L.
      • Kennedy F.M.
      • Gadgil R.Y.
      • Wiek C.
      • Linka R.M.
      • Zhang F.
      • Andreassen P.R.
      • Hanenberg H.
      • Leffak M.
      Deficiency of the Fanconi anemia E2 ubiqitin conjugase UBE2T only partially abrogates Alu-mediated recombination in a new model of homology dependent recombination.
      ). Therefore, the loss of the dTomato gene raised the possibility that the replication-dependent DSBs that were resistant to recombination and deleted all or part of chromosome 18 containing the dTomato gene were also inimical to cell survival. To test this hypothesis, cells were allowed to recover for 4, 8, or 10 days following HU treatment (Fig. 3, G–I). The abundance of green cells in the culture suggests that an acentric fragment of chromosome 18 including the dTomato reporter gene may have been lost due to DSBs at the (CTG/CAG)100 microsatellite (see “Discussion”). The progressive loss of the green cells from the population (lower right quadrant) during the 4–10-day time course suggests that unrepaired replication-dependent DSBs had a lethal effect on these cells.

      DNA DSBs are localized downstream of the (CTG/CAG)100 microsatellite

      To determine the location of the replication-dependent DSBs, DNA was isolated from (CTG)100 cells treated with HU or I-Sce1 and subjected to linear amplification ligation–mediated PCR (lamPCR) (
      • Li T.W.
      • Weeks K.M.
      Structure-independent and quantitative ligation of single-stranded DNA.
      ,
      • Paruzynski A.
      • Arens A.
      • Gabriel R.
      • Bartholomae C.C.
      • Scholz S.
      • Wang W.
      • Wolf S.
      • Glimm H.
      • Schmidt M.
      • von Kalle C.
      Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.
      ). The lamPCR primer was complementary to the single-copy eGFP gene (Fig. 4A) and designed to hybridize to the lagging-strand template DNA and leading-strand nascent DNA relative to the c-myc origin.
      Figure thumbnail gr4
      Figure 4PCR mapping of I-Sce1 and replication-dependent DSBs in DF/myc(CTG)100 cells. A, diagram of the ectopic (CTG)100 insert showing the primer used for lamPCR (see “Experimental procedures”). Cells were transfected with an I-Sce1 expression plasmid or empty vector and incubated for 24 h before DNA isolation. Alternatively, cells were treated with 0.2 mm HU for 48 h. Genomic DNA was isolated and subjected to two rounds of lamPCR using the 5′-biotinylated lamPCR primer indicated. The biotinylated PCR products were captured on streptavidin-tagged magnetic beads and ligated to a 5′-phosphate, 3′-dideoxy adapter oligonucleotide (Circligase). Nested primers were used for exponential PCR amplification of the ligated template followed by gel electrophoresis. B, DF/myc(CTG)100 cells were treated with I-Sce1, and DSBs were mapped by lamPCR; B′, darker exposure of B showing endogenous DSB; C, DF/myc(CTG)100 cells were treated with HU, and DSBs were mapped by lamPCR. Arrows, bands indicating DSBs. Asterisks, putative extension products on unbroken leading-strand nascent DNA. These bands are not reproducible. D, DF/myc(CTG)100 cells were treated with nontargeting siControl siRNA or siRNA targeting Mus81 and analyzed by Western blotting. Flow cytometry was performed on cells treated with siControl (E), siControl plus HU (F), Mus81 siRNA (G), or Mus81 siRNA plus HU (H). Although the starting (CTG)100 culture had an increased percentage of green cells (cf. ), the effects of HU treatment and the rescue by Mus81 knockdown were reproducible in three independent experiments ().
      The I-Sce1 site 25 bp 3′ to the (CTG/CAG)100 microsatellite and ∼500 bp from the lamPCR primer was used as a landmark. As expected, when I-Sce1 was expressed in (CTG)100 cells, a major lamPCR band of ∼500 bp was observed (Fig. 4B). Lower-molecular weight bands (∼200–350 bp) were also observed in the undigested (Fig. 4B, lane 1) and I-Sce1–digested (Fig. 4B, lane 2) reactions, which we attribute to multiple leading-strand initiations near the c-myc origin (
      • Trivedi A.
      • Waltz S.E.
      • Kamath S.
      • Leffak M.
      Multiple initiations in the c-myc replication origin independent of chromosomal location.
      ,
      • Waltz S.E.
      • Trivedi A.A.
      • Leffak M.
      DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells.
      ) that are not dependent on exogenous replication stress.
      A discrete band of approximately the same size as the I-Sce1–generated lamPCR band could also be seen in a longer exposure of the PCR products from untreated cells (Fig. 4B′, lane 1). We propose that this band is due to DSBs close to the 3′ end of the (CTG/CAG)100 repeat and the I-Sce1 site that are generated during endogenous replication stress. The breadth of this band suggests that DSBs resulting during unperturbed growth are primarily the result of nuclease cleavage within a limited region near a stalled fork and not due to random torsional breakage throughout the (CTG/CAG)100 microsatellite.
      When (CTG)100 cells were treated with HU, a band of ∼500 bp again appeared (Fig. 4C), suggesting that DSBs induced by HU treatment occur at or near the sites of DSBs due to endogenous replication stress and I-Sce1 cleavage. HU treatment also suppressed the 200–350 bp bands caused by nascent strand DNA annealing to the leftward-facing lamPCR primer. We conclude that endogenous replication stress, HU-induced fork stress, and I-Sce1 cleavage all produce DSBs at or near the 3′ end of the (CTG/CAG)100 microsatellite at the ectopic site in (CTG)100 cells.

      Mus81 knockdown decreases (CTG/CAG)100 DSBs during replication stress

      We have shown that (CTG/CAG)n repeats form hairpin structures in vivo that cause replication fork stalling (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ,
      • Liu G.
      • Chen X.
      • Leffak M.
      Oligodeoxynucleotide Binding to (CTG)·(CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability.
      ,
      • Liu G.
      • Chen X.
      • Gao Y.
      • Lewis T.
      • Barthelemy J.
      • Leffak M.
      Altered replication in human cells promotes DMPK (CTG)n·(CAG)n repeat instability.
      ). Inasmuch as the Mus81 nuclease has been strongly implicated in the cleavage of stalled replication forks (
      • Mayle R.
      • Campbell I.M.
      • Beck C.R.
      • Yu Y.
      • Wilson M.
      • Shaw C.A.
      • Bjergbaek L.
      • Lupski J.R.
      • Ira G.
      DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.
      ,
      • Bétous R.
      • Glick G.G.
      • Zhao R.
      • Cortez D.
      Identification and characterization of SMARCAL1 protein complexes.
      ,
      • Dehé P.M.
      • Gaillard P.H.L.
      Control of structure-specific endonucleases to maintain genome stability.
      ,
      • Duda H.
      • Arter M.
      • Gloggnitzer J.
      • Teloni F.
      • Wild P.
      • Blanco M.G.
      • Altmeyer M.
      • Matos J.
      A mechanism for controlled breakage of under-replicated chromosomes during mitosis.
      ,
      • Franchitto A.
      • Pirzio L.M.
      • Prosperi E.
      • Sapora O.
      • Bignami M.
      • Pichierri P.
      Replication fork stalling in WRN-deficient cells is overcome by prompt activation of a MUS81-dependent pathway.
      ,
      • Fugger K.
      • Chu W.K.
      • Haahr P.
      • Kousholt A.N.
      • Beck H.
      • Payne M.J.
      • Hanada K.
      • Hickson I.D.
      • Sørensen C.S.
      FBH1 co-operates with MUS81 in inducing DNA double-strand breaks and cell death following replication stress.
      ,
      • Xing M.
      • Wang X.
      • Palmai-Pallag T.
      • Shen H.
      • Helleday T.
      • Hickson I.D.
      • Ying S.
      Acute MUS81 depletion leads to replication fork slowing and a constitutive DNA damage response.
      ,
      • Fu H.
      • Martin M.M.
      • Regairaz M.
      • Huang L.
      • You Y.
      • Lin C.M.
      • Ryan M.
      • Kim R.
      • Shimura T.
      • Pommier Y.
      • Aladjem M.I.
      The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage.
      ,
      • Pepe A.
      • West S.C.
      MUS81-EME2 promotes replication fork restart.
      ), we wished to test whether knockdown of Mus81 (Fig. 4D) would affect the (CTG/CAG)100 DSBs. In this experiment, roughly 18% of (CTG)100 cells had suffered DSBs during unperturbed clonal growth (Fig. 4E). HU treatment of these cells significantly increased the percentage of green (DSB) cells in the population to greater than 40–50% (Fig. 4F and Fig. S4, p = 0.018). Consistent with the cleavage of stalled forks by Mus81, knockdown of the nuclease reproducibly resulted in a significant decrease in the percentage of cells with endogenous DSBs (cf. Fig. 4 (E and G) and Fig. S4, p = 0.049). Additionally, Mus81 knockdown dramatically decreased the percentage of DSBs induced by HU treatment from ∼50% to 25% of cells (cf. Fig. 4 (F and H), p = 0.003). Whereas these results are consistent with reports that Mus81 is a structure-selective nuclease that cleaves stalled replication forks (
      • Pepe A.
      • West S.C.
      MUS81-EME2 promotes replication fork restart.
      ,
      • Regairaz M.
      • Zhang Y.W.
      • Fu H.
      • Agama K.K.
      • Tata N.
      • Agrawal S.
      • Aladjem M.I.
      • Pommier Y.
      Mus81-mediated DNA cleavage resolves replication forks stalled by topoisomerase I-DNA complexes.
      ,
      • Pepe A.
      • West S.C.
      Substrate specificity of the MUS81-EME2 structure selective endonuclease.
      ,
      • Lemaçon D.
      • Jackson J.
      • Quinet A.
      • Brickner J.R.
      • Li S.
      • Yazinski S.
      • You Z.
      • Ira G.
      • Zou L.
      • Mosammaparast N.
      • Vindigni A.
      MRE11 and EXO1 nucleases degrade reversed forks and elicit MUS81-dependent fork rescue in BRCA2-deficient cells.
      ,
      • Quinet A.
      • Lemaçon D.
      • Vindigni A.
      Replication fork reversal: players and guardians.
      ), and our results show that Mus81 is involved in dTomato marker loss, we note that our experiments have not shown that it is specifically the nuclease activity of Mus81 that is responsible for the DSBs.

      G quadruplex formation induces DSBs at the PKD1 polypurine/polypyrimidine microsatellite

      The polycystic kidney disease type 1 (PKD1) locus harbors a polypurine-polypyrimidine (Pu/Py)88 tract of 88 base pairs in intron 21 (
      • Blaszak R.T.
      • Potaman V.
      • Sinden R.R.
      • Bissler J.J.
      DNA structural transitions within the PKD1 gene.
      ), which is capable of forming intramolecular DNA triplex (H-DNA) and G quadruplex structures (
      • Patel H.P.
      • Lu L.
      • Blaszak R.T.
      • Bissler J.J.
      PKD1 intron 21: triplex DNA formation and effect on replication.
      ,
      • Guédin A.
      • Gros J.
      • Alberti P.
      • Mergny J.L.
      How long is too long? Effects of loop size on G-quadruplex stability.
      ). These structures have been strongly implicated in replication fork stalling and collapse (
      • Voineagu I.
      • Freudenreich C.H.
      • Mirkin S.M.
      Checkpoint responses to unusual structures formed by DNA repeats.
      ,
      • Bacolla A.
      • Tainer J.A.
      • Vasquez K.M.
      • Cooper D.N.
      Translocation and deletion breakpoints in cancer genomes are associated with potential non-B DNA-forming sequences.
      ,
      • Zhao J.
      • Bacolla A.
      • Wang G.
      • Vasquez K.M.
      Non-B DNA structure-induced genetic instability and evolution.
      ,
      • Belotserkovskii B.P.
      • De Silva E.
      • Tornaletti S.
      • Wang G.
      • Vasquez K.M.
      • Hanawalt P.C.
      A triplex-forming sequence from the human c-MYC promoter interferes with DNA transcription.
      ). Replication of the polypurine strand is blocked by non-B structure formation in vitro, and the PKD1 (Pu/Py)88 tract selectively inhibits replication when the polypurine strand is the lagging-strand template in vivo (
      • Liu G.
      • Myers S.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
      ). To determine whether G quadruplex formation would sensitize this microsatellite to DSBs, we integrated the PKD1 (Pu/Py)88 repeat at the ectopic chromosomal site, in either the (Pu)88 or (Py)88 lagging-strand orientation when replicated from the c-myc origin (Fig. 5A).
      Figure thumbnail gr5
      Figure 5The PKD1 microsatellite is broken after replication stress and G-quadruplex formation, dependent on replication polarity. A, diagram of the ectopic (Pu/Py)88 inserts; B, DF/myc(Pu)88 control cells; C, DF/myc(Pu)88 cells treated with HU; D, DF/myc(Pu)88 cells treated with TMS; E, DF/myc(Pu)88 cells treated with TMS and HU; F, DF/myc(Py)88 control cells; G, DF/myc(Py)88 cells treated with HU; H, DF/myc(Py)88 cells treated with TMS; I, DF/myc(Py)88 cells treated with TMS and HU.
      The starting culture of DF/myc/(Pu)88 cells (referred to as (Pu)88 cells) showed a significantly higher percentage of cells than the (Py)88 cells in the lower right (green) quadrant, resulting from DSBs occurring in the absence of exogenous stress (Fig. 5B and Fig. S6, p = 0.0001). Treatment of the (Pu)88 cells with HU did not significantly increase the percentage of green cells (Fig. 5C, p = 0.354); however, treatment of (Pu)88 cells with the G quadruplex–stabilizing drug telomestatin (TMS) (
      • De Cian A.
      • Guittat L.
      • Shin-Ya K.
      • Riou J.F.
      • Mergny J.L.
      Affinity and selectivity of G4 ligands measured by FRET.
      ,
      • Gomez D.
      • Paterski R.
      • Lemarteleur T.
      • Shin-Ya K.
      • Mergny J.L.
      • Riou J.F.
      Interaction of telomestatin with the telomeric single-strand overhang.
      ) markedly increased chromosome fragility at the ectopic site (Fig. 5D, p = 0.0045), and this effect was enhanced by co-administration of HU (Fig. 5E and Fig. S6, p = 0.024). These data suggest that replication fork slowing acts synergistically when G quadruplex formation is induced by the exogenous ligand. However, it remains to be seen whether endogenous levels of replication stress promote G4 versus H-DNA formation in the (Pu)88 repeat in vivo.
      We showed previously that the PKD1 microsatellite in the (Pu)88 lagging-strand orientation blocked replication fork progress in vivo from the c-myc origin and elicited a constitutive DNA damage response, which was not observed when the ectopic site repeat was in the (Py)88 orientation (
      • Liu G.
      • Myers S.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
      ). To test the effect of replication polarity on the stability of the PKD1 microsatellite, we also analyzed the stability of the ectopic site when (Py)88 was in the lagging-strand orientation. As shown in Fig. 5F, a significantly smaller percentage of DF/myc/(Py)88 cells (referred to as (Py)88 cells) than (Pu)88 cells were initially green in the absence of exogenous replication stress (cf. Fig. 5 (B and F) and Fig. S6, p = 0.0001). This result suggests that the ectopic (Pu/Py)88 tract is more sensitive to endogenous DSBs when the purine-rich strand is replicated as the lagging-strand template (i.e. in the fork-stalling orientation for replication). Nevertheless, compared with the effects of TMS on (Pu)88 cells, TMS had a reduced but statistically significant effect on the percentage of green (Py)88 cells in the absence (Fig. 5H, p = 0.006) or presence (Fig. 5I, p = 0.004) of HU, which is likely due to the presence of the NHE III1 G4 prone sequence in the c-myc replication origin core (
      • Del Mundo I.M.A.
      • Zewail-Foote M.
      • Kerwin S.M.
      • Vasquez K.M.
      Alternative DNA structure formation in the mutagenic human c-MYC promoter.
      ,
      • Mathad R.I.
      • Hatzakis E.
      • Dai J.
      • Yang D.
      c-MYC promoter G-quadruplex formed at the 5‘-end of NHE III1 element: insights into biological relevance and parallel-stranded G-quadruplex stability.
      ,
      • Yang D.
      • Hurley L.H.
      Structure of the biologically relevant G-quadruplex in the c-MYC promoter.
      ). In contrast, HU treatment did not have a significant effect on the flow cytometry profile of (Py)88 cells in the absence (Fig. 5G and Fig. S6, p = 0.355) or presence of TMS (Fig. 5I and Fig. S6, p = 0.483).

      Indirect induction of (CTG/CAG)100 DSBs

      TMS is a highly selective intramolecular G quadruplex ligand (
      • De Cian A.
      • Guittat L.
      • Shin-Ya K.
      • Riou J.F.
      • Mergny J.L.
      Affinity and selectivity of G4 ligands measured by FRET.
      ,
      • Gomez D.
      • Paterski R.
      • Lemarteleur T.
      • Shin-Ya K.
      • Mergny J.L.
      • Riou J.F.
      Interaction of telomestatin with the telomeric single-strand overhang.
      ,
      • Kim M.Y.
      • Gleason-Guzman M.
      • Izbicka E.
      • Nishioka D.
      • Hurley L.H.
      The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures.
      ), which inhibits telomerase and causes telomere shortening in vivo (
      • Tauchi T.
      • Shin-Ya K.
      • Sashida G.
      • Sumi M.
      • Nakajima A.
      • Shimamoto T.
      • Ohyashiki J.H.
      • Ohyashiki K.
      Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: involvement of ATM-dependent DNA damage response pathways.
      ). The results of TMS treatment of (Pu)88 and (Py)88 cells imply that DSBs occur preferentially when G4 prone sequences are present on lagging-strand templates and that G quadruplex formation contributes to fork stalling and replication-dependent DSBs.
      Therefore, it was surprising that treatment of (CTG)100 cells with TMS resulted in DSBs between the dTomato and eGFP reporter genes (Fig. 6, A and B). The effect of HU on these cells was not additive to the effect of TMS (Fig. 6C), in contrast to the dramatic effect of HU on (CTG)100 cells in the absence of TMS (Fig. 2). These results suggest that the induction of DSBs by TMS or HU at the ectopic (CTG/CAG)100 microsatellite may both be related to replication fork stalling. To confirm that the TMS effect in (CTG)100 cells was due to the (CTG/CAG)100 repeat, we treated DF/myc cells with TMS and observed a significantly decreased appearance of green cells (Fig. 6 (D–F), p = 0.011).
      Figure thumbnail gr6
      Figure 6TMS indirectly induces (CTG/CAG)100 DSBs. A, DF/myc(CTG)100 cells, untreated; B, DF/myc(CTG)100 cells treated with TMS; C, DF/myc(CTG)100 cells treated with TMS and HU; D, DF/myc cells, untreated; E, DF/myc cells treated with TMS; F, DF/myc cells treated with TMS and HU; G, CD spectra of the indicated DNAs with or without TMS. Note the overlap of the (CTG)100 versus (CTG)100 + TMS spectra, (CAG)100 versus (CAG)100 + TMS spectra, and scrambled DNA versus scrambled DNA + TMS spectra.
      The effect of TMS on DF/myc control cells was not statistically significantly different from its effect on (Py)88 cells (p = 0.149), suggesting that as in (Py)88 cells, the residual effect of TMS on the DF/myc control cells may be due to the NHE III1 G quadruplex–forming sequence in the 2.4 kb c-myc replication origin DNA (
      • Belotserkovskii B.P.
      • De Silva E.
      • Tornaletti S.
      • Wang G.
      • Vasquez K.M.
      • Hanawalt P.C.
      A triplex-forming sequence from the human c-MYC promoter interferes with DNA transcription.
      ,
      • Mathad R.I.
      • Hatzakis E.
      • Dai J.
      • Yang D.
      c-MYC promoter G-quadruplex formed at the 5‘-end of NHE III1 element: insights into biological relevance and parallel-stranded G-quadruplex stability.
      ,
      • Yang D.
      • Hurley L.H.
      Structure of the biologically relevant G-quadruplex in the c-MYC promoter.
      ,
      • Kim M.Y.
      • Gleason-Guzman M.
      • Izbicka E.
      • Nishioka D.
      • Hurley L.H.
      The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures.
      ,
      • Siddiqui-Jain A.
      • Grand C.L.
      • Bearss D.J.
      • Hurley L.H.
      Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription.
      ,
      • Bedrat A.
      • Lacroix L.
      • Mergny J.L.
      Re-evaluation of G-quadruplex propensity with G4Hunter.
      ).
      To test the possibility that TMS induces unexpected structural changes in the (CTG/CAG) microsatellite, we used CD to monitor the effect of TMS on CTG and CAG oligonucleotides (Fig. 6G). As anticipated, TMS caused a dramatic shift in the CD spectrum of a 22-mer oligonucleotide derived from the c-myc G quadruplex–forming promoter sequence (
      • Kim M.Y.
      • Gleason-Guzman M.
      • Izbicka E.
      • Nishioka D.
      • Hurley L.H.
      The different biological effects of telomestatin and TMPyP4 can be attributed to their selectivity for interaction with intramolecular or intermolecular G-quadruplex structures.
      ,
      • Siddiqui-Jain A.
      • Grand C.L.
      • Bearss D.J.
      • Hurley L.H.
      Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription.
      ,
      • Bedrat A.
      • Lacroix L.
      • Mergny J.L.
      Re-evaluation of G-quadruplex propensity with G4Hunter.
      ). However, TMS had no discernible effect on the CD spectra of a scrambled DNA negative control, or (CTG)12 or (CAG)12 oligonucleotides, although the (CTG)12 and (CAG)12 sequences are known to form hairpins in vitro (
      • Figueroa A.A.
      • Cattie D.
      • Delaney S.
      Structure of even/odd trinucleotide repeat sequences modulates persistence of non-B conformations and conversion to duplex.
      ). This is consistent with the lack of stabilization of dsDNA by telomestatin or similar G4 ligands (
      • Pilch D.S.
      • Barbieri C.M.
      • Rzuczek S.G.
      • Lavoie E.J.
      • Rice J.E.
      Targeting human telomeric G-quadruplex DNA with oxazole-containing macrocyclic compounds.
      ,
      • Nakamura T.
      • Okabe S.
      • Yoshida H.
      • Iida K.
      • Ma Y.
      • Sasaki S.
      • Yamori T.
      • Shin-Ya K.
      • Nakano I.
      • Nagasawa K.
      • Seimiya H.
      Targeting glioma stem cells in vivo by a G-quadruplex-stabilizing synthetic macrocyclic hexaoxazole.
      ). We conclude that TMS does not have a direct effect on (CTG/CAG) DNA structure in vitro and, therefore, that the effects of TMS on (CTG/CAG) stability in vivo are more likely to be an indirect result of activation of the DNA stress response (see “Discussion”), consistent with the observation that TMS leads to phosphorylation of the DNA damage response proteins Chk1, Chk2, and H2AX (
      • Hasegawa D.
      • Okabe S.
      • Okamoto K.
      • Nakano I.
      • Shin-Ya K.
      • Seimiya H.
      G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells.
      ) (Fig. S5).
      The ectopic (Pu)88 repeat is not sensitive to HU, whereas the ectopic (CTG)100 microsatellite is sensitive to multiple forms of replication stress, suggesting that the (Pu)88 G4 structure is responsible for DSBs. Nevertheless, the effects of TMS on (CTG)100 cells raise the possibility that TMS may contribute to DSBs at the ectopic G4 sequences in (Pu/Py)88 cells both in trans through the DNA damage response and directly by binding to G quadruplex–prone DNA.

      Translesion DNA synthesis enzymes stabilize (CTG/CAG)100 against replication stress

      Rad18 is important for translesion synthesis (TLS) in yeast and human cells (
      • Bailly V.
      • Lauder S.
      • Prakash S.
      • Prakash L.
      Yeast DNA repair proteins Rad6 and Rad18 form a heterodimer that has ubiquitin conjugating, DNA binding, and ATP hydrolytic activities.
      ,
      • Yoon J.H.
      • Prakash S.
      • Prakash L.
      Requirement of Rad18 protein for replication through DNA lesions in mouse and human cells.
      ), where Rad18 monoubiquitination of proliferating cell nuclear antigen (PCNA) can recruit TLS DNA polymerases η, κ, ι, λ, ζ, and Rev1 to sites of replication fork stalling at non-B DNA structures (
      • Yang W.
      • Gao Y.
      Translesion and repair DNA polymerases: diverse structure and mechanism.
      ,
      • Bétous R.
      • Rey L.
      • Wang G.
      • Pillaire M.J.
      • Puget N.
      • Selves J.
      • Biard D.S.
      • Shin-Ya K.
      • Vasquez K.M.
      • Cazaux C.
      • Hoffmann J.S.
      Role of TLS DNA polymerases η and κ in processing naturally occurring structured DNA in human cells.
      ,
      • Bergoglio V.
      • Boyer A.S.
      • Walsh E.
      • Naim V.
      • Legube G.
      • Lee M.Y.
      • Rey L.
      • Rosselli F.
      • Cazaux C.
      • Eckert K.A.
      • Hoffmann J.S.
      DNA synthesis by Pol η promotes fragile site stability by preventing under-replicated DNA in mitosis.
      ,
      • Day T.A.
      • Palle K.
      • Barkley L.R.
      • Kakusho N.
      • Zou Y.
      • Tateishi S.
      • Verreault A.
      • Masai H.
      • Vaziri C.
      Phosphorylated Rad18 directs DNA polymerase η to sites of stalled replication.
      ,
      • Garg P.
      • Burgers P.M.
      Ubiquitinated proliferating cell nuclear antigen activates translesion DNA polymerases η and REV1.
      ,
      • Prakash S.
      • Johnson R.E.
      • Prakash L.
      Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.
      ,
      • Watanabe K.
      • Tateishi S.
      • Kawasuji M.
      • Tsurimoto T.
      • Inoue H.
      • Yamaizumi M.
      Rad18 guides polη to replication stalling sites through physical interaction and PCNA monoubiquitination.
      ). Because (CTG/CAG) sequences have been shown to form hairpin structures in vivo (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ,
      • Axford M.M.
      • Wang Y.H.
      • Nakamori M.
      • Zannis-Hadjopoulos M.
      • Thornton C.A.
      • Pearson C.E.
      Detection of slipped-DNAs at the trinucleotide repeats of the myotonic dystrophy type I disease locus in patient tissues.
      ), we wished to determine whether knockdown of Rad18 or the TLS polymerases Pol η or Pol κ would sensitize the (CTG/CAG)100 microsatellite to replication stress.
      Compared with cells transfected with control siRNA (Fig. 7, A, F, and K), combined control siRNA and HU treatment led to (CTG/CAG)100 DSBs in 35–50% of cells (Fig. 7 (B, G, and L) and Fig. S7, p = 5 × 10−6). Depletion of Rad18 (Fig. 7C) led to a reproducible increase in the percentage of cells with DSBs (green cells) (cf. Fig. 7 (A and D) and Fig. S7, p = 0.046), and Rad18 knockdown substantially increased the percentage of cells with DSBs when combined with HU treatment (cf. Fig. 7 (B and E) and Fig. S7, p = 0.037). Considered together, these results suggest that Rad18/TLS stabilizes the (CTG/CAG)100 microsatellite against the fork-slowing effects of endogenous and exogenous replication stress.
      Figure thumbnail gr7
      Figure 7Translesion synthesis pathways affect (CTG/CAG)100 stability. DF/myc(CTG)100 cells were subjected to the following treatments: nontargeting control siRNA (A, F, and K), nontargeting siRNA and HU (B, G, and L); Rad18 siRNA (C and D); Rad18 siRNA plus HU (E); Pol η siRNA (H and I), Pol η siRNA plus HU (J), Pol κ siRNA (M and N), Pol κ siRNA plus HU (O), and Western blotting (C, H, and M).
      TLS polymerases have been implicated in the bypass of DNA hairpin structures (e.g. by Escherichia coli Pol V synthesis across abasic DNA sites (
      • Maor-Shoshani A.
      • Ben-Ari V.
      • Livneh Z.
      Lesion bypass DNA polymerases replicate across non-DNA segments.
      ) and Saccharomyces cerevisiae Pol ζ/Rev1 primer extension by DNA template switching at hairpins (
      • Northam M.R.
      • Moore E.A.
      • Mertz T.M.
      • Binz S.K.
      • Stith C.M.
      • Stepchenkova E.I.
      • Wendt K.L.
      • Burgers P.M.
      • Shcherbakova P.V.
      DNA polymerases ζ and Rev1 mediate error-prone bypass of non-B DNA structures.
      )). In the current experiments, depletion of Pol η (cf. Fig. 7 (F and I) and Fig. S7) resulted in a statistically significant increase in green cells when compared with siControl (p = 0.041), whereas knockdown of Rev1 did not (not shown).
      In addition, the effect of HU treatment was amplified by siRNA depletion of Rad18 (Fig. 7 (B and E) and Fig. S7, p = 0.022) or Pol η (Fig. 7 (G and J) and Fig. S7, p = 0.042), whereas Rev1 knockdown did not increase the effect of HU treatment (not shown). These results suggest that Pol η is one of the TLS polymerases involved in the restart of (CTG)100 stalled forks.
      In contrast to the effects of knockdown of Rad18 or Pol η, depletion of Pol κ in the absence of HU treatment dramatically increased the fraction of cells containing DSBs (cf. Fig. 7 (K and N) and Fig. S7, p = 0.0007). Taken together, the modest effect of Rad18 knockdown versus the strong effect of Pol κ knockdown suggests that Pol κ may also have a fork restart function independent of Rad18 (
      • Lewis J.S.
      • Spenkelink L.M.
      • Schauer G.D.
      • Yurieva O.
      • Mueller S.H.
      • Natarajan V.
      • Kaur G.
      • Maher C.
      • Kay C.
      • O'Donnell M.E.
      • van Oijen A.M.
      Tunability of DNA polymerase stability during eukaryotic DNA replication.
      ,
      • Schmutz V.
      • Janel-Bintz R.
      • Wagner J.
      • Biard D.
      • Shiomi N.
      • Fuchs R.P.
      • Cordonnier A.M.
      Role of the ubiquitin-binding domain of Polη in Rad18-independent translesion DNA synthesis in human cell extracts.
      ,
      • Okada T.
      • Sonoda E.
      • Yamashita Y.M.
      • Koyoshi S.
      • Tateishi S.
      • Yamaizumi M.
      • Takata M.
      • Ogawa O.
      • Takeda S.
      Involvement of vertebrate polκ in Rad18-independent postreplication repair of UV damage.
      ). Surprisingly, HU treatment did not augment the effect of Pol κ knockdown (Fig. 7O and Fig. S7, p = 0.185) These results indicate that Rad18, Pol η, and Pol κ are involved in resolving non-B DNA (
      • Gao Y.
      • Mutter-Rottmayer E.
      • Zlatanou A.
      • Vaziri C.
      • Yang Y.
      Mechanisms of post-replication DNA repair.
      ). In the presence of HU, Pol κ may interact with the stalled fork in a nonproductive manner; thus, when replication is inhibited by HU, fewer structures that lead to DSBs are formed when Pol κ is depleted.

      Replication stress causes (CTG/CAG) BIR

      Non-B DNA structure–prone repeats can induce mutagenesis at a distance in mammalian cells (
      • Wang G.
      • Vasquez K.M.
      Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability.
      ). These mutational events are thought to result from replication fork stalling at microsatellite repeats, fork breakage, and subsequent error-prone repair in a process termed repeat-induced mutagenesis (RIM) or BIR (
      • Wang G.
      • Vasquez K.M.
      Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability.
      ,
      • Shah K.A.
      • Mirkin S.M.
      The hidden side of unstable DNA repeats: mutagenesis at a distance.
      ,
      • Kononenko A.V.
      • Ebersole T.
      • Vasquez K.M.
      • Mirkin S.M.
      Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.
      ).
      Our results have shown that the expanded (CTG/CAG) microsatellite stalls replication forks and induces replication-dependent DSBs. To test for RIM/BIR induced by the (CTG/CAG)100 microsatellite, we integrated a modified reporter plasmid, (CTG)100eGFP/TK, at the ectopic site such that the eGFP, FRT, and TK sequences become fused during FLP-mediated integration (Fig. 8A). We postulated that if BIR occurs following a hydroxyurea-induced, replication-dependent double-strand break at the (CTG/CAG)100 sequence, invasion of the broken end into the sister chromatid would result in mutagenesis of the neighboring TK gene ∼1 kb downstream (Fig. 8B).
      Figure thumbnail gr8
      Figure 8Repeat-induced mutagenesis. A, the DF/myc(CTG)100eGFP/TK ectopic site construct designed to detect BIR/RIM; B, model of repeat-induced mutagenesis of the thymidine kinase gene triggered by a replication-dependent DSB at the ectopic (CTG/CAG)100 repeat; C, GCVR colonies arising from BIR/RIM of the TK gene. Error bars, S.D. (n = 3 experiments).
      Untreated DF/myc(CTG/CAG)100eGFP/TK cells produced ganciclovir (GCV)-resistant clones at a frequency of approximately three per 105 cells (Fig. 8C). These data are comparable with the frequency of GCV-resistant clones stemming from DSBs at an ectopic (CGG/CCG)153 repeat in a clonal population of murine erythroid leukemia cells (
      • Kononenko A.V.
      • Ebersole T.
      • Vasquez K.M.
      • Mirkin S.M.
      Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.
      ). As in the case of the (CGG/CCG)153 murine erythroid leukemia cells, GCV resistance likely arose by endogenous replication stress and BIR during extended clonal outgrowth of the dual-fluorescence cell line.
      When DF/myc(CTG/CAG)100eGFP/TK cells were treated with 0.2 mm HU followed by GCV selection, the frequency of GCV-resistant colonies rose to ∼7–8 colonies/105 cells (Fig. 8C, p = 4 × 10−5). Thus, acute treatment with HU produced a similar number of GCR-resistant cells as extended (>1-year) clonal outgrowth.
      To confirm that the appearance of GCV-resistant cells is the result of BIR, we knocked down PolD3, which is necessary for BIR in yeast (
      • Sakofsky C.J.
      • Malkova A.
      Break induced replication in eukaryotes: mechanisms, functions, and consequences.
      ) and human cells (
      • Costantino L.
      • Sotiriou S.K.
      • Rantala J.K.
      • Magin S.
      • Mladenov E.
      • Helleday T.
      • Haber J.E.
      • Iliakis G.
      • Kallioniemi O.P.
      • Halazonetis T.D.
      Break-induced replication repair of damaged forks induces genomic duplications in human cells.
      ,
      • Kononenko A.V.
      • Ebersole T.
      • Vasquez K.M.
      • Mirkin S.M.
      Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.
      ), or knocked down BRCA2, which mitigates DSBs under conditions of replication stress and promotes Rad51-dependent BIR in human cells (
      • Costantino L.
      • Sotiriou S.K.
      • Rantala J.K.
      • Magin S.
      • Mladenov E.
      • Helleday T.
      • Haber J.E.
      • Iliakis G.
      • Kallioniemi O.P.
      • Halazonetis T.D.
      Break-induced replication repair of damaged forks induces genomic duplications in human cells.
      ,
      • Bhowmick R.
      • Minocherhomji S.
      • Hickson I.D.
      RAD52 facilitates mitotic DNA synthesis following replication stress.
      ) (Fig. 8C). When DF/myc(CTG/CAG)100eGFP/TK cells were exposed to shRNA, knockdown of PolD3 (Fig. 8C, p = 0.015) or BRCA2 (Fig. 8C, p = 0.002) significantly decreased the frequency of GCV-resistant cells following HU treatment to the background levels of TK mutants accumulated during prolonged clonal outgrowth, supporting the view that DSBs at the (CTG/CAG) repeat lead to break-induced replication.
      We previously used inverse PCR (iPCR) to show that replication stress leads to breakage at an ectopic (CTG/CAG)102 repeat in myc(CTG/CAG)102 cells (
      • Barthelemy J.
      • Hanenberg H.
      • Leffak M.
      FANCJ is essential to maintain microsatellite structure genome-wide during replication stress.
      ), as well as DSBs at endogenous microsatellites across the genome (
      • Barthelemy J.
      • Hanenberg H.
      • Leffak M.
      FANCJ is essential to maintain microsatellite structure genome-wide during replication stress.
      ). The (CTG/CAG)102 construct differed from the construct in DF/myc(CTG/CAG)100eGFP/TK cells in that there was no eGFP/TK fusion or GCV selection for cells undergoing BIR. DNA sequence analysis of iPCR products with nonallelic breakpoint junctions initiated within the ectopic site also showed that the broken site underwent nonrandom chromosomal translocations similar to genome rearrangements attributed to BIR in tumor cells (
      • Barthelemy J.
      • Hanenberg H.
      • Leffak M.
      FANCJ is essential to maintain microsatellite structure genome-wide during replication stress.
      ).
      Although there was no GCV selection for BIR in the myc(CTG/CAG)102 cells, we reanalyzed the iPCR DNA-sequencing data to focus on the distribution of mutations upstream and downstream of the ectopic repeat that had been repaired by homology-mediated templating of the sister chromatid (Fig. 9). The great majority (>95%) of mutations were single base substitutions. As predicted by BIR models, base substitutions were dramatically greater downstream of the (CTG/CAG)102 repeat than upstream (Fig. 9), consistent with the rightward replication of the repeat from the c-myc origin and with lamPCR mapping of the DSB at the downstream edge of the (CTG/CAG)100 repeat. Subtracting the frequency of nucleotide substitutions (PCR and sequencing errors, in vivo mutations) upstream of the repeat as background, the average frequency of nucleotide substitution downstream of the (CTG/CAG)102 repeat was ∼1.5 × 10−7 substitutions per bp. This value is at least 2–3 orders of magnitude greater than recent estimates of the natural mutation frequency in humans (
      • Sakofsky C.J.
      • Malkova A.
      Break induced replication in eukaryotes: mechanisms, functions, and consequences.
      ,
      • Nachman M.W.
      • Crowell S.L.
      Estimate of the mutation rate per nucleotide in humans.
      ,
      • Lynch M.
      Rate, molecular spectrum, and consequences of human mutation.
      ).
      Figure thumbnail gr9
      Figure 9High frequency of base substitutions due to BIR. A, base substitution analysis at the ectopic site following sister chromatid templated BIR. DNA was isolated from myc(CTG/CAG)102 cells treated with 0.2 μm aphidicolin, digested with MseI, and intramolecularly circularized. The circularized DNA was amplified by inverse PCR and analyzed by high-throughput sequencing. Nonallelic recombination junctions have been published previously (
      • Barthelemy J.
      • Hanenberg H.
      • Leffak M.
      FANCJ is essential to maintain microsatellite structure genome-wide during replication stress.
      ). B, interpretative map of BIR at the ectopic site. C, quantitation of base substitutions at C, T, and G residues within the (CTG) repeat.
      Within the (CTG)102 repeat, we observed expansions, contractions, inversions, and base substitutions. Interestingly, there was a strong third base periodicity of nucleotide substitutions at dG residues in the (CTG)102 template, which peaked dramatically near the center of the microsatellite (Fig. 9B). These data are consistent with the observation that dG:dC base pairs are preferential targets for single-base substitution mutations in tumor cells (
      • Bacolla A.
      • Temiz N.A.
      • Yi M.
      • Ivanic J.
      • Cer R.Z.
      • Donohue D.E.
      • Ball E.V.
      • Mudunuri U.S.
      • Wang G.
      • Jain A.
      • Volfovsky N.
      • Luke B.T.
      • Stephens R.M.
      • Cooper D.N.
      • Collins J.R.
      • et al.
      Guanine holes are prominent targets for mutation in cancer and inherited disease.
      ) and that a loop at the center of a single large (CTG)102 hairpin is a hotspot for mutagenesis during the process of BIR. Considered together, our data indicate that DSBs at (CTG/CAG) repeats lead to highly mutagenic break-induced replication.

      Discussion

      (CTG/CAG) microsatellite DSBs detected by flow cytometry

      Microsatellite repeats prone to forming non-B DNA structures undergo expansion, contraction, and double-strand breakage in a variety of yeast and mammalian cell systems (
      • Mirkin S.M.
      Expandable DNA repeats and human disease.
      ,
      • Sundararajan R.
      • Gellon L.
      • Zunder R.M.
      • Freudenreich C.H.
      Double-strand break repair pathways protect against CAG/CTG repeat expansions, contractions and repeat-mediated chromosomal fragility in Saccharomyces cerevisiae.
      ,
      • Kim J.C.
      • Harris S.T.
      • Dinter T.
      • Shah K.A.
      • Mirkin S.M.
      The role of break-induced replication in large-scale expansions of (CAG)n/(CTG)n repeats.
      ,
      • Kononenko A.V.
      • Ebersole T.
      • Vasquez K.M.
      • Mirkin S.M.
      Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.
      ,
      • Gomes-Pereira M.
      • Foiry L.
      • Nicole A.
      • Huguet A.
      • Junien C.
      • Munnich A.
      • Gourdon G.
      CTG trinucleotide repeat “big jumps”: large expansions, small mice.
      ,
      • Shishkin A.A.
      • Voineagu I.
      • Matera R.
      • Cherng N.
      • Chernet B.T.
      • Krasilnikova M.M.
      • Narayanan V.
      • Lobachev K.S.
      • Mirkin S.M.
      Large-scale expansions of Friedreich's ataxia GAA repeats in yeast.
      ,
      • Saini N.
      • Zhang Y.
      • Nishida Y.
      • Sheng Z.
      • Choudhury S.
      • Mieczkowski P.
      • Lobachev K.S.
      Fragile DNA motifs trigger mutagenesis at distant chromosomal loci in Saccharomyces cerevisiae.
      ,
      • Zhao J.
      • Wang G.
      • Del Mundo I.M.
      • McKinney J.A.
      • Lu X.
      • Bacolla A.
      • Boulware S.B.
      • Zhang C.
      • Zhang H.
      • Ren P.
      • Freudenreich C.H.
      • Vasquez K.M.
      Distinct mechanisms of nuclease-directed DNA-structure-induced genetic instability in cancer genomes.
      ,
      • Usdin K.
      • House N.C.
      • Freudenreich C.H.
      Repeat instability during DNA repair: insights from model systems.
      ,
      • Cherng N.
      • Shishkin A.A.
      • Schlager L.I.
      • Tuck R.H.
      • Sloan L.
      • Matera R.
      • Sarkar P.S.
      • Ashizawa T.
      • Freudenreich C.H.
      • Mirkin S.M.
      Expansions, contractions, and fragility of the spinocerebellar ataxia type 10 pentanucleotide repeat in yeast.
      ). Here, we used a dual-fluorescence reporter gene system to analyze DSBs in human cells. We show that DNA double-strand breaks occur at a relatively high frequency in an ectopic (CTG/CAG)100 microsatellite expanded beyond the WT range of repeats found in the human DMPK gene. These DSBs occur in unstressed cells and were dramatically increased in cells treated with four qualitatively different replication stressors (hydroxyurea (
      • Barthelemy J.
      • Hanenberg H.
      • Leffak M.
      FANCJ is essential to maintain microsatellite structure genome-wide during replication stress.
      ,
      • Singh A.
      • Xu Y.J.
      The Cell Killing Mechanisms of Hydroxyurea.
      ,
      • Feng W.
      • Di Rienzi S.C.
      • Raghuraman M.K.
      • Brewer B.J.
      Replication stress-induced chromosome breakage is correlated with replication fork progression and is preceded by single-stranded DNA formation.
      ), aphidicolin (
      • Liu G.
      • Chen X.
      • Leffak M.
      Oligodeoxynucleotide Binding to (CTG)·(CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability.
      ), hydrogen peroxide (
      • Venkatachalam G.
      • Surana U.
      • Clément M.V.
      Replication stress-induced endogenous DNA damage drives cellular senescence induced by a sub-lethal oxidative stress.
      ), and telomestatin (
      • Hasegawa D.
      • Okabe S.
      • Okamoto K.
      • Nakano I.
      • Shin-Ya K.
      • Seimiya H.
      G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells.
      ,
      • Bétous R.
      • Rey L.
      • Wang G.
      • Pillaire M.J.
      • Puget N.
      • Selves J.
      • Biard D.S.
      • Shin-Ya K.
      • Vasquez K.M.
      • Cazaux C.
      • Hoffmann J.S.
      Role of TLS DNA polymerases η and κ in processing naturally occurring structured DNA in human cells.
      )), in agreement with the view that endogenous and exogenous replication stress leads to DNA DSBs. The low background level of DSBs at the ectopic site (CTG/CAG)23 repeat suggests that expanded (CTG/CAG)100 tracts promote replication-dependent breakage.
      Replication-dependent DSBs at repeated sequences have been attributed to the propensity of these repeats to form noncanonical DNA structures (
      • Voineagu I.
      • Freudenreich C.H.
      • Mirkin S.M.
      Checkpoint responses to unusual structures formed by DNA repeats.
      ,
      • Voineagu I.
      • Narayanan V.
      • Lobachev K.S.
      • Mirkin S.M.
      Replication stalling at unstable inverted repeats: interplay between DNA hairpins and fork stabilizing proteins.
      ,
      • Nambiar M.
      • Goldsmith G.
      • Moorthy B.T.
      • Lieber M.R.
      • Joshi M.V.
      • Choudhary B.
      • Hosur R.V.
      • Raghavan S.C.
      Formation of a G-quadruplex at the BCL2 major breakpoint region of the t(14;18) translocation in follicular lymphoma.
      ,
      • Lukusa T.
      • Fryns J.P.
      Human chromosome fragility.
      ,
      • Wang G.
      • Vasquez K.M.
      Non-B DNA structure-induced genetic instability.
      ). Consistent with this view, it has been shown that (CTG/CAG) repeats form hairpin structures in vivo (
      • Liu G.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication-dependent instability at (CTG)·(CAG) repeat hairpins in human cells.
      ,
      • Liu G.
      • Chen X.
      • Leffak M.
      Oligodeoxynucleotide Binding to (CTG)·(CAG) microsatellite repeats inhibits replication fork stalling, hairpin formation, and genome instability.
      ,
      • Avila Figueroa A.
      • Delaney S.
      Mechanistic studies of hairpin to duplex conversion for trinucleotide repeat sequences.
      ). It is intriguing, therefore, that the cellular repair machinery treats a restriction enzyme–generated DSB differently than a replication-dependent DSB. We speculate that localized Mus81-dependent cleavage near the downstream edge of the (CTG/CAG)100 repeat is due to a noncanonical DNA structure that is refractory to replication and repair. Similar conclusions regarding the breakage and repair of structured ends have been obtained with an AT-rich repeat derived from the FRA16D common fragile site (
      • Zhang H.
      • Freudenreich C.H.
      An AT-rich sequence in human common fragile site FRA16D causes fork stalling and chromosome breakage in S. cerevisiae.
      ,
      • Wang H.
      • Li Y.
      • Truong L.N.
      • Shi L.Z.
      • Hwang P.Y.
      • He J.
      • Do J.
      • Cho M.J.
      • Li H.
      • Negrete A.
      • Shiloach J.
      • Berns M.W.
      • Shen B.
      • Chen L.
      • Wu X.
      CtIP maintains stability at common fragile sites and inverted repeats by end resection-independent endonuclease activity.
      ).
      The abundance of green cells (∼50%) after different forms of replication stress is consistent with models in which both ends of a replication-dependent DSB persist in the population (
      • Tubbs A.
      • Sridharan S.
      • van Wietmarschen N.
      • Maman Y.
      • Callen E.
      • Stanlie A.
      • Wu W.
      • Wu X.
      • Day A.
      • Wong N.
      • Yin M.
      • Canela A.
      • Fu H.
      • Redon C.
      • Pruitt S.C.
      • et al.
      Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse.
      ); based on the abundance of dTomato eGFP+ cells, we propose that the eGFP side of the DSB is replicated by a leftward moving replication fork (Figs. 8B and 10) (
      • Mayle R.
      • Campbell I.M.
      • Beck C.R.
      • Yu Y.
      • Wilson M.
      • Shaw C.A.
      • Bjergbaek L.
      • Lupski J.R.
      • Ira G.
      DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.
      ) that produces two DSBs that are functionally single-ended.
      Figure thumbnail gr10
      Figure 10Proposed model for loss of the dTomato marker gene after replication stress. A, ideogram of chromosome 18 showing the FRT site at 18p11.22. B, integrated (CTG)100 construct. C, enlarged (not to scale) diagram of converging replication forks and the Mus81 cleavage substrate. D, proposed pathway for loss of the dTomato marker gene and generation of eGFP+ (green) cells.
      Among other possibilities, the subsequent instability of the green cells may be due to the structure of the non-B DNA end per se, inhibition of the major pathways of repair (homologous recombination and nonhomologous end joining) with the downstream end, a nontelomeric structure of the downstream eGFP DSB end, and loss of DNA from the acentric upstream side of the DSB.
      In contrast to the results presented here for the PKD1 microsatellite, Wenger et al. (
      • Wenger S.L.
      • Giangreco C.A.
      • Tarleton J.
      • Wessel H.B.
      Inability to induce fragile sites at CTG repeats in congenital myotonic dystrophy.
      ) reported the inability to detect fragile sites by cytogenetic G banding in blood cell cultures from congenital DM1 patients containing repeats as large as (CTG/CAG)1000 after treatment with replication stressors including 0.2 μm aphidicolin. Aside from differences in cell type, Wenger et al. (
      • Wenger S.L.
      • Giangreco C.A.
      • Tarleton J.
      • Wessel H.B.
      Inability to induce fragile sites at CTG repeats in congenital myotonic dystrophy.
      ) treated cells with bromodeoxyuridine, 5′-deoxy-5-fluorouridine, or aphidicolin for 24 h immediately before chromosome spreading, whereas the present experiments treated cells for 4 days prior to 4–10-day recovery in drug-free medium and flow cytometry. It is possible therefore that in the present experiments, DSBs occur during prolonged replication stress or during replication restart following drug treatment.
      We observed as well that treatment of cells with telomestatin induced DSBs at the (CTG/CAG)100 ectopic site in vivo. Telomestatin is a known replication stressor of intramolecular G quadruplex–prone sequences, especially telomeres (
      • Tauchi T.
      • Shin-Ya K.
      • Sashida G.
      • Sumi M.
      • Nakajima A.
      • Shimamoto T.
      • Ohyashiki J.H.
      • Ohyashiki K.
      Activity of a novel G-quadruplex-interactive telomerase inhibitor, telomestatin (SOT-095), against human leukemia cells: involvement of ATM-dependent DNA damage response pathways.
      ,
      • Hasegawa D.
      • Okabe S.
      • Okamoto K.
      • Nakano I.
      • Shin-Ya K.
      • Seimiya H.
      G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells.
      ,
      • Kim M.Y.
      • Vankayalapati H.
      • Shin-Ya K.
      • Wierzba K.
      • Hurley L.H.
      Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular g-quadruplex.
      ,
      • Sun D.
      • Guo K.
      • Rusche J.J.
      • Hurley L.H.
      Facilitation of a structural transition in the polypurine/polypyrimidine tract within the proximal promoter region of the human VEGF gene by the presence of potassium and G-quadruplex-interactive agents.
      ,
      • Temime-Smaali N.
      • Guittat L.
      • Sidibe A.
      • Shin-Ya K.
      • Trentesaux C.
      • Riou J.F.
      The G-quadruplex ligand telomestatin impairs binding of topoisomerase IIIα to G-quadruplex-forming oligonucleotides and uncaps telomeres in ALT cells.
      ); however, no change in (CTG/CAG) oligonucleotide structure due to TMS could be detected in vitro by CD. These results suggest that telomestatin action at telomeres or other G quadruplex–prone sequences (
      • Hasegawa D.
      • Okabe S.
      • Okamoto K.
      • Nakano I.
      • Shin-Ya K.
      • Seimiya H.
      G-quadruplex ligand-induced DNA damage response coupled with telomere dysfunction and replication stress in glioma stem cells.
      ) can affect (CTG/CAG) stability in trans. We propose that G quadruplex formation elsewhere in the genome causes a diffusible state of replication stress in the cell that promotes fork slowing and non-B structure formation at the ectopic (CTG/CAG)100 repeat.

      DSBs at the structure-prone (Pu/Py)88 microsatellite

      During DNA replication, the lagging-strand template is expected to be more susceptible to structure formation than the leading-strand template, due to its relatively prolonged single-strandedness (
      • Mirkin S.M.
      DNA structures, repeat expansions and human hereditary disorders.
      ,
      • Voineagu I.
      • Narayanan V.
      • Lobachev K.S.
      • Mirkin S.M.
      Replication stalling at unstable inverted repeats: interplay between DNA hairpins and fork stabilizing proteins.
      ). Consistent with this model, replication of the PKD1 intron 21 (Pu)88 sequence as the lagging-strand template leads to replisome stalling; recruitment of RPA, Rad9, and ATR to the stalled fork; and induction of a DNA damage response (
      • Liu G.
      • Myers S.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
      ).
      In the current work, the (Pu/Py)88 repeat displayed replication polarity–dependent instability in the absence of exogenous stress. Administration of the G quadruplex–binding ligand telomestatin strongly enhanced the sensitivity of the (Pu/Py)88 tract to DSBs in the same replication polarity–dependent manner, which we attribute to the stabilization of G quadruplex DNA structures in the lagging-strand template. However, the (Pu/Py)88 tract also has the ability to form triplex H-DNA structures in the absence of telomestatin (
      • Liu G.
      • Myers S.
      • Chen X.
      • Bissler J.J.
      • Sinden R.R.
      • Leffak M.
      Replication fork stalling and checkpoint activation by a PKD1 locus mirror repeat polypurine-polypyrimidine (Pu-Py) tract.
      ,
      • Blaszak R.T.
      • Potaman V.
      • Sinden R.R.
      • Bissler J.J.
      DNA structural transitions within the PKD1 gene.
      ,
      • Patel H.P.
      • Lu L.
      • Blaszak R.T.
      • Bissler J.J.
      PKD1 intron 21: triplex DNA formation and effect on replication.
      ,
      • Van Raay T.J.
      • Burn T.C.
      • Connors T.D.
      • Petry L.R.
      • Germino G.G.
      • Klinger K.W.
      • Landes G.M.
      A 2.5 kb polypyrimidine tract in the PKD1 gene contains at least 23 H-DNA-forming sequences.
      ). Therefore, whereas the present results indicate that induced G quadruplex formation can stall replication forks and cause DSBs, the sensitivity of the lagging-strand (Pu)88 tract to DSBs in unperturbed cells could also be the result of H-DNA formation. This possibility is currently under investigation.
      In contrast to the DSB sensitivity of the ectopic PKD1 IVS21 (Pu/Py)88 tract, PCR analysis of 57 patients with autosomal dominant polycystic kidney disease (ADPKD1) showed no hotspot for mutation in the PKD1 gene, although mutations were 2–3 times more frequent in the exons surrounding IVS21 than in exons 1–8 (
      • Rossetti S.
      • Strmecki L.
      • Gamble V.
      • Burton S.
      • Sneddon V.
      • Peral B.
      • Roy S.
      • Bakkaloglu A.
      • Komel R.
      • Winearls C.G.
      • Harris P.C.
      Mutation analysis of the entire PKD1 gene: genetic and diagnostic implications.
      ). Similarly, in samples from 15 tuberous sclerosis (TSC) patients in which deletions in the upstream TSC2 gene extended into PKD1, multiplex ligation-dependent probe amplification did not show clustering of breakpoints near the IVS21 (Pu/Py)88 tract (
      • Kozlowski P.
      • Bissler J.
      • Pei Y.
      • Kwiatkowski D.J.
      Analysis of PKD1 for genomic deletion by multiplex ligation-dependent probe assay: absence of hot spots.
      ). One explanation for these observations may be a strong selection against PKD1 DSBs, which includes origin choice (
      • Mirkin E.V.
      • Mirkin S.M.
      To switch or not to switch: at the origin of repeat expansion disease.
      ,
      • Gerhardt J.
      • Tomishima M.J.
      • Zaninovic N.
      • Colak D.
      • Yan Z.
      • Zhan Q.
      • Rosenwaks Z.
      • Jaffrey S.R.
      • Schildkraut C.L.
      The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells.
      ,
      • Prioleau M.N.
      • MacAlpine D.M.
      DNA replication origins-where do we begin?.
      ,
      • Stevanoni M.
      • Palumbo E.
      • Russo A.
      The replication of frataxin gene is assured by activation of dormant origins in the presence of a GAA-repeat expansion.
      ) to avoid lagging-strand replication of the PKD1 (Pu)88 sequence.

      Translesion polymerases mediate (CTG/CAG) stability

      The TLS polymerases comprise a group of functionally divergent enzymes that can bypass non-B DNA and template lesions that stall replicative polymerases (
      • Gao Y.
      • Mutter-Rottmayer E.
      • Zlatanou A.
      • Vaziri C.
      • Yang Y.
      Mechanisms of post-replication DNA repair.
      ). Rad18-dependent ubiquitination of the PCNA scaffold allows TLS polymerase exchange and access to the unreplicated lesion (
      • Bétous R.
      • Rey L.
      • Wang G.
      • Pillaire M.J.
      • Puget N.
      • Selves J.
      • Biard D.S.
      • Shin-Ya K.
      • Vasquez K.M.
      • Cazaux C.
      • Hoffmann J.S.
      Role of TLS DNA polymerases η and κ in processing naturally occurring structured DNA in human cells.
      ,
      • Prakash S.
      • Johnson R.E.
      • Prakash L.
      Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.
      ,
      • Zhao L.
      • Washington M.T.
      Translesion synthesis: insights into the selection and switching of DNA polymerases.
      ,
      • Vujanovic M.
      • Krietsch J.
      • Raso M.C.
      • Terraneo N.
      • Zellweger R.
      • Schmid J.A.
      • Taglialatela A.
      • Huang J.W.
      • Holland C.L.
      • Zwicky K.
      • Herrador R.
      • Jacobs H.
      • Cortez D.
      • Ciccia A.
      • Penengo L.
      • et al.
      Replication fork slowing and reversal upon DNA damage require PCNA polyubiquitination and ZRANB3 DNA translocase activity.
      ,
      • Vaisman A.
      • Woodgate R.
      Translesion DNA polymerases in eukaryotes: what makes them tick?.
      ). Multiple TLS polymerases can bind to ubiquitinated PCNA (
      • Gao Y.
      • Mutter-Rottmayer E.
      • Zlatanou A.
      • Vaziri C.
      • Yang Y.
      Mechanisms of post-replication DNA repair.
      ); thus, in the absence of one TLS polymerase, alternative postreplication repair pathways can be employed (
      • Prakash S.
      • Johnson R.E.
      • Prakash L.
      Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.
      ,
      • Gao Y.
      • Mutter-Rottmayer E.
      • Zlatanou A.
      • Vaziri C.
      • Yang Y.
      Mechanisms of post-replication DNA repair.
      ). In the dual-fluorescence assay, siRNA knockdown of Rad18 or Pol η caused a marked increase in ectopic instability when combined with HU treatment. In contrast, depletion of Pol κ led to a large increase in DSBs in otherwise unstressed cells but minimized the effect of HU treatment. We suggest that Rad18 and Pol η assist in the replication of the (CTG/CAG)100 repeat, particularly during HU-induced fork slowing, whereas depletion of Pol κ in the presence of HU decreases the formation of a subset of difficult-to-replicate structures. Intriguingly, translesion synthesis has also been implicated in break-induced replication (
      • Wong R.P.
      • Garcia-Rodriguez N.
      • Zilio N.
      • Hanulova M.
      • Ulrich H.D.
      Processing of DNA polymerase-blocking lesions during genome replication is spatially and temporally segregated from replication forks.
      ,
      • Sakofsky C.J.
      • Ayyar S.
      • Deem A.K.
      • Chung W.H.
      • Ira G.
      • Malkova A.
      Translesion polymerases drive microhomology-mediated break-induced replication leading to complex chromosomal rearrangements.
      ). Our results are consistent with reports that Pol η and Pol κ are involved in the replication of common fragile sites (
      • Barnes R.P.
      • Hile S.E.
      • Lee M.Y.
      • Eckert K.A.
      DNA polymerases η and κ exchange with the polymerase delta holoenzyme to complete common fragile site synthesis.
      ) and that knockdown of these polymerases enhances DSBs in HeLa cells transfected with plasmids containing c-myc G4-prone DNA (
      • Bétous R.
      • Rey L.
      • Wang G.
      • Pillaire M.J.
      • Puget N.
      • Selves J.
      • Biard D.S.
      • Shin-Ya K.
      • Vasquez K.M.
      • Cazaux C.
      • Hoffmann J.S.
      Role of TLS DNA polymerases η and κ in processing naturally occurring structured DNA in human cells.
      ).

      (CTG/CAG)100 break-induced replication

      Early replicating fragile sites (ERFSs) are detected as DNA breaks in the absence of exogenous replication stress but are increased on release from hydroxyurea treatment or ATR inhibition (
      • Barlow J.H.
      • Faryabi R.B.
      • Callén E.
      • Wong N.
      • Malhowski A.
      • Chen H.T.
      • Gutierrez-Cruz G.
      • Sun H.W.
      • McKinnon P.
      • Wright G.
      • Casellas R.
      • Robbiani D.F.
      • Staudt L.
      • Fernandez-Capetillo O.
      • Nussenzweig A.
      Identification of early replicating fragile sites that contribute to genome instability.
      ). Tubbs et al. (
      • Tubbs A.
      • Sridharan S.
      • van Wietmarschen N.
      • Maman Y.
      • Callen E.
      • Stanlie A.
      • Wu W.
      • Wu X.
      • Day A.
      • Wong N.
      • Yin M.
      • Canela A.
      • Fu H.
      • Redon C.
      • Pruitt S.C.
      • et al.
      Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse.
      ) recently showed that a subset of ERFSs close to replication origins containing poly(dA:dT) tracts are highly sensitive to DSBs in lymphocyte cultures treated with HU. The 2:1 ratio of DNA ends on opposite sides of the DNA breaks suggested that both arms of the replication fork are broken into DSBs, in contrast to the single-ended DSB model of RIM and BIR (
      • Sakofsky C.J.
      • Malkova A.
      Break induced replication in eukaryotes: mechanisms, functions, and consequences.
      ,
      • Kramara J.
      • Osia B.
      • Malkova A.
      Break-induced replication: the where, the why, and the how.
      ,
      • Donnianni R.A.
      • Symington L.S.
      Break-induced replication occurs by conservative DNA synthesis.
      ,
      • Ruff P.
      • Donnianni R.A.
      • Glancy E.
      • Oh J.
      • Symington L.S.
      RPA stabilization of single-stranded DNA is critical for break-induced replication.
      ,
      • Saini N.
      • Ramakrishnan S.
      • Elango R.
      • Ayyar S.
      • Zhang Y.
      • Deem A.
      • Ira G.
      • Haber J.E.
      • Lobachev K.S.
      • Malkova A.
      Migrating bubble during break-induced replication drives conservative DNA synthesis.
      ).
      The ectopic (CTG/CAG)100 repeat resembles an ERFS in its proximity to an origin (
      • Tubbs A.
      • Sridharan S.
      • van Wietmarschen N.
      • Maman Y.
      • Callen E.
      • Stanlie A.
      • Wu W.
      • Wu X.
      • Day A.
      • Wong N.
      • Yin M.
      • Canela A.
      • Fu H.
      • Redon C.
      • Pruitt S.C.
      • et al.
      Dual roles of poly(dA:dT) tracts in replication initiation and fork collapse.
      ), its early replication (
      • Kemp M.G.
      • Ghosh M.
      • Liu G.
      • Leffak M.
      The histone deacetylase inhibitor trichostatin A alters the pattern of DNA replication origin activity in human cells.
      ), and its sensitivity to several forms of replication stress (
      • Barlow J.H.
      • Faryabi R.B.
      • Callén E.
      • Wong N.
      • Malhowski A.
      • Chen H.T.
      • Gutierrez-Cruz G.
      • Sun H.W.
      • McKinnon P.
      • Wright G.
      • Casellas R.
      • Robbiani D.F.
      • Staudt L.
      • Fernandez-Capetillo O.
      • Nussenzweig A.
      Identification of early replicating fragile sites that contribute to genome instability.
      ). However, breakage at the (CTG)100 may differ from the model for ERFS DSBs on both arms of a fork based on RIM/BIR mutagenesis at the flanking TK sequence upon HU treatment (i.e. the 3′ end of a replication-dependent DSB generated by (CTG)100 fork collapse can invade and mutagenize the TK gene of an intact sister chromatid).
      In the dual-fluorescence system, the frequency of GCVR colonies is comparable with recently reported values for BIR initiated by (CGG/CCG)153 repeats in murine cells (
      • Kononenko A.V.
      • Ebersole T.
      • Vasquez K.M.
      • Mirkin S.M.
      Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.
      ) and may reflect the rate of error-prone DNA synthesis during break-induced replication. However, additional factors (efficiency of mismatch repair, incidence of sister chromatid (versus nonallelic) invasion, efficiency of synthesis on the sister chromatid template, frequency of template switching) may also affect the observed frequency of mutagenesis.
      A variety of replication stressors, including HU, induced breaks between the dTomato and eGFP reporter genes at the ectopic site. HU also induced a (CTG/CAG)100 DSB localized to the edge of the repeat downstream of the c-myc replication origin. The induction of GCV-resistant clones by HU treatment and the decrease in TK mutant clones following knockdown of PolD3 or BRCA2 suggest that HU causes BIR in this system. Considered with the strong preferential occurrence of mutations downstream of the ectopic repeat in (CTG/CAG)102 cells, these data are consistent with a model in which replication stress at the (CTG/CAG) microsatellite leads to DSBs in forks originating at the c-myc origin, resulting in BIR and mutagenesis of the downstream TK gene.
      We propose that Mus81 cleavage of the stalled rightward moving fork results in a covalently open or closed hairpin end (
      • Zhang Y.
      • Saini N.
      • Sheng Z.
      • Lobachev K.S.
      Genome-wide screen reveals replication pathway for quasi-palindrome fragility dependent on homologous recombination.
      ) of the dTomato chromosome fragment (Fig. 10). Alternatively, Mus81 cleavage may occur as the leftward moving fork stalls at a hairpin structure or template-switched/chicken-foot reversed fork structure. The abundance of green cells after HU treatment implies that the eGFP gene downstream of the (CTG/CAG)100 repeat DSB is replicated by a leftward moving replication fork and is preserved in cells that have lost the dTomato gene (
      • Mayle R.
      • Campbell I.M.
      • Beck C.R.
      • Yu Y.
      • Wilson M.
      • Shaw C.A.
      • Bjergbaek L.
      • Lupski J.R.
      • Ira G.
      DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.
      ).
      We speculate that the non-B DNA structure of the dTomato end blocks repair and yields two DSBs that are functionally single-ended. The unligated DSB also leads to loss of the acentric dTomato chromosome fragment.
      A small fraction of eGFP ends may undergo BIR and generate GCVS cells that are green (mutant dTomato) or yellow, whereas the majority of green cells die, inasmuch as a single unresolved DSB can cause apoptosis (
      • Huang L.C.
      • Clarkin K.C.
      • Wahl G.M.
      Sensitivity and selectivity of the DNA damage sensor responsible for activating p53-dependent G1 arrest.
      ,
      • Rich T.
      • Allen R.L.
      • Wyllie A.H.
      Defying death after DNA damage.
      ). BIR of the dTomato end gives cells that are GCVR (TK mutant) and red (eGFP mutant) or yellow.
      Recently, Mayle et al. (
      • Mayle R.
      • Campbell I.M.
      • Beck C.R.
      • Yu Y.
      • Wilson M.
      • Shaw C.A.
      • Bjergbaek L.
      • Lupski J.R.
      • Ira G.
      DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage.
      ) showed that knockout of Mus81 in yeast could increase BIR mutagenesis. In this system, a mutant form of the FLP recombinase was used to generate a long-lived DNA nick that could be converted to a seDSB when traversed by a replication fork. The authors concluded that Mus81 cleavage of the BIR D-loop normally reduced BIR mutagenesis. In contrast, the present results suggest that earlier cleavage by Mus81 at stalled forks may also increase DSBs. Further experiments are under way to analyze the structures of the right and left DSB ends and test the effects of enzymes involved in BIR in processing DSBs in this system.

      Experimental procedures

      Cell culture

      HeLa/406 acceptor cells contain a single FRT site (
      • Liu G.
      • Malott M.
      • Leffak M.
      Multiple functional elements comprise a mammalian chromosomal replicator.
      ). Cell lines were derived by co-transfecting HeLa/406 cells with dual-fluorescence donor plasmids and the FLP recombinase expression vector pOG44 (
      • O'Gorman S.
      • Fox D.T.
      • Wahl G.M.
      Recombinase-mediated gene activation and site-specific integration in mammalian cells.
      ). Cells were maintained on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 5% CO2 at 37 °C. GCV-resistant cell growth in control (no HU)- or HU-treated cells was assayed in 96-well plates using resazurin (Biotium, catalog no. 30025) according to the manufacturer's directions (
      • Kanagaraj R.
      • Huehn D.
      • MacKellar A.
      • Menigatti M.
      • Zheng L.
      • Urban V.
      • Shevelev I.
      • Greenleaf A.L.
      • Janscak P.
      RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription.
      ) after 14 days of GCV selection, as described (
      • Kononenko A.V.
      • Ebersole T.
      • Vasquez K.M.
      • Mirkin S.M.
      Mechanisms of genetic instability caused by (CGG)n repeats in an experimental mammalian system.
      ).

      Hydroxyurea, aphidicolin, and telomestatin treatment assays

      Cells were treated at a final hydroxyurea concentration of 0.2 mm, aphidicolin at a final concentration of 0.2 μm, and TMS at a final concentration of 0.5 μm. The reagents were added to the medium 24 h after plating the cells and maintained until the start of recovery (2–4 days). Cells were treated with 200 μm H2O2 for 15 min. Treatment and recovery of cells was in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 5% CO2 at 37 °C.

      I-Sce1 transfection

      DF2/myc/(CTG/CAG)100 cells were transfected with Lipofectamine 2000 (Invitrogen, catalog no. 11668-019) and 2 μg of I-Sce1 plasmid (Addgene no. 26477) in a 6-well plate, 24 h postplating. During transfection, medium without antibiotic was used. 8 days after transfection, cells were harvested for analysis by flow cytometry.

      siRNA/shRNA treatment

      The siRNAs used to knock down translesion synthesis polymerases were generously provided by Yanzhe Gao (University of North Carolina). Cells were transfected with Lipofectamine 2000 (Invitrogen 11668-019) and 100 nm (final concentration) of siRNA in a 6-well plate, 24 h postplating. Control experiments were performed using AllStars negative control siRNA (Qiagen, catalog no. 1027281). Cells were allowed to recover 48 h post-transfection for 4 days and then analyzed by flow cytometry. PolD3 was knocked down in the presence of doxycycline (1.25 μg/ml) in cells stably transfected with a SMARTvector Inducible Lentiviral PolD3 shRNA (V3SH11252-226758280, Dharmacon/Horizon).

      Western blotting

      Whole cell lysates from treated or untreated cells were prepared. After SDS-PAGE, membranes were probed using a 1:1000 dilution of antibodies against pChk1 (Cell Signaling, catalog no. 2341), BRCA2 antibody (Calbiochem, catalog no. 3146957; 1:1000), or PolD3 (Invitrogen, catalog no. PA536951; 1:500). Primary antibodies against Pol, Pol κ (1:2000 dilution), Rad18, or Rev1 (1:5000 dilution) were generously provided by Yanzhe Gao (University of North Carolina).

      Flow cytometry

      Cell were trypsinized and centrifuged at 300 × g for 3 min. Medium was aspirated, and cells were washed with cold PBS. After a final wash with PBS, cells were analyzed using a C-Flow® Plus Accuri cytometer. All of the results that compare the effect of treatments on a single cell line within a figure were obtained contemporaneously from sister subcultures split from the same cell population.

      Statistical analysis

      Student's two-tailed t test was used to analyze the statistical significance of the experimental results versus the corresponding paired controls using the “percent green cells” shown in Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9, Figure 10 and Figs. S1S7 and generate p values (GraphPad Prism 8). A value of p < 0.05 was taken to indicate statistical significance.
      Unpaired Student's two-tailed t tests were used to compare (Pu)88 versus (Py)88 cells (Fig. 5) and (CTG)100 versus DF/myc cells treated with TMS with or without HU (Fig. 6), as these cells were derived from separate clonal outgrowths using different integrant constructs.

      DRAQ-7 flow cytometry

      After treatment of cells with replication stress–inducing agent followed by recovery, cells were centrifuged at 500 × g for 3 min. Medium was aspirated, and cells were washed with cold PBS and spun down at 500 × g for 3 min. Cells were permeabilized with 70% ethanol at −20 °C for 20 min or overnight. Cells were centrifuged and washed and resuspended in 1 ml of PBS with RNase A (0.75 μg/ml final concentration) and incubated at 37 °C for 20 min. Finally, DRAQ7 dye (
      • Wlodkowic D.
      • Akagi J.
      • Dobrucki J.
      • Errington R.
      • Smith P.J.
      • Takeda K.
      • Darzynkiewicz Z.
      Kinetic viability assays using DRAQ7 probe.
      ) (Abcam, catalog no. ab109202) was added at a final concentration of 7.5 μm. Cells were incubated in the dark for 25 min and analyzed using a CFlow® Plus Accuri cytometer.

      CD

      CD spectra were collected using a Jasco J-815 CD spectropolarimeter (Jasco Inc., Easton, MD). Spectra were recorded from 320 to 220 nm with a bandwidth of 1.0 nm, scan rate of 50 nm/min, and time constant of 1 s. All DNA samples were dissolved in 10 mm Tris, 1 mm EDTA, pH 7.4, and diluted in water to a working concentration of 10 μm. Telomestatin was added to DNA samples at a final concentration of 50 μm. The CD spectra represent the average of four scans taken at 25 °C and baseline-corrected for buffer. The oligonucleotides used for CD were as follows: c-myc G4, 5′-TGA GGG TGG GGA GGG TGG GTA A, (CTG)12, and (CAG)12.

      Ligation-mediated lamPCR

      LamPCR was performed based on previously described conditions with the following modifications (
      • Li T.W.
      • Weeks K.M.
      Structure-independent and quantitative ligation of single-stranded DNA.
      ,
      • Paruzynski A.
      • Arens A.
      • Gabriel R.
      • Bartholomae C.C.
      • Scholz S.
      • Wang W.
      • Wolf S.
      • Glimm H.
      • Schmidt M.
      • von Kalle C.
      Genome-wide high-throughput integrome analyses by nrLAM-PCR and next-generation sequencing.
      ). Genomic DNA was isolated from untransfected DF2/myc/(CTG/CAG)100 cells, 24 h after transfection with the I-Sce1 expression plasmid, or after 4 days of 0.2 mm HU treatment. Linear amplification was performed using 5 μg of DNA and the downstream biotinylated primer 3′ F0-biot ((biotin)5′-GTCAGCTTGCCGTAGGTGG-3′) for 50 cycles. A second aliquot (0.5 μl) of HotStarTaq was added, and amplification was performed for an additional 50 cycles.
      The linear amplification products were captured on streptavidin beads, washed, and ligated overnight to the adapter oligonucleotide (5′-pATCGACAACAACTCTCCTCCTCCGTGCGddC-3′) (
      • Li T.W.
      • Weeks K.M.
      Structure-independent and quantitative ligation of single-stranded DNA.
      ). The beads were washed, and the ligated products were amplified with the adapter reverse complement primer (5′-CGCACGGAGGAGGAGAGTTGTTGTCGAT-3′) and the nested downstream primer 3′F1 (5′-GCTGAACTTGTGGCCGTTTA-3′). Products were electrophoresed on 1.5% agarose gels.

      Data availability

      All data are contained in the article and supporting information.

      Acknowledgments

      We thank Yanzhe Gao (University of North Carolina) for reagents, David Hitch (Wright State University) for intellectual and technical contributions, and Eric Brown (University of Pennsylvania) for comments on this work.

      Supplementary Material

      References

        • Lander E.S.
        • Linton L.M.
        • Birren B.
        • Nusbaum C.
        • Zody M.C.
        • Baldwin J.
        • Devon K.
        • Dewar K.
        • Doyle M.
        • FitzHugh W.
        • Funke R.
        • Gage D.
        • Harris K.
        • Heaford A.
        • Howland J.
        • et al.
        Initial sequencing and analysis of the human genome.
        Nature. 2001; 409 (11237011): 860-921
        • Subramanian S.
        • Mishra R.K.
        • Singh L.
        Genome-wide analysis of microsatellite repeats in humans: their abundance and density in specific genomic regions.
        Genome Biol. 2003; 4 (12620123): R13
        • Neil A.J.
        • Kim J.C.
        • Mirkin S.M.
        Precarious maintenance of simple DNA repeats in eukaryotes.
        Bioessays. 2017; 39 (28703879): 1700077
        • Kim J.C.
        • Mirkin S.M.
        The balancing act of DNA repeat expansions.
        Curr. Opin. Genet. Dev. 2013; 23 (23725800): 280-288
        • López Castel A.
        • Cleary J.D.
        • Pearson C.E.
        Repeat instability as the basis for human diseases and as a potential target for therapy.
        Nat. Rev. Mol. Cell Biol. 2010; 11 (20177394): 165-170
        • Mirkin S.M.
        DNA structures, repeat expansions and human hereditary disorders.
        Curr. Opin. Struct. Biol. 2006; 16 (16713248): 351-358
        • Mirkin S.M.
        Expandable DNA repeats and human disease.
        Nature. 2007; 447 (17581576): 932-940
        • Voineagu I.
        • Freudenreich C.H.
        • Mirkin S.M.
        Checkpoint responses to unusual structures formed by DNA repeats.
        Mol. Carcinog. 2009; 48 (19306277): 309-318
        • Wang G.
        • Vasquez K.M.
        Impact of alternative DNA structures on DNA damage, DNA repair, and genetic instability.
        DNA Repair (Amst.). 2014; 19 (24767258): 143-151
        • Shastri N.
        • Tsai Y.C.
        • Hile S.
        • Jordan D.
        • Powell B.
        • Chen J.
        • Maloney D.
        • Dose M.
        • Lo Y.
        • Anastassiadis T.
        • Rivera O.
        • Kim T.
        • Shah S.
        • Borole P.
        • Asija K.
        • et al.
        Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition.
        Mol. Cell. 2018; 72 (30293786): 222-238.e11
        • Schmidt M.H.
        • Pearson C.E.
        Disease-associated repeat instability and mismatch repair.
        DNA Repair (Amst.). 2016; 38 (26774442): 117-126
        • Kumari D.
        • Hayward B.
        • Nakamura A.J.
        • Bonner W.M.
        • Usdin K.
        Evidence for chromosome fragility at the frataxin locus in Friedreich ataxia.
        Mutat. Res. 2015; 781 (26379101): 14-21
        • Usdin K.
        Bending the rules: unusual nucleic acid structures and disease pathology in the repeat expansion diseases.
        in: Wells R.D. Ashizawa T. Genetic Instabilities and Neurological Diseases. Elsevier, Amsterdam2006: 617-635
        • Usdin K.
        • Grabczyk E.
        DNA repeat expansions and human disease.
        Cell. Mol. Life Sci. 2000; 57 (10950307): 914-931
        • Genetic Modifiers of Huntington's Disease (GeM-HD) Consortium
        CAG repeat not polyglutamine length determines timing of Huntington's disease onset.
        Cell. 2019; 178 (31398342): 887-900.e14
        • Zhao X.N.
        • Lokanga R.
        • Allette K.
        • Gazy I.
        • Wu D.
        • Usdin K.
        A MutSβ-dependent contribution of MutSα to repeat expansions in fragile X premutation mice?.
        PLoS Genet. 2016; 12 (27427765): e1006190
        • Kumari D.
        • Usdin K.
        Sustained expression of FMR1 mRNA from reactivated fragile X syndrome alleles after treatment with small molecules that prevent trimethylation of H3K27.
        Hum. Mol. Genet. 2016; 25 (27378697): 3689-3698
        • Gerhardt J.
        • Bhalla A.D.
        • Butler J.S.
        • Puckett J.W.
        • Dervan P.B.
        • Rosenwaks Z.
        • Napierala M.
        Stalled DNA replication forks at the endogenous GAA repeats drive repeat expansion in Friedreich's ataxia cells.
        Cell Rep. 2016; 16 (27425605): 1218-1227
        • Lee J.A.
        • Carvalho C.M.
        • Lupski J.R.
        A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders.
        Cell. 2007; 131 (18160035): 1235-1247
        • Cocquempot O.
        • Brault V.
        • Babinet C.
        • Herault Y.
        Fork stalling and template switching as a mechanism for polyalanine tract expansion affecting the DYC mutant of HOXD13, a new murine model of synpolydactyly.
        Genetics. 2009; 183 (19546318): 23-30
        • Zhang F.
        • Khajavi M.
        • Connolly A.M.
        • Towne C.F.
        • Batish S.D.
        • Lupski J.R.
        The DNA replication FoSTeS/MMBIR mechanism can generate genomic, genic and exonic complex rearrangements in humans.
        Nat. Genet. 2009; 41 (19543269): 849-853
        • Koumbaris G.
        • Hatzisevastou-Loukidou H.
        • Alexandrou A.
        • Ioannides M.
        • Christodoulou C.
        • Fitzgerald T.
        • Rajan D.
        • Clayton S.
        • Kitsiou-Tzeli S.
        • Vermeesch J.R.
        • Skordis N.
        • Antoniou P.
        • Kurg A.
        • Georgiou I.
        • Carter N.P.
        • et al.
        FoSTeS, MMBIR and NAHR at the human proximal Xp region and the mechanisms of human Xq isochromosome formation.
        Hum. Mol. Genet. 2011; 20 (21349920): 1925-1936
        • Verdin H.
        • D'Haene B.
        • Beysen D.
        • Novikova Y.
        • Menten B.
        • Sante T.
        • Lapunzina P.
        • Nevado J.
        • Carvalho C.M.
        • Lupski J.R.
        • De Baere E.
        Microhomology-mediated mechanisms underlie non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory domain.
        PLoS Genet. 2013; 9 (23516377): e1003358
        • Hsiao M.C.
        • Piotrowski A.
        • Callens T.
        • Fu C.
        • Wimmer K.
        • Claes K.B.
        • Messiaen L.
        Decoding NF1 intragenic copy-number variations.
        Am. J. Hum. Genet. 2015; 97 (26189818): 238-249
        • Vogt J.
        • Wernstedt A.
        • Ripperger T.
        • Pabst B.
        • Zschocke J.
        • Kratz C.
        • Wimmer K.
        PMS2 inactivation by a complex rearrangement involving an HERV retroelement and the inverted 100-kb duplicon on 7p22.1.
        Eur. J. Hum. Genet. 2016; 24 (27329736): 1598-1604
        • Deshpande M.
        • Gerhardt J.
        Break-induced replication sparks CGG-repeat instability.
        Nat. Struct. Mol. Biol. 2018; 25 (30061599): 643-644