RESEARCH ARTICLE

Polη, a Y-family translesion synthesis polymerase, promotescellular tolerance of Myc-induced replication stressKiminori Kurashima1,*, Takayuki Sekimoto1,*, Tsukasa Oda1, Tsuyoshi Kawabata2, Fumio Hanaoka3 andTakayuki Yamashita1,‡

ABSTRACTGrowth of precancerous and cancer cells relies on their tolerance ofoncogene-induced replication stress (RS). Translesion synthesis (TLS)plays an essential role in the cellular tolerance of various types ofRS and bypasses replication barriers by employing specializedpolymerases. However, limited information is available about the roleof TLS polymerases in oncogene-induced RS. Here, we report thatPolη, a Y-family TLS polymerase, promotes cellular tolerance ofMyc-inducedRS. Polηwas recruited toMyc-inducedRSsites, andPolηdepletion enhanced theMyc-induced slowing and stalling of replicationforks and the subsequent generation of double-strand breaks (DSBs).Overexpression of a catalytically dead Polη also promoted Myc-induced DSB formation. In the absence of Polη, Myc-induced DSBformation depended on MUS81–EME2 (the S-phase-specificendonuclease complex), and concomitant depletion of MUS81–EME2 and Polη enhanced RS and cell death in a synergisticmanner. Collectively, these results indicate that Polη facilitates forkprogression during Myc-induced RS, thereby helping cells tolerate theresultant deleterious effects. Additionally, the present study highlightsthe possibility of a synthetic sickness or lethality between Polηand MUS81–EME2 in cells experiencing Myc-induced RS.

KEYWORDS: c-Myc, Y-family polymerase, Polη, Oncogene-inducedreplication stress, MUS81–EME2, Synthetic sickness,Synthetic lethality

INTRODUCTIONComplete and correct replication of DNA is essential formaintaining genome integrity and cell growth. However, obstaclessuch as DNA lesions generated by genotoxic agents often inhibit thereplication machinery. The resultant slowing or stalling of thereplication fork is referred to as ‘replication stress’ (RS). RS causesDNA damage, promoting genomic instabilities and cell death and/or senescence. To counteract these pathological effects, normal cellshave evolved multiple RS response pathways that mediate cell cyclecheckpoints and rescue fork slowing/stalling (Berti and Vindigni,2016; Zeman and Cimprich, 2014).

Oncogene-induced RS (OIRS) is a major source of genomicinstability driving tumor development, but paradoxically, it has adeleterious effect on precancerous and cancer cells (Gaillard et al.,2015; Halazonetis et al., 2008). In this context, RS responsepathways support the growth and survival of neoplastic cells,thereby providing potential targets for cancer therapy (Boyer et al.,2016; Puigvert et al., 2016). For example, inhibitors of ATR, acentral player in cell cycle checkpoint signaling, exhibit tumor-selective cytotoxic effects and are currently being investigated inclinical trials (Karnitz and Zou, 2015). For these reasons, increasedattention has focused on the molecular mechanisms of the OIRSresponse pathways, such as Rad51-mediated fork reversal, POLD3-mediated break-induced replication and RecQ helicase-mediatedfork remodeling (Chen et al., 2015; Costantino et al., 2014; Murfuniet al., 2013; Neelsen et al., 2013).

The c-Myc protein (Myc) is a transcriptional regulator of diversegenes involved in cell cycle progression, metabolism and apoptosis(Dang, 2013; McMahon, 2014). Overexpression of Myc drivesoncogenesis in many tumor types, not only by stimulating cellproliferation but also by promoting genomic instabilities (Rohban andCampaner, 2015). Although an early study attributed this effect toMyc-induced oxidative DNA damage (Vafa et al., 2002), morerecent studies have indicated that abnormal replication dynamics,accompanied with origin hyperactivation, are responsible forMyc-induced RS and DNA damage (Dominguez-Sola et al., 2007;Srinivasan et al., 2013). Myc-induced origin hyperactivation might bemediated by increased transcription of licensing factors such as Cdt1or MCM helicases (Perna et al., 2012; Valovka et al., 2013). Anothermechanism of origin hyperactivation that has been proposed is thatMyc activates origin firing through direct interaction with thepre-replicative complex in a transcription-independent manner(Dominguez-Sola et al., 2007; Srinivasan et al., 2013). Importantly,this proposed mechanism was experimentally replicated in cell-freeXenopus extracts, where RS can occur without transcription(Dominguez-Sola et al., 2007; Srinivasan et al., 2013). Additionally,the effect of Myc overexpression can be mimicked in vitro and in vivoby overexpression of Cdc45, a cofactor for the replicative helicasecomplex (Srinivasan et al., 2013). Thus, Myc-induced RS provides animportant model for studying OIRS response pathways.

Translesion synthesis (TLS) plays an essential role in preventingfork stalling at the DNA lesions induced by genotoxic agents. TLSutilizes specialized polymerases to bypass such lesions (VaismanandWoodgate, 2017). However, little is known of the role played byTLS in OIRS. TheY-family polymerases (Y-Pols) – Polη, Polι, Polκand REV1 – form the major group of TLS polymerases (Sale et al.,2012). Although Y-Pols each contain common low-stringencycatalytic domains, eachmember has a distinct enzymatic feature. Forinstance, Polη performs particularly efficient TLS across the UV-induced DNA lesion known as a cyclobutane pyrimidine dimer.Inherited deficiency of Polη causes a variant form of xerodermaReceived 17 October 2017; Accepted 10 May 2018

1Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation,Gunma University, 371-8512 Gunma, Japan. 2Department of Stem Cell Biology,Atomic Bomb Disease Institute, Nagasaki University, 852-8523 Nagasaki, Japan.3Department of Life Science, Faculty of Science, Gakushuin University, 171-8588Tokyo, Japan.*These authors contributed equally to this work

‡Author for correspondence (y-taka@gunma-u.ac.jp)

T.S., 0000-0001-6966-6706; T.O., 0000-0002-8941-161X; T.Y., 0000-0003-2074-9310

1

© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

pigmentosum (XP-V), a human genetic disease characterized bysunlight hypersensitivity and skin cancer susceptibility (Johnsonet al., 1999; Masutani et al., 1999). Of interest, several studies reportthat Y-Pols catalyze DNA synthesis in various situations otherthan canonical TLS. These situations include homologousrecombination, nucleotide excision repair, re-replication andreplication of ‘hard-to-replicate’ regions, such as common fragilesites (CFSs) (Bergoglio et al., 2013; McIlwraith et al., 2005; Ogiet al., 2010; Rey et al., 2009; Sekimoto et al., 2015). In the presentstudy, we aimed to explore the role of Y-Pols in Myc-induced RS.Our data indicate that Polη alleviates Myc-induced RS, thuscontributing to cellular tolerance of the resultant deleteriouseffects. Notably, we provide evidence that the S-phase-specificendonuclease complex MUS81–EME2 (Pepe and West, 2014a),limits Myc-induced RS and cell death in the absence of Polη. Thisfinding suggests that MUS81–EME2 is a potential target forinducing synthetic sickness or lethality as a treatment approach forPolη-deficient tumors in XP-V patients.

RESULTSPolη knockdown enhances Myc-induced growth arrest andcell deathTo explore the role of Y-Pols in Myc-induced RS, we used U2OScells stably expressing human Myc fused to the tamoxifen-sensitivehormone-binding domain of the estrogen receptor α (Myc–ER)protein (denoted U2OS/Myc–ER cells). Treating these cells with4-hydroxytamoxifen (4OHT) increased the nuclear levels ofMyc–ER and chromatin-bound Myc–ER by ∼7-fold more thanthe levels of endogenous Myc at 48 h (Fig. S1A,B) and decreasedthe cell growth and survival at 72–96 h (Fig. S1C). These resultsare consistent with the data reported previously (Maya-Mendozaet al., 2014). The Myc–ER levels in whole-cell lysates (WCLs) andthe chromatin-bound fraction in the 4OHT-treated U2OS/Myc–ERcells were similar to the Myc protein levels in the Myc-drivenmyeloma tumor cell lines KMS12PE and KMM1 (Fig. S1D). Theflow cytometric analysis showed that 4OHT-treated U2OS/Myc–ER cells entered S-phase and actively synthesized DNA at 24–48 hpost-treatment. This was followed by reduced DNA synthesis, asmeasured by iododeoxyuridine (IdU) uptake and increased cellcycle arrest through the G1 to G2 phases (Fig. S1E). There was alsoincreased apoptotic cell death, as shown by the increased subG1fraction. To explore the roles of the Y-Pol members inMyc-induced RS, we transfected cells with siRNAs targetingeach Y-Pol member 24 h before the 4OHT treatment. We assessedthe effects of these treatments on the cell numbers and on thecell cycle progression. Our results revealed that the siRNAseffectively depleted each polymerase (Fig. 1A). Of note, thesiRNAs specifically targeting Polη (encoded by POLH) (siPolη)significantly suppressed increases in cell numbers in the 4OHT-treated cells (Fig. 1B). This effect was associated with an increasedproportion of G2 and subG1 cells and a decreased proportion of G1cells (Fig. 1C). Smaller changes in the proportion of cells in G1 andG2 following siPolη transfection were noted in the vehicle-treatedcells, which is presumably attributable to Polη depletion-inducedinstability in CFSs (Bergoglio et al., 2013; Rey et al., 2009). Theincrease in apoptosis was confirmed by the increased proportion ofcells that were positive for cleaved caspase-3 (Fig. 1D). Overall, ourdata suggest that Polη plays a critical role in cellular tolerance ofMyc-induced RS. Of note, depletion of Polκ (encoded by POLK)had smaller but similar effects on the cell cycle, raising thepossibility that Polη is not the only TLS polymerase with thisfunction (Fig. 1B–D).

Polη is recruited to Myc-induced replication sitesTo further test the idea that Polη is involved in the Myc-induced RSresponse pathway, we studied whether Polη is recruited toreplication sites in Myc-activated cells. To test this hypothesis, weproduced a transformant of U2OS/Myc–ER cells that stablyexpressed GFP-tagged Polη (GFP–Polη) at ∼20-fold higher levelsthan endogenous Polη (U2OS/Myc–ER/GFP–Polη). U2OS/Myc–ER/GFP–Polη cells were pulse-labeled with bromodeoxyuridine(BrdU) and then immunostained to visualize BrdU and GFP–Polη.In these cells, 4OHT treatment progressively increased localizationof GFP–Polη in active (BrdU-labeled) replication foci in 20–70% ofthe DNA-synthesizing cells at 24–96 h post-treatment (Fig. 2A,B).This recruitment was rarely (<3%) observed in the control cells.Importantly, analysis of the U2OS/Myc–ER cells revealed that mostof the endogenous Polη foci also overlapped with BrdU foci in∼30% of the replicating cells after 4OHT treatment (Fig. 2C,D).Collectively, these data indicate that Polη is recruited to Myc-induced replication sites.

The following four situations are proposed to result in themajority of OIRS: (1) nucleotide deficiency via excessiveconsumption (Bester et al., 2011), (2) replication–transcriptionconflicts (Jones et al., 2013; Kotsantis et al., 2016), (3) oxidativeDNA damage (Vafa et al., 2002), and (4) hyperactivation of originfiring (Dominguez-Sola et al., 2007; Srinivasan et al., 2013). Tostudy which of these mechanisms is involved in Myc-induced Polηrecruitment to replication foci in the U2OS/Myc–ER/GFP–Polηcells, we performed the following series of experiments. First, westudied the effects of exogenously supplying dADP, dGDP, dCDPand dUMP to the culture medium to test whether nucleotidedeprivation in the cells was responsible for the Polη accumulationin replication foci. This treatment significantly suppressedhydroxyurea (HU)-induced Polη foci formation but had littleeffect on Myc-induced Polη foci formation (Fig. S2A), indicatingthat nucleotide deficiency does not explain Myc-induced Polη fociformation. These results are consistent with previous observationsshowing that Myc activation upregulates the expression ofnucleotide biosynthetic enzymes, thus rescuing the OIRS causedby nucleotide deficiency (Bester et al., 2011; Liu et al., 2008;Mannava et al., 2008).

Next, we performed knockdowns of alternative splicing factor(ASF; encoded by SRSF1) or topoisomerase I (Topo I; subunitencoded by TOP1) using siRNAs in order to induce replication–transcription conflicts (Fig. S2B). These treatments increased thenumber of γH2AX foci, especially in replicating cells, in a mannerhighly sensitive to RNase H1 expression (Fig. S2C), as reportedpreviously (Tuduri et al., 2009). However, they had little effecton Polη foci formation (Fig. S2D), indicating that replication–transcription conflicts do not induce Polη foci formation.Furthermore, the Myc-induced formation of Polη foci was notaffected by expression of RNase H1 (Fig. S2E). Taken together, theseresults indicate that replication–transcription conflicts do not accountfor Myc-induced Polη foci formation. These results are consistentwith the finding that Myc-induced RS is observed in an in vitroreplication system using Xenopus egg extracts in which transcriptionis absent (Dominguez-Sola et al., 2007; Srinivasan et al., 2013).

To test the involvement of oxidative DNA damage in Myc-induced RS, we examined the effects of N-acetyl cysteine (NAC), ascavenger of reactive oxygen species (ROS), on the Myc-inducedfoci formation of GFP–Polη. The presence of NAC in the culturemedium effectively decreased hydrogen peroxide-induced Polηfoci, but had little effect on Myc-induced Polη foci (Fig. S2F).This indicates that oxidative stress is not involved in this process,

2

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

a result consistent with the previous observation that detectionof Myc-induced RS precedes increased ROS (Maya-Mendozaet al., 2014).

Finally, we tested whether hyperactivation of origin firing isresponsible for Myc-induced RS (Fig. 3). To this end, we produceda knockdown of the Cdc45 cofactor of the replicative helicase

Fig. 1. See next page for legend.

3

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

complex and examined its effect on Myc-induced replication andGFP–Polη localization, because Cdc45 is reported to play a criticalrole in Myc-induced origin firing (Srinivasan et al., 2013). Becausetransfecting Cdc45 siRNAs at a high concentration (30 nM)induced severe depletion of Cdc45 and resulted in a markedsuppression of DNA synthesis (data not shown), we conducted the

experiment at a lower concentration (7.5 nM). The mild reduction inCdc45 levels induced by this treatment had little effect on DNAsynthesis but significantly suppressed Polη localization inreplication foci (Fig. 3A–C). We next studied the reciprocal effectof Cdc45 overexpression on Polη foci formation. The resultsrevealed that Polη recruitment to replication foci was markedlyenhanced in the Cdc45-expressing cells compared with the Cdc45non-expressing cells (Fig. 3D–F). Collectively, these results suggestthat Cdc45-mediated hyperactivation of origin firing plays a criticalrole in Myc-induced RS, a finding consistent with a previous study(Srinivasan et al., 2013).

Rad18-mediated monoubiquitylation of PCNA is required forPolη recruitment to Myc-induced replication sitesRS activates Rad18/Rad6-mediated monoubiquitylation ofproliferating cell nuclear antigen (PCNA) at the amino acid residueK164. The subsequent interaction betweenmonoubiquitylated PCNA(Ub–PCNA) and Polη plays a central role in recruiting Polη toreplication foci (Kannouche et al., 2004; Watanabe et al., 2004). Tounderstand themolecularmechanisms underlying Polη recruitment toMyc-induced replication sites, we performed a series of analyses of4OHT-treated U2OS/Myc–ER/GFP–Polη cells, comparing someaspects of the analyses with what was seen in UV-irradiated cells.First, we monitored the levels of Ub–PCNA and GFP–Polη, and theinteraction between GFP–Polη and Ub–PCNA in the chromatin

Fig. 1. Polη knockdown enhances Myc-induced growth arrest andapoptosis. (A) U2OS/Myc–ER cells were transfected with siRNAs targetingeach Y-Pol member [Polη, Polι, Polκ (POLH, POLI and POLK, respectively),REV1], or with negative control (N.C.) siRNA. After 48 h, WCLs were preparedand immunoblotted. The asterisk indicates a non-specific signal. (B–D) U2OS/Myc–ER cells were transfected with the siRNAs as described above, andsubsequently treated with vehicle or 4OHT (0.5 µM) at 24 h after transfection.(B) Relative increases in cell number at 48 and 72 h after drug treatment areshown as fold increases over the 0 h values. The values represent themean±s.d. of three independent experiments. (C) Cells were fixed at 48 hafter drug treatment, and the DNA content was analyzed by flow cytometry.Left, representative cell cycle profiles. Right, bar graphs showing the cellpercentages in the subG1, G1, S or G2 growth phases. The values representthe mean±s.d. of three independent experiments. PI, propidium iodide.(D) Cells were fixed at 48 h after drug treatment and immunostained with ananti-cleaved caspase-3 antibody. Nuclei were stained with DAPI. Left,representative images from the cleaved caspase-3 staining experiments.Right, bar graph showing the cleaved caspase-3-positive cell percentages.The values represent the mean±s.d. of three independent experiments.Scale bars: 10 µm. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001.

Fig. 2. Polη is localized in replication foci in Myc-activated cells. (A,B) U2OS/Myc–ER/GFP–Polη cells were treated with vehicle or 4OHT for the timesindicated. The cells were labeledwith BrdU, extractedwith CSK buffer and fixed, then immunostained with anti-BrdU and anti-Polη antibodies. Nuclei were stainedwith DAPI. (A) Representative images of BrdU foci and GFP–Polη foci at 96 h after drug treatment. Scale bars: 10 µm. (B) Bar graph showing the percentagesof BrdU foci-positive cells, GFP–Polη foci-positive cells and cells in which GFP–Polη foci colocalized with BrdU foci [BrdU/GFP–Polη foci (+)]. The valuesrepresent the mean±s.d. of three independent experiments. (C,D) U2OS/Myc–ER cells were treated with vehicle or 4OHT for 96 h. The cells were labeled withBrdU and immunostained as described in A and B. (C) Representative images of BrdU foci and endogenous Polη foci. Scale bars: 10 µm. (D) Bar graph showingthe percentages of BrdU foci-positive cells, endogenous Polη foci-positive cells and cells in which endogenous Polη foci colocalized with BrdU foci [BrdU/Polηfoci (+)]. The results were reproduced in two independent experiments of which one is shown.

4

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

fraction in 4OHT-treated U2OS/Myc–ER/GFP–Polη cells. Theresults showed that the levels of Ub–PCNA and GFP–Polη andtheir interaction increasedwith similar time courses to that seen for the

formation of GFP–Polη foci (Fig. 4A,B). Second, we analyzedmonoubiquitylation of the FLAG- and hemagglutinin (HA)-taggedPCNA (K164R) mutant after 4OHT treatment and UV irradiation.

Fig. 3. Polη foci formation is caused by replication origin hyperactivation. (A–C) U2OS/Myc–ER/GFP–Polη cells were transfected with the siRNAs indicated(7.5 nM) and subsequently treated with vehicle or 4OHT at 24 h post transfection. At 48 h after drug treatment, WCLs were prepared and the cells were fixed andimmunostained as described in Fig. 2. (A) Representative images of BrdU foci and GFP–Polη foci. Scale bars: 10 µm. (B) WCL immunoblots. The relativeamounts of Cdc45 normalized against GAPDH are shown. (C) Bar graph showing cell percentages, as described in Fig. 2B. The values represent the mean±s.d.of three independent experiments. (D–F) U2OS/GFP–Polη cells lentivirally transduced with C-terminally FLAG-tagged Cdc45 (Cdc45-F) or transduced withempty vector (E.V.). At 48 h post transduction, WCLs were prepared for immunoblotting while the cells were labeled with EdU and then fixed. EdU was visualizedby click chemistry, and the cells were then immunostained with anti-Cdc45 and anti-Polη antibodies. Nuclei were stained with DAPI. (D) Representative images ofEdU, GFP–Polη and Cdc45-F staining. Scale bars: 10 µm. (E) WCL immunoblots. (F) Cells transduced with Cdc45-F were grouped into Cdc45-F-positive[Cdc45-F (+)] and Cdc45-F negative [Cdc45-F (−)] subpopulations according to the Cdc45-F immunostaining level. Bar graph showing cell percentages, asdescribed in Fig. 2B (EdU instead of BrdU). The values represent the mean±s.d. of three independent experiments.

5

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

We found that the mutant protein was not monoubiquitylated uponeither treatment, indicating that the K164 amino acid residue is thetarget for monoubiquitylation after both treatments (Fig. 4C). Third,the siRNA-mediated depletion of Rad18 suppressed increases in theUb–PCNA levels and the proportion of Polη foci-positive cellsinduced by 4OHT treatment and UV irradiation (Fig. 4D,E; data

not shown). Taken together, these results indicate that increasedRad18-mediated production of Ub–PCNA is responsible forMyc-induced Polη recruitment to replication sites. We also notedthat diubiquitylated PCNAwas detected in the 4OHT-treated cells aswell as in the aphidicolin-treated cells (Fig. 4F), raising the possibilitythat PCNA polyubiquitylation-mediated Polη-independent lesion

Fig. 4. Rad18-mediated monoubiquitylation of PCNA is required for its interaction with Polη and for Polη recruitment to Myc-induced replication sites.(A) U2OS/Myc–ER/GFP–Polη cells treated with 4OHT were extracted with CSK buffer, fixed and immunostained with an anti-Polη antibody at the time pointsindicated. The percentages of the GFP–Polη foci-positive cells are shown. Data represent the mean±s.d. of three independent experiments. (B) U2OS/Myc–ER/GFP–Polη cells were treated with vehicle or 4OHT. The CSK buffer-insoluble fractions were prepared at the times indicated. They were immunoprecipitated(IP) and then immunoblotted (top panels), or directly immunoblotted (bottom panels). As a positive control, extracts were prepared 16 h after UV irradiation(5 J/m2). The relative amounts of immunoprecipitated GFP–Polη are shown under the top blot. (C) U2OS/Myc–ER/GFP–Polη cells stably expressing FLAG- andHA-tagged wild-type PCNA (WT), or mutant PCNA (K164R), were treated with vehicle or 4OHT for 48 h. The CSK buffer-insoluble fractions were immunoblotted.(D,E) U2OS/Myc–ER/GFP–Polη cells were transfected with the siRNAs indicated and subsequently treated with vehicle or 4OHT at 24 h post transfection.(D) At 72 h after drug treatment, the insoluble fractions in CSK buffer and the WCLs were immunoblotted. UV-irradiated cells served as the positive control.(E) At 72 h after drug treatment, the cells were immunostained as shown in A. The percentages of GFP–Polη foci-positive cells are shown. The values representthe mean±s.d. of three independent experiments. (F) U2OS/Myc–ER/GFP–Polη cells were treated with vehicle (−) or 4OHT (+). The CSK buffer-insolublefractions were prepared at the times indicated and immunoblotted. As a positive control, extracts were prepared 6 or 16 h after treatment with 10 µg/ml aphidicolin.Ub–PCNA and diubiquitylated (Ub2-PCNA) are indicated.

6

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

bypass pathways, such as template switching (Mailand et al., 2013),may be involved in the Myc-induced RS response.

Polη depletion promotes Myc-induced DSB formationMyc-induced RS leads to the formation of double-strand breaks(DSBs) in DNA molecules (Dominguez-Sola et al., 2007; Maya-Mendoza et al., 2014; Srinivasan et al., 2013). Hence, we next studiedthe impact of Polη depletion on Myc-induced DSB formation byperforming various different assays. First, immunoblot analysesshowed that γH2AX levels increased progressively from 24–96 hafter 4OHT-treatment of U2OS/Myc–ER cells (Fig. S3A), aspreviously reported (Maya-Mendoza et al., 2014). These increaseswere significantly enhanced by siRNA-mediated depletion of Polη(Fig. 5A). Next, we employed the neutral comet assay to directlyestimate the number of DSBs. Consistent with the above results, thenumber of DSBs increased in the 4OHT-treated U2OS/Myc–ERcells, and this increase was further promoted by Polη depletion(Fig. 5B; Fig. S3B). Finally, we asked whether the DSB-promotingeffect of Polη depletion is related to potentiation of Myc-inducedRS. To address this question, we performed an assay specificfor replication-associated DNA damage: γH2AX/PCNA doublestaining. We found that Polη depletion synergistically enhanced theMyc-induced increase in nuclear γH2AX intensity in PCNA-positivecells (Fig. 5C; Fig. S3C). In this experiment, most of the cells withhigh intensities of γH2AX were detected in PCNA-positive cellsduring S-phase (Fig. 5D). Although siPolη #4 had a larger effectthan siPolη #3 on γH2AX levels throughout the experiments in thissection, this result cannot be explained by a difference in knockdownefficiency (Fig. 5A), and is presumably caused by extra non-specificeffects. In summary, taken together, our results in this section indicatethat Polη depletion promotes DSB formation resulting from Myc-induced RS.

Myc-induced cell cycle arrest and γH2AX induction arepotentiated in Polη-deficient fibroblastsTo confirm the role of Polη in the cellular response to Myc-inducedRS, we expressedMyc–ER in anXP-V patient-derived Polη-deficientfibroblast clone (XPV-Neo/Myc–ER) and in its counterpartcomplemented with the wild-type (WT) Polη (XPV-Polη/Myc–ER)(Fig. S4A). 4OHT treatment increased the levels of chromatin-boundMyc–ER in both cell lines. This treatment also increased the levels ofUb–PCNA and Polη in the XPV-Polη/Myc–ER cells; however, theincrease in Ub–PCNA levels was slight in the XPV-Neo/Myc–ERcells, presumably because Ub–PCNA is unstable in the absence ofPolη (Durando et al., 2013) (Fig. S4A). In∼7% of the 4OHT-treated,but not in the vehicle-treated XPV-Polη/Myc–ER cells, Polη wasdetected in replication foci (Fig. S4B). These observations suggestthat Polη is recruited to Myc-induced RS sites in these fibroblasts,similar to what is seen in the U2OS/Myc–ER cells. Cytometricanalysis showed that 4OHT treatment negligibly affected the XPV-Polη/Myc–ER and the XPV-Neo/Myc–ER cell populations in termsof their cell cycles (Fig. S4C). Notably, our comparison of the cellpopulations with high levels of chromatin-bound of Myc–ERrevealed that the G2 arrest was a more dramatic feature in the XPV-Neo/Myc–ER cells than in the XPV-Polη/Myc–ER cells (Fig. S4D).Furthermore, in the cell populations with high levels of chromatin-bound Myc–ER, the 4OHT-treated XPV-Neo/Myc–ER cellscontained significantly higher levels of γH2AX than those of the4OHT-treated XPV-Polη/Myc–ER cells (Fig. S4E,F). As expected,the highest levels of γH2AX in the XPV-Neo/Myc–ER cells wereobserved in the late S and G2 phases (Fig. S4G), suggesting thatan association exists between Myc-induced RS and G2 arrest in

these cells. Taken together, these results indicate that Polη alleviatesMyc-induced RS in the fibroblasts.

Polη depletion promotes Myc-induced fork stallingWe reasoned that the increases in Myc-induced growth arrest,apoptosis and DSB formation in Polη-depleted cells result fromincreased fork slowing and stalling (Dominguez-Sola et al., 2007;Maya-Mendoza et al., 2014; Srinivasan et al., 2013). To verify thisnotion, the effect of Polη depletion on Myc-induced alterationof replication dynamics was analyzed. To this end, we performedDNA fiber analysis in control and siPolη-transfected U2OS/Myc–ER cells. Cells were treated with 4OHTand sequentially pulsed withIdU (10 min) and chlorodeoxyuridine (CldU) (20 min) to label thenewly synthesized DNA (Fig. 6A). Analysis of the samplesobtained at 48 h after drug treatment showed that the mean inter-origin distance was shorter in the 4OHT-treated cells than in thecontrol cells (Fig. S5A,B), indicating that Myc activation promotesorigin firing, as previously reported (Srinivasan et al., 2013).It should be noted that Polη depletion had little effect on the meaninter-origin distances in these cells (Fig. S5B).

Next, we analyzed the effect of Myc activation on fork velocityby measuring the lengths of IdU- and CldU-labeled tracks. Theresults showed that the Myc activation induced by 4OHT decreasedthe fork velocity significantly, and this decrease was enhanced bytransfection with the two different siPolηs (Fig. 6B,C). Becausethese data reflect both fork slowing and stalling, we estimated theextent of fork asymmetry as an indicator of fork stalling. Sister forksprogress bidirectionally at a similar velocity from a fired origin incells without RS (Fig. 6D). We measured the lengths of the CldUtracts for each pair of sister forks, and plotted each pair as theright length versus the left length. This revealed that there was asignificant increase in fork asymmetry at 48 h after 4OHT treatment(Fig. 6E; Fig. S5C). This increase was synergistically potentiated byPolη depletion. Collectively, these data indicate that Polη protectsfork progression from Myc-induced fork slowing and stalling.

Polη catalytic activity is required for suppressing Myc-induced RSBecause Polη interacts with a number of replication-associatedproteins including other DNA polymerases (Akagi et al., 2009;Baldeck et al., 2015; Ohashi et al., 2009; Yuasa et al., 2006), itsnon-catalytic role may be involved in preventing Myc-induced RS.To test whether the catalytic role of Polη is critical for this action, weused a catalytically dead Polη mutant (dead-Polη), which hasD115A and E116A amino acid mutations in the catalytic domain.Overexpression of this mutant would be expected to exert adominant-negative effect on the catalytic function of endogenousPolη, promoting DSB formation in stalled forks, as reportedpreviously (Bergoglio et al., 2013). By means of lentivirus vectors,we transduced GFP-tagged WT-Polη and dead-Polη into U2OS/Myc–ER cells at comparable levels (Fig. 7A). At 48 h after 4OHTtreatment of these transformants, the nuclear foci of γH2AX, PCNAand GFP–WT-Polη or dead-Polη were analyzed. While Mycactivation induced formation of GFP–WT-Polη and dead-Polηnuclear foci to similar levels, γH2AX colocalized with almost all ofthe dead-Polη foci but not with the WT-Polη foci (Fig. 7B,C). Inaddition, almost all of the mutant and WT-Polη foci colocalizedwith PCNA (Fig. 7D). Taken together, these results indicate that thecatalytic activity of Polη is required to prevent Myc-induced DSBformation in the replication sites. This further supports the notionthat Polη-mediated fork progression across Myc-induced barrierslimits DSB formation.

7

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

MUS81–EME2-dependent cleavage of Myc-induced stalledforks limits cell deathThe above results raise the possibility that inhibition of Polηincreases the number of Myc-induced stalled forks, which are then

converted into DSBs. Another possible mechanism is that impairedreplication of CFSs leads to cleavage of the DNA into DSBs,because Polη is involved in replication of CFSs (Bergoglio et al.,2013; Rey et al., 2009). MUS81 is the major structure-specific

Fig. 5. Depletion of Polη promotes Myc-induced DSB formation. U2OS/Myc–ER cells were transfected with the N.C. siRNA or Polη siRNA #3 or #4 andsubsequently treatedwith vehicle (−) or 4OHT (+) at 24 h post transfection. At 48 h after drug treatment, each experimentwas performed. (A)WCLswere preparedandimmunoblotted. The relative amounts of γH2AX normalized against H2AX are shown. The results were reproduced in three independent experiments of which one isshown. (B) Neutral comet assay. Dot plot showing the tail moment distributions (arbitrary units, A.U.). The median scores are represented by horizontal lines andshown under the graph. Statistical differences between the distributions were assessed with a Mann–Whitney rank sum test. The results were reproduced in twoindependent experiments of which one is shown. (C,D) After extraction with 0.2% Triton X-100 and fixation, the cells were immunostained with anti-γH2AX andanti-PCNA antibodies, and nuclei were stained with DAPI (refer to Fig. S3C). (C) Dot plot showing the γH2AX intensity distribution per nucleus in the PCNA-positivecells. The median scores are represented by horizontal lines and shown under the graph. Statistical differences between the distributions were assessed with aMann–Whitney rank sum test. The results were reproduced in two independent experiments of which one is shown. (D) Scatter plots of γH2AX intensity versus DNAcontent. Red and black dots indicate PCNA-positive [PCNA (+)] and -negative [PCNA (−)] cells, respectively. The percentage of PCNA/γH2AX-double positive cells inS-phase (blue region) is indicated.

8

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

endonuclease involved in cleavage of stalled forks and CFSs(Franchitto et al., 2008; Hanada et al., 2007; Naim et al., 2013;Regairaz et al., 2011; Ying et al., 2013). A recent study reported thatthese two processes are mediated by different MUS81 heterodimers,with stalled forks and CFSs being cleaved by MUS81–EME2 andMUS81–EME1, respectively (Pepe andWest, 2014a). To attempt toclarify the role of these heterodimers in DSB formation in cellswhere Myc is activated and Polη is depleted, we transfected siRNAstargetingMUS81, EME1 and EME2 into U2OS/Myc–ER cells. ThesiRNA transfections resulted in significantly suppressed expressionof the targets (Fig. 8A,B). Additionally, the neutral comet assayanalysis revealed that DSB formation in these cells was suppressedby the knockdown of MUS81 or EME2 but not EME1 (Fig. 8C).These results indicate that the MUS81–EME2 complex-mediated

cleavage of stalled forks mainly contributes to DSB formation. Thisis consistent with the notion that Polη rescues Myc-induced forkstalling rather than CFS instability, as shown in the data from theDNA fiber analysis. While the MUS81-mediated DSBs potentiallylead to apoptosis (Forment et al., 2011; Fugger et al., 2013),paradoxically they initiate homologous recombination-mediatedfork repair, promoting fork restart and cell survival (Franchitto et al.,2008; Hanada et al., 2007; Murfuni et al., 2013; Regairaz et al.,2011). Therefore, we reasoned that MUS81–EME2 might alleviateMyc-induced RS in the absence of Polη. To address this hypothesis,we assessed the impact of MUS81, EME1 and EME2 depletion onRPA2 foci formation as a measurement of Myc-induced RS and celldeath. The results showed that co-depletion of Polη and MUS81 orEME2, but not EME1, increased RPA2 foci formation and cell

Fig. 6. Polη knockdown enhances Myc-induced fork stalling. U2OS/Myc–ER cells were transfected with the siRNAs indicated and subsequently treated withvehicle or 4OHTat 24 h post transfection. At 48 h after drug treatment, DNA fiber assayswere performed. (A) Schematic representation of dual labeling in the DNAfiber assay. Replication forks were labeled with IdU (red) for 10 min followed by CldU (green) for 20 min. (B) Representative images of replication tracks. Aschematic representation of the track length is shown underneath. Scale bars: 5 µm. (C) The fork velocities were measured and plotted. At least 150 forks wereexamined in each sample. The lines are the median scores. Differences between distributions were assessed with a Mann–Whitney rank sum test. The resultswere reproduced in two independent experiments of which one is shown. (D) Representative images of symmetric and asymmetric forks. A schematicrepresentation of an fork where replication is continuing (ongoing) and a stalled fork is shown under each panel. Scale bars: 5 µm. (E) Bar graphs show thepercentages of the asymmetric forks (refer to Fig. S5C). To score the asymmetric forks, at least 50 forks were measured. The values represent the mean±s.d. ofthree independent experiments. ***P<0.005; ****P<0.001; N.S., not significant.

9

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

death in a more than additive manner (Fig. 8D,E; Fig. S6). Thissuggests that MUS81–EME2 functions as a back-up for Polη toprevent Myc-induced RS.

DISCUSSIONMyc-induced RS plays a critical role in genomic instability duringtumorigenesis and provides potential target molecules for cancertreatment. However, the regulatory mechanisms underlying thecellular responses to Myc-induced RS are poorly understood(Rohban and Campaner, 2015). In the present study, we exploredthe roles played by Y-Pols, and found that depletion of Polηsignificantly impaired cellular tolerance of Myc-induced RS. Aninitial clue about the possible involvement of Polη came from theobservation that the growth of 4OHT-treated but not control U2OS/Myc–ER cells was suppressed to a significantly larger extentfollowing Polη depletion compared with depletion of the otherY-Pol members. This effect of Polη depletion reflects the activationof cell cycle checkpoints and the apoptosis program in associationwith DSB formation, as was evaluated by γH2AXmeasurement andcomet assays. Similarly, Myc–ER activation-induced accumulationof G2-phase cells and γH2AX increases were more pronouncedin the XP-V-derived Polη-deficient fibroblast line than in itsPolη-complemented counterpart.

Two major models are conceivable for the molecular mechanismsby which Polη promotes cellular tolerance of Myc-induced RS(Fig. S7). In model A, Polη suppresses RS by promoting the stabilityof CFSs localized in ‘hard-to-replicate’DNA regions with secondarystructures, as recently reported (Bergoglio et al., 2013; Rey et al.,2009), but does not directly affectMyc-induced RS.Myc-induced RSmay promote instabilities in the CFSs (Gaillard et al., 2015). InmodelB, in addition to the function shown in model A, Polη directlysuppresses Myc-induced RS. Several lines of evidence in the presentstudy indicate that Polη diminishes Myc-induced RS, as shown inmodel B. First, Myc activation induced PCNA monoubiquitylation,and Polη was recruited to the Myc-induced, but not the control,replication foci through the interaction with Ub–PCNA. Second,Polη depletion and Myc activation were able to induce replication-associated DSB formation during S-phase in a synergistic manner.Consistent with this, overexpression of dead-Polη in the Myc-activated cells caused pronounced accumulation of γH2AX in thePolη-positive replication foci. The effect of dead-Polη was morepronounced than that of Polη depletion, presumably becausepersistent localization of the mutant protein at fork stalling sitesmore efficiently inhibits the functions of endogenous Polη, leadingto fork collapse. Of note, a similar difference has been observedwith CFS instability (Bergoglio et al., 2013). Most importantly,Polη depletion had little effect on fork progression in the vehicle-treated cells, a finding consistent with previous data (Rey et al.,2009), while promoting Myc-induced fork slowing and, moreimpressively, fork stalling by ∼2-fold. These effects are betterexplained by model B than by model A. Overall, our resultsindicate that Polη-mediated fork progression alleviates the Myc-induced RS.

The molecular nature of Myc-induced replication barriers awaitsclarification. Nevertheless, our results show that Myc-induced Polηfoci formation was not affected by the exogenous supply ofnucleotides, or by the introduction of RNase H1 or cellulartreatment with NAC, but was inhibited by Cdc45 depletion.Furthermore, overexpression of Cdc45 promoted Polη accumulationin the replication foci. Overall, these results suggest that among themajor mechanisms proposed for OIRS, origin hyperactivation, butnot nucleotide deficiency, replication–transcription conflicts orincreased oxidative DNA damage, is responsible for Myc-inducedRS, a finding consistent with the results from previous studies(Bester et al., 2011; Dominguez-Sola et al., 2007; Liu et al., 2008;Mannava et al., 2008; Maya-Mendoza et al., 2014; Srinivasan

Fig. 7. Polηhas acatalytic role in preventingMyc-inducedRS.U2OS/Myc–ER cells were lentivirally transduced with an empty vector (E.V.), GFP-taggedWT-Polη (GFP–WT-Polη) or GFP-tagged dead-Polη (GFP–dead-Polη) andsubsequently treated with vehicle or 4OHT at 48 h post infection. (A) WCLswere prepared at 48 h after drug treatment and immunoblotted. (B,C) The cellswere fixed and immunostained with anti-Polη and anti-γH2AX antibodies at48 h after drug treatment. Nuclei were stained with DAPI. (B) Representativeimages of GFP–Polη foci and γH2AX foci. Scale bars: 10 µm. (C) Bar graphshowing the percentage of γH2AX foci-positive cells, GFP–Polη foci-positivecells or the cells in which the GFP–Polη foci colocalized with >70% of γH2AXfoci [GFP–Polη/γH2AX foci (+)]. The values represent the mean±s.d. of threeindependent experiments. (D) The cells were extracted with CSK buffer, fixedand immunostained with anti-Polη and anti-PCNA antibodies at 48 h after drugtreatment. Nuclei were then stained with DAPI. Representative images ofGFP–Polη foci and PCNA foci are shown. Scale bars: 10 µm.

10

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

et al., 2013). In good agreement with these observations, Srinvasanet al. recently reported that Cdc45 functions as a downstreameffector of Myc-induced RS in a cell-free replication system usingXenopus egg extracts, where nucleotide deficiency, transcription

and oxidative DNA damage do not occur (Srinivasan et al., 2013).The authors have proposed that Myc and Cdc45 simultaneouslyactivate an excessive number of origins, which causes insufficiencyof replication fork processing factors, such as helicases and

Fig. 8. See next page for legend.

11

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

topoisomerases, leading to fork slowing or stalling (Srinivasanet al., 2013). We postulate that Polη diminishes such RS.Because each TLS polymerase has its own characteristic substrate

specificity, it is interesting to question which of them preferentiallybypasses the replication obstacles caused by different oncogenes.Different oncogenic signals have distinct effects on RS byregulating their effects on multiple cellular processes, includingmitogenic signals, cell cycle progression, metabolism and genetranscription (Bester et al., 2011;Maya-Mendoza et al., 2014;Mironet al., 2015; Neelsen et al., 2013; Yang et al., 2017). During thepreparation of this manuscript, Yang et al. reported that Polκ but notPolη is involved in cellular tolerance of cyclin E- and Ras-inducedRS, but the authors excluded the involvement of Y-Pols in Myc-induced RS because of the lack of Myc-induced PCNAmonoubiquitylation in their system (Yang et al., 2017).Notwithstanding, our results indicate that Polη plays a major rolein cellular tolerance of Myc-induced RS in U2OS/Myc–ER cells,although Polκ had a similar but smaller effect. The discrepancybetween their results and ours may be partly explained bydifferences in the experimental conditions that were used (e.g.different cell types, oncogenes and gene expression methods).However, more interesting perhaps is the possibility that theactivities of Polη and Polκ each favor a distinctive, but partlyoverlapping range of replication obstacles. Supporting thispossibility, recent studies have reported that cyclin E- and Ras-induced RS is caused by increased replication–transcriptionconflicts (Jones et al., 2013; Kotsantis et al., 2016), whereas ourdata suggest that Myc-induced RS is not. Certainly, the differentialfunctions of TLS polymerases in OIRS will be an important topicfor future studies.We showed that Myc-induced DSB generation in the absence of

Polη depends on MUS81–EME2. MUS81-mediated DSBformation potentially induces cell cycle arrest and/or apoptosis(Forment et al., 2011; Fugger et al., 2013). However, many studieshave documented that it also initiates homologous recombination-mediated fork repair, leading to replication restart, thus promotingcell growth and survival as an overall effect (Franchitto et al., 2008;Hanada et al., 2007; Murfuni et al., 2013; Regairaz et al., 2011). Itshould also be noted that in most of the earlier studies, it wasaccepted that MUS81–EME1 contributed to fork restart as well as

CFS fragility (Forment et al., 2011; Franchitto et al., 2008; Naimet al., 2013; Regairaz et al., 2011; Ying et al., 2013). However, arecent study reported that MUS81–EME2 promotes restart of HU-induced fork stalling in S-phase, whereas MUS81–EME1 mediatesCFS expression in mitotic phase (Pepe and West, 2014a). Thus, theobservations that MUS81–EME2 but not MUS81–EME1 isinvolved in DSB formation in Polη-depleted Myc-activated cellsare consistent with our notion that stalled forks but not CFSs are themajor source of DSBs in these cells. Taken together with all theavailable information, our observations raise the possibility thatMUS81–EME2 serves as a fail-safe for Polη to prevent Myc-induced RS. In agreement with this possibility, co-depletion ofMUS81 or EME2 with Polη increased RS, as estimated bymeasuring the increase in the number of RPA2 foci and the levelof cell death, in a more than additive manner. It is not known whichreplication intermediates are cleaved by MUS81–EME2 in Myc-activated cells. In vitro studies documented that, like MUS81–EME1, MUS81–EME2 targets a wide spectrum of replicationintermediates (Amangyeld et al., 2014; Pepe and West, 2014b).Thus, one possibility is that MUS81–EME2 makes incisions in thevicinity of the single-strand DNA gaps generated at stalled sitesarising from helicase-polymerase uncoupling and/or upstreamrepriming. Alternatively, stalled forks might be processed intodifferent structures, such as reversed forks, which are putativesubstrates of MUS81 in the context of OIRS, as previously reported(Neelsen et al., 2013). There is increasing evidence showing thatMUS81 participates in different OIRS response pathways(Minocherhomji et al., 2015; Sotiriou et al., 2016). Ourobservations have added a partnership with Polη to the roles ofMUS81.

The present work provides potential insights into oncogenesis.Of interest, several clinical studies have shown that high Polηexpression in pre-treatment tumor samples is a predictor of poorclinical response to treatment with platinum-based regimens (Ceppiet al., 2009; Teng et al., 2010; Zhou et al., 2013). Therefore, it ispossible that neoplastic cells with high Polη expression levels mightbe selectively expanded during tumor development, because of theirtolerance of OIRS-derived deleterious effects. This hypothesisis clearly worthy of future study. Another question is whetherMUS81–EME2 inhibition has a synthetic sickness or lethality effecton Polη-deficient precancerous or cancer cells in XP-V patients.

MATERIALS AND METHODSPlasmidspBabepuro-myc-ER (plasmid 19128; Ricci et al., 2004) was purchasedfrom Addgene (Cambridge, MA). cDNAs encoding Myc–ER, full-lengthhuman RNase H1 and C-terminally FLAG-tagged full-length human Cdc45(Cdc45-F) were inserted into the blasticidin-selectable lentiviral vectorCSII-CMV-MCS-IRES2-Bsd (kindly provided by Dr Hiroyuki Miyoshi,RIKEN, Japan). cDNAs encoding N-terminally FLAG- and HA-taggedfull-length human PCNA (FH-PCNA) and a PCNA mutant carrying amissense mutation (K164R) were inserted into a neomycin-selectablelentiviral vector (CSII-CMV-MCS-IRES-Neo), which we constructed byreplacing the blasticidin-resistance gene in CSII-CMV-MCS-IRES2-Bsdwith a neomycin-resistance gene. This mutation was generated using theQuikChange mutagenesis kit (Agilent Technologies, Santa Clara, CA).CSII-CMV-MCS-IRES2-Bsd/GFP-Polη and CSII-CMV-MCS-IRES-Bsd/GFP-dead-Polη were as described previously (Sekimoto et al., 2015).

CellsU2OS cells (a human osteosarcoma cell line) and XPV cells (XP2SASV3, aPolη-deficient SV40 transformed human fibroblast cell line) weremaintained in Dulbecco’s modified Eagle’s medium containing 10% fetalbovine serum at 37°C under 5% CO2. KMS12PE cells and KMM1 cells

Fig. 8. Knockdown of MUS81 or EME2, but not EME1, suppresses DSBformation and increases RPA2 foci formation and cell death in Myc-activated and Polη-depleted cells. U2OS/Myc–ER cells were transfectedwith the siRNAs indicated and subsequently treated with vehicle (−) or 4OHT(+) at 24 h post transfection. (A) At 48 h after drug treatment, WCLs wereprepared and immunoblotted. The asterisk indicates a non-specific signal.(B) Total RNAs were isolated and EME2 mRNA amounts were measured byRT-qPCR. (C) At 48 h after drug treatment, neutral comet assays wereperformed. Dot plot showing the tail moment distributions (arbitrary units,A.U.). Themedian scores are represented by horizontal lines and shown underthe graph. Statistical differences between distributions were assessed with aMann–Whitney rank sum test. The results were reproduced in two independentexperiments of which one is shown. (D) At 24 and 48 h after drug treatment, thecells were extracted with CSK buffer, fixed and immunostained with anti-RPA2antibody, and the nuclei were stained with DAPI (refer to Fig. S6A). Bar graphsshow the percentages of RPA2 foci-positive cells containing 1–5, 6–9 or >10bright foci per nucleus. The results were reproduced in two independentexperiments of which one is shown. (E) Cells were stained with PI and Hoechst33342 at 24, 48 and 72 h after drug treatment (refer to Fig. S6B). The deadcells were defined as cells double-positive for PI-positive and Hoechst 33342.Bar graphs show the percentages of the dead cells. The values represent themean±s.d. of three independent experiments. *P<0.05; **P<0.01; ***P<0.005;****P<0.001; N.S., not significant.

12

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

(human myeloma cell lines) were maintained in RPMI-1640 mediumcontaining 10% heat-inactivated fetal bovine serum at 37°C under 5% CO2.These cells were recently authenticated and tested for contamination. U2OScells stably expressing Myc–ER (U2OS/Myc–ER) were obtained throughinfection with the pBabepuro-myc-ER recombinant retroviral vector.Infected U2OS cells were selected with 1 µg/ml puromycin (Sigma, StLouis, MO) and then cloned. U2OS/Myc–ER and U2OS cells stablyexpressing GFP–Polη (U2OS/Myc–ER/GFP–Polη and U2OS/GFP–Polη,respectively) were obtained through infection with the recombinantlentiviral vector CSII-CMV-MCS-IRES2-Bsd/GFP-Polη. Infected cellswere selected with 20 µg/ml blasticidin S (Wako, Osaka, Japan) and thencloned. U2OS/Myc–ER/GFP–Polη cells stably expressing FH-PCNA orFH-PCNA (K164R) were obtained through infection with the recombinantlentiviral vector CSII-CMV-MCS-IRES-Neo/FH-PCNA or CSII-CMV-MCS-IRES-Neo/FH-PCNA (K164R), respectively. Infected cells wereselected with 1 mg/ml of G418 (Wako). The XPV-Neo and XPV-Polη cellsstably expressing Myc–ER (XPV-Neo/Myc–ER and XPV-Polη/Myc–ER,respectively) were obtained through infection with the CSII-CMV-MCS-IRES-Bsd/Myc–ER recombinant lentiviral vectors. Infected cells wereselected with blasticidin S (10 µg/ml).

AntibodiesAntibodies used in this study are listed in Table S1.

siRNAsThe siRNAs and the negative control (N.C.) siRNA (MISSION siRNAUniversal Negative control #1) were purchased from Sigma. The targetsequences of the siRNAs used in this study are shown in Table S2. EachsiRNA was transfected at final concentration of 30 nM (unless indicatedotherwise) using Lipofectamine RNAiMAX according to the manufacturer’sinstructions (Thermo Fisher Scientific, Waltham, MA).

Chemicals4OHT, aphidicolin, NAC, dADP, dGDP, dCDP and dUMP were purchasedfrom Sigma, and HU and hydrogen peroxide were purchased from Wako.

Cell extracts, immunoprecipitation and immunoblottingWCLs and cytoskeleton (CSK) buffer-insoluble fractions were prepared asdescribed previously (Sekimoto et al., 2010, 2015). Immunoprecipitationand immunoblotting were performed using standard procedures. Proteinsignals were quantified by densitometry using National Institutes of Healthimage software (ImageJ).

Immunofluorescence, fluorescence microscopy and imagecytometrySoluble cell components were removed by extraction in CSK buffer [10 mMPIPES pH 6.8, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 0.2% Triton X-100, 300 mM sucrose and protease inhibitor cocktail (Roche)], or inphosphate-buffered saline containing 0.2% Triton X-100 for 5 min. Cellswere immunostained as described previously (Sekimoto et al., 2010, 2015).Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI)Fluoromount-G (SouthernBiotech, Birmingham, AL). To analyzecolocalization of Polη and the replication focus, the cells were pulse-labeled with 10 μMBrdU for 30 min (U2OS/Myc–ER/GFP–Polη andU2OS/Myc–ER) or for 60 min (XPV-Polη/Myc–ER), respectively, prior toextraction and fixing. Replication foci were visualized using the BrdUlabeling detection kit I (Roche, Basel, Switzerland), followed by staining withan anti-Polη antibody. To detect GFP–Polη foci, and Cdc45-F expression andreplication foci, the cells were pulse-labeled with 10 µMEdU for 15 min priorto fixation. Replication foci were visualized with the Click-iT Plus EdUAlexaFluor 555 imaging kit (Thermo Fisher Scientific), followed by staining withanti-Polη and anti-Cdc45 antibodies. Immunofluorescence images wereobtained with a one-box fluorescence microscope (BZ-9000, Keyence,Osaka, Japan) with a 40× CFI Plan-Apochromat λ NA 0.95 objective lens(Nikon, Tokyo, Japan), or with a fluorescence microscope (DMIRB, Leica,Wetzlar, Germany) with a DFC360FX camera, a 40× N PLAN L NA 0.55objective lens and Leica Application Suite software. Cells containing more

than ten Polη nuclear foci were considered positive. The signal intensities ofDAPI, γH2AX and Myc–ER were determined using Volocity 3D imageanalysis software (PerkinElmer, Waltham, MA, USA). At least 200 nucleiwere examined. Image cytometry was performed with a CellomicsCellInsight High Content Analyzer (Thermo Fisher Scientific) with a 20×LUCPlanFLN NA 0.45 objective lens (Olympus). The data acquired wereanalyzed using Cellomics vHCS View software (Thermo Fisher Scientific)and FlowJo software (Tree Star, Ashland, OR).

Flow cytometryFlow cytometric analyses were performed as described previously(Sekimoto et al., 2015). Data were acquired using FACSVerse (BDBiosciences, Franklin Lakes, NJ), and then analyzed with FlowJo software.

Neutral comet assaysNeutral comet assays were performed using the CometAssay kit (Trevigen,Gaithersburg, MD, USA) according to the manufacturer’s instructions.Immunofluorescence images were obtained with a fluorescence microscope(Leica DMIRB). The tail moment (defined as the product of tail length andthe fraction of total DNA in the tail) was determined using Comet Analyzersoftware (YUWORKS, Tokyo, Japan).

DNA fiber assaysDNA fiber assays were performed primarily as described previously(Luebben et al., 2014; Sekimoto et al., 2015). Cells were labeled with100 μM IdU (Sigma) for 10 min and then with 100 μM CldU (Sigma) for20 min. The labeled cells were trypsinized and mixed with a 10-fold excessof unlabeled cells. After cell lysis, the DNA fibers released from the cellswere extended by tilting slides, after which they were immunostained. Theimages were captured with a fluorescence microscope (Keyence BZ-9000),processed with Photoshop software (Adobe Systems, San Jose, CA) andthen analyzed using ImageJ. Micrometers were converted to kilo base pairsby multiplying the number of micrometers by 3.5 kb.

Real-time quantitative PCRTotal RNA was isolated with the RNeasy Mini kit (Qiagen, Hilden,Germany) according to the manufacturer’s instructions. Reversetranscription reactions were performed on 1 µg of total RNA with theReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Real-time quantitativePCR (RT-qPCR) was performed using the THUNDERBIRD Probe qPCRMix (Toyobo), which contains gene-specific primers and a probe(PrimeTime Std qPCR Assay, Integrated DNA technologies, Coralville,IA). The results were analyzed with the PikoReal RT-PCR system (ThermoFisher Scientific).

Cell death assaysCells were stained with 2 µg/ml propidium iodide (PI) (Sigma) and 2 µMHoechst 33342 (AAT Bioquest, Sunnyvale, CA) for 10 min, and the imageswere captured with a fluorescence microscope (Leica DMIRB). PI- andHoechst 33342 double-stained cells were defined as dead cells.

Statistical analysesStatistical analyses were performed using a two-tailed Student t-test unlessotherwise indicated. TheMann–Whitney rank sum test was performed usingthe R software package.

AcknowledgementsWe thank K. Tomizawa for technical help and H. Miyoshi for generous giftsof materials. We also thank Sandra Cheesman, PhD, from Edanz Group(www.edanzediting.com/ac) for editing a draft of this manuscript.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: K.K., T.S., T.O., F.H., T.Y.; Methodology: K.K., T.S., T.O., T.K.;Investigation: K.K., T.S.; Resources: F.H.; Writing - original draft: K.K., T.S., T.Y.;

13

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

Writing - review & editing: K.K., T.S., T.O., T.Y.; Supervision: T.Y.; Projectadministration: T.Y.; Funding acquisition: K.K., T.S., T.Y.

FundingThis study was supported in part by Japan Society for the Promotion of Science(JSPS) KAKENHI Grant Numbers JP15K18438 (to K.K.), JP15K06825 (to T.S.) andJP15K06826 (to T.Y.).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.212183.supplemental

ReferencesAkagi, J., Masutani, C., Kataoka, Y., Kan, T., Ohashi, E., Mori, T., Ohmori, H. andHanaoka, F. (2009). Interaction with DNA polymerase eta is required for nuclearaccumulation of REV1 and suppression of spontaneousmutations in human cells.DNA Repair (Amst) 8, 585-599.

Amangyeld, T., Shin, Y.-K., Lee, M., Kwon, B. and Seo, Y.-S. (2014). HumanMUS81-EME2 can cleave a variety of DNA structures including intact Hollidayjunction and nicked duplex. Nucleic Acids Res. 42, 5846-5862.

Baldeck, N., Janel-Bintz, R., Wagner, J., Tissier, A., Fuchs, R. P., Burkovics, P.,Haracska, L., Despras, E., Bichara, M., Chatton, B. et al. (2015). FF483-484motif of human Poleta mediates its interaction with the POLD2 subunit of Poldeltaand contributes to DNA damage tolerance. Nucleic Acids Res. 43, 2116-2125.

Bergoglio, V., Boyer, A.-S., Walsh, E., Naim, V., Legube, G., Lee, M. Y., Rey, L.,Rosselli, F., Cazaux, C., Eckert, K. A. et al. (2013). DNA synthesis by Pol etapromotes fragile site stability by preventing under-replicated DNA in mitosis.J. Cell Biol. 201, 395-408.

Berti, M. and Vindigni, A. (2016). Replication stress: getting back on track. Nat.Struct. Mol. Biol. 23, 103-109.

Bester, A. C., Roniger, M., Oren, Y. S., Im, M. M., Sarni, D., Chaoat, M.,Bensimon, A., Zamir, G., Shewach, D. S. and Kerem, B. (2011). Nucleotidedeficiency promotes genomic instability in early stages of cancer development.Cell 145, 435-446.

Boyer, A.-S., Walter, D. and Sorensen, C. S. (2016). DNA replication and cancer:From dysfunctional replication origin activities to therapeutic opportunities.Semin.Cancer Biol. 37-38, 16-25.

Ceppi, P., Novello, S., Cambieri, A., Longo, M., Monica, V., Lo Iacono, M.,Giaj-Levra, M., Saviozzi, S., Volante, M., Papotti, M. et al. (2009). Polymeraseeta mRNA expression predicts survival of non-small cell lung cancer patientstreated with platinum-based chemotherapy. Clin. Cancer Res. 15, 1039-1045.

Chen, E., Ahn, J. S., Sykes, D. B., Breyfogle, L. J., Godfrey, A. L., Nangalia, J.,Ko, A., DeAngelo, D. J., Green, A. R. and Mullally, A. (2015). RECQL5suppresses oncogenic JAK2-induced replication stress and genomic instability.Cell Rep. 13, 2345-2352.

Costantino, L., Sotiriou, S. K., Rantala, J. K., Magin, S., Mladenov, E., Helleday,T., Haber, J. E., Iliakis, G., Kallioniemi, O. P. and Halazonetis, T. D. (2014).Break-induced replication repair of damaged forks induces genomic duplicationsin human cells. Science 343, 88-91.

Dang, C. V. (2013). MYC, metabolism, cell growth, and tumorigenesis. Cold SpringHarb. Perspect Med. 3, a014217.

Dominguez-Sola, D., Ying, C. Y., Grandori, C., Ruggiero, L., Chen, B., Li, M.,Galloway, D. A., Gu, W., Gautier, J. and Dalla-Favera, R. (2007). Non-transcriptional control of DNA replication by c-Myc. Nature 448, 445-451.

Durando, M., Tateishi, S. and Vaziri, C. (2013). A non-catalytic role of DNApolymerase eta in recruiting Rad18 and promoting PCNA monoubiquitination atstalled replication forks. Nucleic Acids Res. 41, 3079-3093.

Forment, J. V., Blasius, M., Guerini, I. and Jackson, S. P. (2011). Structure-specific DNA endonuclease Mus81/Eme1 generates DNA damage caused byChk1 inactivation. PLoS One 6, e23517.

Franchitto, A., Pirzio, L. M., Prosperi, E., Sapora, O., Bignami, M. and Pichierri,P. (2008). Replication fork stalling in WRN-deficient cells is overcome by promptactivation of a MUS81-dependent pathway. J. Cell Biol. 183, 241-252.

Fugger, K., Chu, W. K., Haahr, P., Kousholt, A. N., Beck, H., Payne, M. J.,Hanada, K., Hickson, I. D. and Sorensen, C. S. (2013). FBH1 co-operates withMUS81 in inducing DNA double-strand breaks and cell death following replicationstress. Nat. Commun. 4, 1423.

Gaillard, H., Garcia-Muse, T. and Aguilera, A. (2015). Replication stress andcancer. Nat. Rev. Cancer 15, 276-289.

Halazonetis, T. D., Gorgoulis, V. G. and Bartek, J. (2008). An oncogene-inducedDNA damage model for cancer development. Science 319, 1352-1355.

Hanada, K., Budzowska, M., Davies, S. L., van Drunen, E., Onizawa, H.,Beverloo, H. B., Maas, A., Essers, J., Hickson, I. D. and Kanaar, R. (2007).The structure-specific endonuclease Mus81 contributes to replication restartby generating double-strand DNA breaks. Nat. Struct. Mol. Biol. 14,1096-1104.

Johnson, R. E., Kondratick, C. M., Prakash, S. and Prakash, L. (1999). hRAD30mutations in the variant form of xeroderma pigmentosum. Science 285, 263-265.

Jones, R. M., Mortusewicz, O., Afzal, I., Lorvellec, M., Garcıa, P., Helleday, T.and Petermann, E. (2013). Increased replication initiation and conflicts withtranscription underlie Cyclin E-induced replication stress. Oncogene 32,3744-3753.

Kannouche, P. L., Wing, J. and Lehmann, A. R. (2004). Interaction of human DNApolymerase eta with monoubiquitinated PCNA: a possible mechanism for thepolymerase switch in response to DNA damage. Mol. Cell 14, 491-500.

Karnitz, L. M. and Zou, L. (2015). Molecular pathways: targeting ATR in cancertherapy. Clin. Cancer Res. 21, 4780-4785.

Kotsantis, P., Silva, L. M., Irmscher, S., Jones, R. M., Folkes, L., Gromak, N. andPetermann, E. (2016). Increased global transcription activity as a mechanism ofreplication stress in cancer. Nat. Commun. 7, 13087.

Liu, Y.-C., Li, F., Handler, J., Huang, C. R. L., Xiang, Y., Neretti, N., Sedivy, J. M.,Zeller, K. I. and Dang, C. V. (2008). Global regulation of nucleotide biosyntheticgenes by c-Myc. PLoS One 3, e2722.

Luebben, S. W., Shima, N. and Kawabata, T. (2014). Methods for the detection ofgenome instability derived from replication stress in primary mouse embryonicfibroblasts. Methods Mol. Biol. 1194, 341-352.

Mailand, N., Gibbs-Seymour, I. and Bekker-Jensen, S. (2013). Regulation ofPCNA-protein interactions for genome stability. Nat. Rev. Mol. Cell Biol. 14,269-282.

Mannava, S., Grachtchouk, V., Wheeler, L. J., Im, M., Zhuang, D., Slavina, E. G.,Mathews, C. K., Shewach, D. S. and Nikiforov, M. A. (2008). Direct role ofnucleotide metabolism in C-MYC-dependent proliferation of melanoma cells. CellCycle 7, 2392-2400.

Masutani, C., Kusumoto, R., Yamada, A., Dohmae, N., Yokoi, M., Yuasa, M.,Araki, M., Iwai, S., Takio, K. and Hanaoka, F. (1999). The XPV (xerodermapigmentosum variant) gene encodes human DNA polymerase eta. Nature 399,700-704.

Maya-Mendoza, A., Ostrakova, J., Kosar, M., Hall, A., Duskova, P., Mistrik, M.,Merchut-Maya, J. M., Hodny, Z., Bartkova, J., Christensen, C. et al. (2014).Myc and Ras oncogenes engage different energy metabolism programs andevoke distinct patterns of oxidative and DNA replication stress. Mol. Oncol. 9,601-616.

McIlwraith, M. J., Vaisman, A., Liu, Y., Fanning, E., Woodgate, R. andWest, S. C.(2005). Human DNA polymerase eta promotes DNA synthesis from strandinvasion intermediates of homologous recombination. Mol. Cell 20, 783-792.

McMahon, S. B. (2014). MYC and the control of apoptosis. Cold Spring Harb.Perspect. Med. 4, a014407.

Minocherhomji, S., Ying, S., Bjerregaard, V. A., Bursomanno, S., Aleliunaite,A., Wu, W., Mankouri, H. W., Shen, H., Liu, Y. and Hickson, I. D. (2015).Replication stress activates DNA repair synthesis in mitosis.Nature 528, 286-290.

Miron, K., Golan-Lev, T., Dvir, R., Ben-David, E. and Kerem, B. (2015).Oncogenes create a unique landscape of fragile sites. Nat. Commun. 6, 7094.

Murfuni, I., Nicolai, S., Baldari, S., Crescenzi, M., Bignami, M., Franchitto, A.and Pichierri, P. (2013). The WRN and MUS81 proteins limit cell death andgenome instability following oncogene activation. Oncogene 32, 610-620.

Naim, V., Wilhelm, T., Debatisse, M. and Rosselli, F. (2013). ERCC1 and MUS81-EME1 promote sister chromatid separation by processing late replicationintermediates at common fragile sites during mitosis. Nat. Cell Biol. 15,1008-1015.

Neelsen, K. J., Zanini, I. M. Y., Herrador, R. and Lopes, M. (2013). Oncogenesinduce genotoxic stress by mitotic processing of unusual replicationintermediates. J. Cell Biol. 200, 699-708.

Ogi, T., Limsirichaikul, S., Overmeer, R. M., Volker, M., Takenaka, K., Cloney,R., Nakazawa, Y., Niimi, A., Miki, Y., Jaspers, N. G. et al. (2010). Three DNApolymerases, recruited by different mechanisms, carry out NER repair synthesisin human cells. Mol. Cell 37, 714-727.

Ohashi, E., Hanafusa, T., Kamei, K., Song, I., Tomida, J., Hashimoto, H., Vaziri,C. and Ohmori, H. (2009). Identification of a novel REV1-interacting motifnecessary for DNA polymerase kappa function. Genes Cells 14, 101-111.

Pepe, A. and West, S. C. (2014a). MUS81-EME2 promotes replication fork restart.Cell Rep. 7, 1048-1055.

Pepe, A. and West, S. C. (2014b). Substrate specificity of the MUS81-EME2structure selective endonuclease. Nucleic Acids Res. 42, 3833-3845.

Perna, D., Faga, G., Verrecchia, A., Gorski, M. M., Barozzi, I., Narang, V., Khng,J., Lim, K. C., Sung, W.-K., Sanges, R. et al. (2012). Genome-wide mapping ofMyc binding and gene regulation in serum-stimulated fibroblasts. Oncogene 31,1695-1709.

Puigvert, J. C., Sanjiv, K. and Helleday, T. (2016). Targeting DNA repair, DNAmetabolism and replication stress as anti-cancer strategies. FEBS J. 283,232-245.

Regairaz, M., Zhang, Y.-W., Fu, H., Agama, K. K., Tata, N., Agrawal, S., Aladjem,M. I. and Pommier, Y. (2011). Mus81-mediated DNA cleavage resolvesreplication forks stalled by topoisomerase I-DNA complexes. J. Cell Biol. 195,739-749.

Rey, L., Sidorova, J. M., Puget, N., Boudsocq, F., Biard, D. S. F., Monnat, R. J.Jr., Cazaux, C. and Hoffmann, J. S. (2009). Human DNA polymerase eta isrequired for common fragile site stability during unperturbed DNA replication.Mol.Cell. Biol. 29, 3344-3354.

14

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience

Ricci M. S., Jin Z., Dews M., Yu D., Thomas-Tikhonenko A., Dicker D. T. andEl-Deiry W. S. (2004). Direct repression of FLIP expression by c-Myc is a majordeterminant of TRAIL sensitivity. Mol. Cell Biol. 24, 8541-8555.

Rohban, S. and Campaner, S. (2015). Myc induced replicative stressresponse: How to cope with it and exploit it. Biochim. Biophys. Acta 1849,517-524.

Sale, J. E., Lehmann, A. R. and Woodgate, R. (2012). Y-family DNA polymerasesand their role in tolerance of cellular DNA damage. Nat. Rev. Mol. Cell Biol. 13,141-152.

Sekimoto, T., Oda, T., Pozo, F. M., Murakumo, Y., Masutani, C., Hanaoka, F. andYamashita, T. (2010). The molecular chaperone Hsp90 regulates accumulationof DNA polymerase eta at replication stalling sites in UV-irradiated cells.Mol. Cell37, 79-89.

Sekimoto, T., Oda, T., Kurashima, K., Hanaoka, F. and Yamashita, T. (2015).Both high-fidelity replicative and low-fidelity Y-family polymerases are involved inDNA rereplication. Mol. Cell. Biol. 35, 699-715.

Sotiriou, S. K., Kamileri, I., Lugli, N., Evangelou, K., Da-Re, C., Huber, F.,Padayachy, L., Tardy, S., Nicati, N. L., Barriot, S. et al. (2016). MammalianRAD52 functions in break-induced replication repair of collapsed DNA replicationforks. Mol. Cell 64, 1127-1134.

Srinivasan, S. V., Dominguez-Sola, D., Wang, L. C., Hyrien, O. and Gautier, J.(2013). Cdc45 is a critical effector of myc-dependent DNA replication stress. CellRep. 3, 1629-1639.

Teng, K. Y., Qiu, M. Z., Li, Z. H., Luo, H. Y., Zeng, Z. L., Luo, R. Z., Zhang, H. Z.,Wang, Z. Q., Li, Y. H. and Xu, R. H. (2010). DNA polymerase eta proteinexpression predicts treatment response and survival of metastatic gastricadenocarcinoma patients treated with oxaliplatin-based chemotherapy.J. Transl. Med. 8, 126.

Tuduri, S., Crabbe, L., Conti, C., Tourriere, H., Holtgreve-Grez, H., Jauch, A.,Pantesco, V., De Vos, J., Thomas, A., Theillet, C. et al. (2009). Topoisomerase Isuppresses genomic instability by preventing interference between replicationand transcription. Nat. Cell Biol. 11, 1315-1324.

Vafa, O., Wade, M., Kern, S., Beeche, M., Pandita, T. K., Hampton, G. M. andWahl, G. M. (2002). c-Myc can induce DNA damage, increase reactive oxygenspecies, and mitigate p53 function: a mechanism for oncogene-induced geneticinstability. Mol. Cell 9, 1031-1044.

Vaisman, A. and Woodgate, R. (2017). Translesion DNA polymerases ineukaryotes: what makes them tick? Crit. Rev. Biochem. Mol. Biol. 52, 274-303.

Valovka, T., Schonfeld, M., Raffeiner, P., Breuker, K., Dunzendorfer-Matt, T.,Hartl, M. and Bister, K. (2013). Transcriptional control of DNA replicationlicensing by Myc. Sci. Rep. 3, 3444.

Watanabe, K., Tateishi, S., Kawasuji, M., Tsurimoto, T., Inoue, H. andYamaizumi, M. (2004). Rad18 guides poleta to replication stalling sites throughphysical interaction and PCNA monoubiquitination. EMBO J. 23, 3886-3896.

Yang, Y., Gao, Y., Mutter-Rottmayer, L., Zlatanou, A., Durando, M., Ding, W.,Wyatt, D., Ramsden, D., Tanoue, Y., Tateishi, S. et al. (2017). DNA repair factorRAD18 and DNA polymerase Polkappa confer tolerance of oncogenic DNAreplication stress. J. Cell Biol. 216, 3097-3115.

Ying, S., Minocherhomji, S., Chan, K. L., Palmai-Pallag, T., Chu,W. K., Wass, T.,Mankouri, H. W., Liu, Y. and Hickson, I. D. (2013). MUS81 promotes commonfragile site expression. Nat. Cell Biol. 15, 1001-1007.

Yuasa, M. S., Masutani, C., Hirano, A., Cohn, M. A., Yamaizumi, M., Nakatani, Y.and Hanaoka, F. (2006). A human DNA polymerase eta complex containingRad18, Rad6 and Rev1; proteomic analysis and targeting of the complex to thechromatin-bound fraction of cells undergoing replication fork arrest. Genes Cells11, 731-744.

Zeman,M. K. andCimprich, K. A. (2014). Causes and consequences of replicationstress. Nat. Cell Biol. 16, 2-9.

Zhou, W., Chen, Y. W., Liu, X., Chu, P., Loria, S., Wang, Y., Yen, Y. and Chou,K. M. (2013). Expression of DNA translesion synthesis polymerase eta inhead and neck squamous cell cancer predicts resistance to gemcitabine andcisplatin-based chemotherapy. PLoS One 8, e83978.

15

RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212183. doi:10.1242/jcs.212183

Journal

ofCe

llScience