DNA-damage-induced degradation of EXO1 exonucleaselimits DNA end resection to ensure accurate DNA repairReceived for publication, December 14, 2016, and in revised form, May 11, 2017 Published, Papers in Press, May 17, 2017, DOI 10.1074/jbc.M116.772475

Nozomi Tomimatsu‡1, Bipasha Mukherjee‡1, Janelle Louise Harris§, Francesca Ludovica Boffo¶,Molly Catherine Hardebeck‡, Patrick Ryan Potts�, Kum Kum Khanna§, and Sandeep Burma‡2

From the ‡Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, §SignalTransduction Laboratory, QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4006, Australia, ¶Department ofMolecular Medicine and Medical Biotechnology, Universita Federico II, Napoli 80131, Italy, and �Department of Cell and MolecularBiology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

Edited by Patrick Sung

End resection of DNA double-strand breaks (DSBs) to gen-erate 3�-single-stranded DNA facilitates DSB repair viaerror-free homologous recombination (HR) while stymie-ing repair by the error-prone non-homologous end joining(NHEJ) pathway. Activation of DNA end resection involvesphosphorylation of the 5� to 3� exonuclease EXO1 by thephosphoinositide 3-kinase-like kinases ATM (ataxia telangi-ectasia-mutated) and ATR (ATM and Rad3-related) and bythe cyclin-dependent kinases 1 and 2. After activation, EXO1must also be restrained to prevent over-resection that isknown to hamper optimal HR and trigger global genomicinstability. However, mechanisms by which EXO1 is re-strained are still unclear. Here, we report that EXO1 is rapidlydegraded by the ubiquitin-proteasome system soon after DSBinduction in human cells. ATR inhibition attenuated DNA-damage-induced EXO1 degradation, indicating that ATR-mediated phosphorylation of EXO1 targets it for degrada-tion. In accord with these results, EXO1 became resistant todegradation when its SQ motifs required for ATR-mediatedphosphorylation were mutated. We show that upon theinduction of DNA damage, EXO1 is ubiquitinated by a mem-ber of the Skp1-Cullin1-F-box (SCF) family of ubiquitinligases in a phosphorylation-dependent manner. Impor-tantly, expression of degradation-resistant EXO1 resulted inhyper-resection, which attenuated both NHEJ and HR andseverely compromised DSB repair resulting in chromosomalinstability. These findings indicate that the coupling of EXO1activation with its eventual degradation is a timing mecha-nism that limits the extent of DNA end resection for accurateDNA repair.

DNA double-strand breaks (DSBs)3 are arguably the mostdangerous of all types of DNA lesions that can arise in the cell.These breaks arise as a result of both exogenous (for e.g. ioniz-ing radiation and chemotherapeutic drugs) and endogenous(for e.g. reactive oxygen species and stalled replication forks)insults. DSBs can be repaired by one of two major pathways ineukaryotes: 1) non-homologous end joining (NHEJ), an error-prone process wherein the DNA ends are directly rejoined afterlimited end processing (1), and 2) homologous recombination(HR), an error-free pathway that uses the undamaged sisterchromatid as a template for repair (2). Correct “repair pathwaychoice” is critical for the maintenance of genomic integrity (forreview, see Refs. 3–5). Recent evidence suggests that cyclin-de-pendent kinases (CDKs) that are active in S and G2 phases reg-ulate repair pathway choice by promoting DNA end resectionthat stymies NHEJ and facilitates HR (for review, see Ref. 6).End resection results in the generation of 3�-ended single-stranded DNA (ssDNA) that is rapidly coated by replicationprotein A (RPA), which is then replaced with Rad51 to generatea nucleoprotein filament that copies information from the sis-ter chromatid. DNA end resection occurs in a two-step manner(for review, see Refs. 7 and 8). First, resection is initiated by theremoval of �50 –100 bases of DNA from the 5� end by theMRX/MRN complex (Mre11-Rad50-Xrs2 in yeast and MRE11-RAD50-NBS1 in mammals) in concert with Sae2/CtIP (9 –13).Next, long range resection is carried out by two alternate path-ways involving either EXO1 alone or the helicase Sgs1/BLMworking in conjunction with EXO1 or the nuclease DNA2 (14 –16). Research from a number of laboratories has establishedthat CDKs 1 and 2 promote the initiation of resection by phos-phorylating Sae2/CtIP (12, 17–21) and NBS1 (22), thereby cou-pling HR to S and G2 phases of the cell cycle.

Recent results from our laboratory established that CDK1and CDK2 also promote long-range resection via phosphoryla-tion of EXO1 (23; for review, see Refs. 8 and 24). EXO1 is a 5� to3� exonuclease with key roles in DNA mismatch repair, mitoticand meiotic recombination, replication, and telomere homeo-

This work was supported, in whole or in part, by National Institutes of HealthGrants RO1CA197796, RO1CA149461, and R21CA202403 (to S. B). Thiswork was also supported by National Aeronautics and Space Administra-tion Grant NNX16AD78G. The authors declare that they have no conflict ofinterest with the contents of this article. The content is solely the respon-sibility of the authors and does not necessarily represent the official viewsof the National Institutes of Health.

This article contains supplemental Tables 1–3 and Figs. 1–5.1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Division of Molecular Radi-

ation Biology, Dept. of Radiation Oncology, University of Texas SouthwesternMedical Center, 2201 Inwood Rd., NC7.208, Dallas, TX 75390. Tel.: 214-648-7440; Fax: 214-648-5995; E-mail: sandeep.burma@utsouthwestern.edu.

3 The abbreviations used are: DSB, double-strand break; NHEJ, non-homo-logous end joining; ssDNA, single-stranded DNA; RPA, replication proteinA; ATM, ataxia telangiectasia-mutated; ATR; ATM and Rad3-related; CPT,camptothecin; CHX, cycloheximide; SCF, Skp1-Cullin1-F-box; DN, domi-nant negative; AUC, area under the curve; RFP, red fluorescent protein; Ub,ubiquitin; CDK, cyclin-dependent kinase.

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stasis (for review, see Refs. 25–27). Research from our labora-tory has established that EXO1 plays a major role in DNA endresection in human cells and not only promotes a switch fromNHEJ to HR but also facilitates a transition from ATM- to ATR-mediated checkpoint signaling (15, 16, 23, 28, 29). The nucleasedomain of EXO1 is highly conserved (30), whereas its C-termi-nal region is divergent and unstructured and mediates interac-tions with multiple DNA repair proteins (25, 31–34). The Cterminus of EXO1 is phosphorylated at four (S/T)P sites byCDKs 1 and 2 in the S/G2 phases of the cell cycle (23). Phospho-rylation of EXO1 by CDKs stimulates DNA end resection bypromoting the recruitment of EXO1 to DNA breaks via inter-actions with BRCA1 (23). The C terminus is also phosphorylat-ed at serine 714 by ATM (35) and ATR (36), which are thecentral kinases triggering the DNA-damage response to DSBsand DNA replication stress, respectively (37, 38). The func-tional consequences of serine 714 phosphorylation are not wellunderstood.

Given that EXO1 is a key HR exonuclease in eukaryotic cells,it is important to understand how such an enzyme is kept on atight rein after it is activated to prevent excessive DNA endresection. Excessive ssDNA would pose a threat to genomicintegrity as they would be prone to breakage and could eventrigger global genomic instability by exhausting the existingpool of RPA (39, 40). Furthermore, extensive DNA end resec-

tion would also cause a switch in the DSB repair mode fromerror-free HR to the highly deleterious single-strand-annealingpathway (41, 42). Here, we describe a mechanism by whichresection is restrained in human cells that involves the degra-dation of EXO1 after DNA damage in a phosphorylation- andubiquitination-dependent manner.

Results

EXO1 is rapidly degraded after DNA damage

In the course of our studies on EXO1 we were intrigued tofind that EXO1 levels rapidly declined in HEK-293 cells treatedwith the topoisomerase I poison camptothecin (CPT), whichinduces replication fork-associated DSBs (43) (Fig. 1a). Areduction in EXO1 levels was also seen in additional human celllines treated with CPT (supplemental Fig. 1a) or other geno-toxic agents (supplemental Fig. 1b). By following EXO1 levelsover a longer time period after CPT treatment, we found thatthe decrease in EXO1 was transient, showing recovery by �16 hpost-CPT treatment (supplemental Fig. 1c). By synchronizingcells in different phases of the cell cycle, we determined thatEXO1 levels were cell cycle-dependent, with the highest levelsobserved in phases where HR is operative, i.e. in S and G2 (sup-plemental Fig. 1d). However, the rapid reduction in EXO1 lev-els after DNA damage could not be attributed to cell-cycle

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Figure 1. EXO1 was rapidly degraded after DNA damage. a, HEK-293 cells were treated with CPT for the indicated times, and EXO1 protein levels wereassessed by Western blotting. Phosphorylation of KAP-1 and p53 were assayed by Western blotting with phospho-specific antibodies to confirm DNA-damageinduction. Actin served as a loading control. b, HEK-293 cells were pretreated with the proteasome inhibitor MG-132 or with DMSO as control for 4 h, thentreated with CPT for the indicated times. EXO1 levels were assessed by Western blotting. c, HEK-293 cells were treated with DMSO, ionizing radiation (IR), CPT,okadaic acid, or calyculin A as indicated immediately after the addition of the protein synthesis inhibitor CHX. EXO1 levels were assessed by Western blottingat the indicated times after CHX treatment. The plot shows relative EXO1 protein levels after CHX treatment (y axis) versus time (x axis) as determined byscanning and quantifying EXO1 bands and normalizing to actin bands using ImageJ software. All experiments were replicated three times. Error bars depict S.E.

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changes, as no major differences in cell-cycle distribution wereobserved for at least 8 h post-CPT treatment (supplemental Fig.1e). No changes in EXO1 transcript levels were seen after CPTtreatment (supplemental Fig. 1f), indicating that post-tran-scriptional mechanisms were involved in the decrease in EXO1levels. Indeed, the decrease in EXO1 appeared to be due toproteasomal degradation as this could be inhibited by treat-ment of cells with the proteasomal inhibitor MG-132 (Fig. 1b).By treating cells with the protein synthesis inhibitor cyclohex-imide (CHX), we found that the half-life of EXO1 was consid-erably shortened upon the induction of DNA damage by ioniz-ing radiation (IR) or CPT (Fig. 1c). Interestingly, treatment ofcells with the phosphatase inhibitors okadaic acid or calyculinA in the absence of DNA damage also resulted in destabilizationof EXO1, indicating that phosphorylation of EXO1 might trig-ger its degradation (Fig. 1c).

Phosphorylation of EXO1 by ATR triggers its degradation

EXO1 is known to be phosphorylated at serine 714 by ATMand ATR in response to ionizing radiation and DNA replicationinhibitors, respectively (35, 36). CPT-induced EXO1 degrada-tion was attenuated by pretreatment of cells with caffeine, abroad-specific inhibitor of both ATM and ATR kinases (44)(Fig. 2a). KU55933, a specific inhibitor ATM (45), or NU7026, aspecific inhibitor of DNA-PKcs (46), had no effect on EXO1degradation (supplemental Fig. 2a). On the other hand, pre-treatment of cells withVE822, a specific inhibitor of ATR (47),or siRNA-mediated knockdown of ATR resulted in stabiliza-

tion of EXO1 (Fig. 2, b and c). Taken together, these observa-tions indicated that EXO1 degradation might be triggered byATR-mediated phosphorylation events. Knockdown of sec-ond-tier kinases that function downstream of ATR and ATM,CHK1 and CHK2, respectively (48), had no effect on EXO1levels (supplemental Fig. 2b), implying that direct phosphory-lation of EXO1 by ATR might trigger its degradation. To testthis possibility, we mutated serine 714 of EXO1 to alanine, andexpressed siRNA-resistant V5-tagged S714A-EXO1 in HEK-293 cells with siRNA-mediated knockdown of endogenousEXO1 (23). However, upon challenging these cells with CPT,we found that mutation of the serine 714 phosphorylation sitedid not affect EXO1 degradation appreciably (Fig. 2d). We rea-soned that EXO1 might be phosphorylated at additional SQsites upon DNA damage. This line of reasoning was based uponthe observation that EXO1 with mutated serine 714 (S714A-EXO1) was still recognized by anti-phospho-(Ser/Thr) ATM/ATR substrate antibody in immunoprecipitation/Westernblotting, whereas EXO1 with mutation of all six potential SQphosphorylation sites in its C-terminal tail (S432A/S652A/S674A/S676A/S694A/S714A; henceforth referred to as 6A-EXO1) was no longer recognized by this antibody (supple-mental Fig. 2c). Moreover, CPT-induced phosphorylation ofS714A-EXO1 was attenuated by pretreatment of cells with theATR inhibitor VE822 (47), thereby implicating ATR in EXO1phosphorylation (supplemental Fig. 2d). Significantly, unlikewild-type EXO1, 6A-EXO1 was very stable after the induction

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Figure 2. Phosphorylation of EXO1 by ATR triggered its degradation. a, HEK-293 cells were treated with the ATM/ATR inhibitor caffeine or with DMSO asthe control for 1 h before treatment with CPT for the indicated times. EXO1 protein levels were assessed by Western blotting. Phosphorylation of KAP-1 and p53were assayed by Western blotting with phospho-specific antibodies to confirm DNA-damage induction. Phosphorylation of CHK1 was assessed to confirm ATRinactivation. b, HEK-293 cells were treated with the ATR inhibitor VE822 or with DMSO as the control for 1 h before treatment with CPT. EXO1 protein levels wereassessed by Western blotting. c, EXO1 levels were assessed after CPT treatment of HEK-293 cells with siRNA-mediated knockdown of ATR. d, HEK-293 cells weredepleted of endogenous EXO1 using siRNA and complemented with V5-tagged, siRNA-resistant wild-type EXO1 (WT) or EXO1 mutated at serine 714 (S714A).Cells were treated with CPT, and EXO1 levels were assessed by Western blotting with anti-V5 antibody. e, HEK-293 cells were depleted of endogenous EXO1using siRNA and complemented with siRNA-resistant EXO1 mutated at all six SQ sites (Ser-432, Ser-652, Ser-674, Ser-676, Ser-694, and Ser-714) in its C-terminaldomain (6A). EXO1 levels after CPT treatment were assessed by Western blotting. All experiments were replicated three times.

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of DNA damage (Fig. 2e). We, therefore, concluded that DNA-damage-induced phosphorylation of the C terminus of EXO1triggers its degradation.

EXO1 is ubiquitinated in a phosphorylation-dependentmanner

As EXO1 appears to be degraded by the ubiquitin-protea-some system (Fig. 1b), we next sought to determine if EXO1phosphorylation upon DNA damage serves as trigger for itsubiquitination and subsequent degradation. To determinewhether EXO1 was directly ubiquitinated in vivo, we co-ex-pressed V5-EXO1 and His6-ubiquitin in HeLa cells (49). Thecells were exposed to CPT, His6-ubiquitin-conjugated proteinswere captured under denaturing conditions using Ni2�-aga-rose beads, and ubiquitinated forms of EXO1 were detected byWestern blotting with anti-V5 antibody. We found that EXO1was ubiquitinated at low levels in unstressed cells and thatEXO1 ubiquitination increased dramatically in response toDNA damage (Fig. 3a). The modified forms of EXO1 were notseen in cells expressing conjugation-defective His6-ubiquitin(�GG) (50), confirming that they represented ubiquitinatedEXO1. Importantly, the DNA-damage-induced increase inEXO1 ubiquitination was not seen in cells treated with the ATRinhibitor caffeine (Fig. 3b) or in cells expressing 6A-EXO1 (Fig.3c), indicating that phosphorylation of EXO1 by ATR serves totarget it for ubiquitination.

In light of the apparent link between EXO1 phosphorylationand ubiquitination, we were interested in the Skp1-Cullin1-F-

box (SCF) family of ubiquitin ligases, which typically recognizeand ubiquitinate phosphorylated substrates (51, 52). SCF ubiq-uitin ligases regulate multiple DNA-damage-response path-ways (51, 53). Notably, a Cullin1-containing SCF has beenreported to target CHK1 for degradation after it is phosphory-lated by ATR in a manner similar to that observed for EXO1 (54,55). To examine if a cullin-based E3 ligase was involved inEXO1 ubiquitination, we first treated cells with MLN4924,which is a general inhibitor of the cullin-RING subtype of ubiq-uitin ligases (56). Treatment with MLN4924 ablated EXO1ubiquitination (Fig. 3d) and resulted in failure of CPT-induceddestabilization of EXO1 (Fig. 3e). To specifically examine therole of Cullin1 in EXO1 ubiquitination, we transfected HeLacells with V5-EXO1 and His6-ubiquitin along with dominantnegative Cullin1 (DN-Cul1) (57) and assayed for EXO1 ubiq-uitination by Ni2�-pulldown under denaturing conditions.Expression of DN-Cul1 attenuated EXO1 ubiquitination (Fig.3f) and suppressed degradation of EXO1 protein triggered byCPT exposure (Fig. 3g). As a control, we co-expressed domi-nant negative Cullin4A and Cullin4B (DN-Cul4) (57) in HeLacells but saw no effect on EXO1 stability (supplemental Fig. 3.).These results implicate a Cullin1-containing SCF complex inphosphorylation-dependent EXO1 degradation.

EXO1 degradation prevents hyper-resection and genomicinstability

We next asked whether EXO1 degradation might serve toprevent unbridled DNA resection, as the generation of exces-

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Figure 3. EXO1 was ubiquitinated in a phosphorylation-dependent manner. a, HeLa cells expressing V5-tagged EXO1 (V5-EXO1) and His6-tagged wild-type (WT)or conjugation-defective (�GG) ubiquitin were treated with CPT for the indicated periods of time. His6-ubiquitin-conjugated proteins were captured under denaturingconditions using Ni2�-agarose beads, and ubiquitinated forms of EXO1 were detected by Western blotting with anti-V5 antibody. b, HeLa cells expressing V5-EXO1and His-Ub were treated with caffeine or DMSO as control before the addition of CPT for 4 h. EXO1 ubiquitination was assessed by Ni2�-capture followed by Westernblotting. c, HeLa cells expressing wild-type (WT) or phosphorylation site-mutant (6A) V5-EXO1 and His-Ub were treated with CPT for 4 h. EXO1 ubiquitination wasassessed by Ni2�-capture followed by Western blotting. d, HeLa cells expressing V5-EXO1 were treated with the cullin-RING ubiquitin ligase inhibitor MLN4924 orDMSO for 4 h before the addition of CPT for 4 h. EXO1 ubiquitination was assessed by Ni2�-capture followed by Western blotting. e, HEK-293 cells were treated withDMSO or MLN4924 for 4 h before treatment with CPT for the indicated times. EXO1 protein levels were assessed by Western blotting. f, HeLa cells expressing V5-EXO1and His-Ub in the presence or absence of dominant negative Cullin1 (DN-Cullin1) were treated with CPT for 4 h. EXO1 ubiquitination was assessed by Ni2�-capturefollowed by Western blotting. Cells were transfected with a V5-�-gal vector as control (Mock). g, HEK-293 cells expressing DN-Cullin1 were treated with CPT for theindicated times. EXO1 levels were assessed by Western blotting. All experiments were replicated three times.

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sive ssDNA would not be conducive to optimal HR (41, 42) andcould even contribute to global genomic instability by seques-tering the nuclear pool of RPA (39, 40). The availability of adegradation-resistant version of EXO1 (6A-EXO1) (Fig. 2d)allowed us to directly answer this question. We first confirmedthat these mutations did not affect the exonuclease activity ofEXO1 per se (supplemental Fig. 4a) or its interactions with theresection proteins CtIP and BLM (23, 58, 59) (supplemental Fig.4b). We next used three complementary assays to measureDNA end resection:quantification of RPA foci (an indirect mea-sure of resection), enumeration of ssDNA/BrdU foci (a directmeasure of resection), and live cell imaging of GFP-RPA accu-mulation at the sites of micro-laser-induced DSBs (whichallows the visualization of end resection in real time) (16, 23,60). We found that expression of 6A-EXO1 in U2OS cellsresulted in increased numbers of CPT-induced RPA (Fig. 4aand supplemental Fig. 4c) and ssDNA/BrdU foci (Fig. 4b) aswell as enhanced recruitment of GFP-RPA to micro-laser-in-duced DNA damage (Fig. 4c). Similar results were obtained bypreventing degradation of endogenous wild-type EXO1 indi-rectly by expressing DN-Cul1 (supplemental Fig. 5, a– c). Thesetwo sets of results clearly demonstrate that EXO1 degradationprevents hyper-resection.

To assess the effect of hyper-resection on DSB repair, weexpressed WT- or 6A-EXO1 in cells harboring integratedreporters used to quantify the repair of I-SceI-induced DSBs byNHEJ (61) or HR (62). As expected, expression of 6A-EXO1resulted in attenuation of NHEJ (Fig. 4d) because DNA endresection stymies NHEJ by preventing the binding of KU (7, 63).Interestingly, expression of 6A-EXO1 also resulted in reducedlevels of HR relative to WT-EXO1 (Fig. 4e), presumably becauseexcessive end resection hinders optimal HR (41, 42). Attenua-tion of both HR and NHEJ was also seen in cells expressingDN-Cul1 (supplemental Fig. 5, d and e). In light of the down-regulation of both NHEJ and HR, we predicted that cellsexpressing degradation-resistant EXO1 would be severely com-promised in the repair of CPT-induced DSBs. We treated cellsexpressing WT- or 6A-EXO1 with CPT and quantified the for-mation and dissolution of 53BP1 or �H2AX foci (64, 65) toevaluate the effects of EXO1 stabilization on DSB repair. Wefound that cells expressing 6A-EXO1 displayed an almostcomplete block in the repair of CPT-induced DSBs (Fig. 4fand supplemental Fig. 4d). A similar block in repair was alsoseen in cells expressing DN-Cul1 (supplemental Fig. 5f). Thelack of repair coupled with the generation of excessivessDNA clearly compromised chromosomal stability, as cellsexpressing 6A-EXO1 or DN-Cul1 exhibited metaphaseswith shattered chromosomes after induction of DNA dam-age with CPT (Fig. 4g and supplemental Fig. 5g). Accord-ingly, cells expressing 6A-EXO1 were more sensitive to CPTcompared with cells expressing WT-EXO1 in a colony sur-vival assay (Fig. 4h). Taken together, these results demon-strate that DNA-damage-induced EXO1 degradation pre-vents excessive DNA end resection and promotes optimalDSB repair, which together contribute to the preservation ofchromosomal integrity and cell survival in the face ofgenomic insults.

Discussion

It is now well understood that several mechanisms exist topromote DNA end resection, a critical step that is essential forHR repair of DSBs, especially replication-associated DSBs likethose induced by CPT (66). However, how resection isrestrained or terminated is not well understood. Our resultsuncover a novel mechanism by which resection is negativelyregulated via the degradation of EXO1, an exonuclease withcritical functions in long-range resection (25, 26). Our resultsindicate that EXO1, although vital for resection and HR, is rap-idly degraded after DNA damage in a phosphorylation- andubiquitination-dependent manner. The rapid degradation ofEXO1 serves to prevent hyper-resection. Hyper-resectioncould promote genomic instability because 1) extensive resec-tion would shift DSB repair from RAD51-dependent HR toRAD52-dependent single-strand annealing, a highly mutagenicDNA repair pathway (41, 42), and 2) excessive ssDNA wouldexhaust the existing pool of RPA thereby triggering globalgenomic instability (39, 40). The net effect of these two phe-nomena would be the accumulation of CPT-induced DNAbreaks and chromosome shattering that we observe in cellsexpressing degradation-resistant EXO1.

In light of these data and our previously published results(23), we envisage that phosphorylation of EXO1 by CDKs inS/G2 primes EXO1 to respond to DSBs, especially replication-associated breaks that can be correctly repaired only by HR.However, upon the induction of such breaks, EXO1 is phos-phorylated by ATR, which marks it for SCF-mediated ubiquiti-nation and subsequent degradation (Fig. 5). A delay betweenEXO1 phosphorylation and its degradation possibly creates atemporal window that is sufficient for EXO1 to optimally resectDNA ends before it is destroyed to prevent excessive resectionor collateral damage to other regions of the genome.

The degradation of EXO1 that we observe appears to bemediated by a Cullin1-containing SCF ubiquitin ligase complex(51, 52). Ubiquitination along with SUMOylation are emergingas important regulators of DNA-damage response (for review,see Refs. 67 and 68). Most published studies have focused on therole of ubiquitination and SUMOylation in the recruitment ofproteins to DSBs, notably CtIP, 53BP1, RIF1, RAD18, andBRCA1 (68 –70). Interestingly, recent reports also implicatethese modifications in the degradation of cell-cycle checkpointor DNA-repair proteins, notably CHK1, CtIP, 53BP1, MDC1,RPA, BRCA1, and FANCA (54, 71–77), and in the release of theNHEJ factor KU from DSBs (78 – 80). Among these proteins, avery close analogy can be drawn between the degradation ofEXO1 and that of the cell-cycle checkpoint kinase CHK1 (54,55). Phosphorylation of CHK1 by ATR serves to initiate cell-cycle checkpoint arrest but also limits the duration of check-point signaling via SCF-mediated degradation of the kinase. Ina similar manner, phosphorylation of EXO1 by ATR limits theextent of DNA end resection by SCF-mediated degradation ofthe potentially genotoxic nuclease. Of relevance to our findings,SCFs have been implicated in the phosphorylation-triggereddegradation of a number of additional DNA repair and cell-cycle checkpoint proteins including BRCA1 (76), KU (78, 79),and Cdc25A (81).

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Lending credence to our observation of DNA-damage-in-duced EXO1 ubiquitination, EXO1 was recently identified as aSCF target in a quantitative proteomic screen for UV-inducedubiquitination events in human cells (82). This screen and oth-ers (57, 83, 84) identified the following ubiquitination sites onEXO1 (Lys-214, Lys-292, Lys-391, Lys-548, and Lys-796). How-ever, mutation of these five sites, singly or in combination, wasnot sufficient to affect EXO1 ubiquitination.4 It is possible thatthere are additional, yet unidentified, ubiquitination sites onEXO1 or that mutation of the above sites simply resulted inpromiscuous ubiquitination on nearby lysines (85). Futurestudies will hopefully shed more light on the exact ubiquitina-tion site(s) on EXO1 that trigger its degradation.

Our results showing that ATR triggers EXO1 degradation inresponse to replication-associated DSBs are of interest in lightof a previous report showing that ATR triggers EXO1 degrada-tion upon treatment of cells with the DNA replication inhibitorhydroxyurea (36). Given that treatment with hydroxyureainduces DSBs due to replication fork collapse (86, 87), it is plau-sible that the actual trigger for EXO1 degradation in the previ-ous study might have been replication fork-associated DSBs.Also, in the previous study, as in ours, mutation of serine 714was not sufficient to stabilize EXO1 (36), which bolsters ourassessment that multiple phosphorylation sites on the C termi-nus of EXO1 are needed for its ubiquitination and subsequentdegradation. In a recent study from the same group, it wasreported that EXO1 is also SUMOylated in response to DNAreplication stress, although mutation of the identified SUMOsites did not affect EXO1 stability (88). It is, therefore, clear that

EXO1 stability is probably regulated by an interplay of post-translational modifications (PTM)–phosphorylation, SUMO-ylation, and ubiquitination– but the exact choreography ofthese PTMs and the exact roles of specific modifications inEXO1 regulation still need to be elucidated.

Regulatory mechanisms impinging on EXO1 are of signifi-cance from a cancer standpoint, as multiple SNPs in EXO1 havebeen identified in patients with colorectal cancers (89 –91), anda specific polymorphism, K589E (rs1047840), has been linkedwith an increased risk of breast (92), gastric (93), oral (94), lung(95), and brain (96) cancer development. Another polymor-phism, N279S (rs4149909), was recently linked to an increasedrisk of breast cancer in a genome-wide association analysis of�120,000 individuals (97). It would be of interest to determinein the future if some of these SNPs affect the degradation ofEXO1 elucidated in our study and whether blocking EXO1 deg-radation might be a viable cancer therapeutic strategy.

Experimental procedures

Cell culture

Cell lines were obtained from the American Type CultureCollection. U2OS cells were maintained in �-MEM (�-mini-mum Eagle’s medium) and HEK-293 and HeLa cells in DMEMmedium supplemented with 10% fetal bovine serum and peni-cillin/streptomycin in a humidified atmosphere with 5% CO2.All cells were mycoplasma free.

Drug treatments

Cells were treated with 1 �M camptothecin (Sigma), 10 �M

etoposide (Selleckchem), or 50 �M hydroxyurea (Sigma) for theindicated periods of time to induce DNA damage. Cells weretreated with 10 �M MG-132 (Selleckchem) for 4 h before treat-

4 N. Tomimatsu, B. Mukherjee, J. L. Harris, F. L. Boffo, M. C. Hardebeck, P. R.Potts, K. K. Khanna, and S. Burma, unpublished data; HR, homologousrecombination.

Figure 4. EXO1 degradation prevented hyper-resection and genomic instability. a, U2OS cells were depleted of endogenous EXO1 using siRNA andcomplemented with V5-tagged, siRNA-resistant wild-type (WT), or phosphorylation site mutant (6A)-EXO1. Cells were treated with CPT or DMSO as control for4 h and immunostained with anti-RPA antibody (red). Nuclei were stained with DAPI (blue). Representative images are shown. The plot shows average numbersof RPA foci per cell after subtracting background (average numbers of foci in DMSO-treated cells). V5-EXO1 expression was verified by Western blotting withanti-V5 antibody. b, U2OS cells expressing WT- or 6A-EXO1 were treated with CPT or DMSO for 4 h and immunostained for BrdU/ssDNA foci (red). Represent-ative images are shown. The plot shows average numbers of BrdU foci per cell after subtracting background. c, U2OS cells expressing WT- or 6A-EXO1 alongwith GFP-RPA were laser-micro-irradiated to induce focal DSBs. Time-lapse images of accumulation of GFP-RPA at DSBs (arrows) are shown. The plot showsrecruitment of GFP-RPA after DNA damage. d, NHEJ efficiency was measured by quantifying RFP expression (by flow cytometry) in HEK293 cells harboring aGFP/RFP reporter after transfection with an I-SceI plasmid. Cells were depleted of endogenous EXO1 using siRNA and complemented with WT- or 6A-EXO1. Theplot shows NHEJ-directed repair relative to WT-EXO1-expressing cells. e, HR efficiency was measured by quantifying GFP expression in HEK-293 cells harboringa DR-GFP reporter after transfection with an I-SceI plasmid. Cells were depleted of endogenous EXO1 using siRNA and complemented with WT- or 6A-EXO1.The plot shows HR-directed repair relative to WT-EXO1 expressing cells. f, U2OS cells depleted of endogenous EXO1 and complemented with WT- or 6A-EXO1were treated with CPT and immunostained for 53BP1 foci (green) at the indicated times post treatment. Representative images are shown. Rates of DSB repairwere determined by scoring 53BP1 foci and plotting average number of 53BP1 foci per cell (y axis) versus time (x axis). g, percentages of metaphase spreads withshattered chromosomes are plotted for CPT-treated U2OS cells with knockdown of endogenous EXO1 and ectopic expression of WT- or 6A-EXO1. Represent-ative metaphases are shown. Scale bar, 5 �m. h, the plot shows clonogenic survival of U2OS cells expressing WT- or 6A-EXO1. Cells were treated with theindicated doses of CPT for 4 h before plating for colony formation. All experiments were replicated three times. Error bars depict S.E. **, p � 0.005; ****, p �0.0001.

Figure 5. Proposed model of EXO1 regulation. As cells enter S phase, EXO1 is primed to function in resection by CDK-mediated phosphorylation (circles). Thetransduction of DNA damage signals by ATR is followed by EXO1 recruitment to DNA breaks and DNA end resection, which promotes DSB repair by HR (23) andcell-cycle checkpoint implementation via ATR (16). Once resection is under way, EXO1 is rapidly ubiquitinated (triangles) and targeted for degradation torestrict its activity and prevent over-resection. Potential phosphorylation sites are numbered.

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ment with camptothecin to block proteasomal degradation ofEXO1. Cells were treated with 50 �g/ml cycloheximide (EMDMillipore) to block protein synthesis. To inhibit protein phos-phatases, cells were treated with 1 �M okadaic acid or 100 nM

calyculin A (Santa Cruz) before treatment with cycloheximide.For inhibition of PI3K-like kinases, cells were treated with 5 mM

caffeine (Sigma), 10 �M KU55933 (Sigma), 10 �M NU7026(Sigma), or 1 �M VE822 (VX970) (Selleckchem) for 1 h beforecamptothecin treatment. To inhibit Cullin-RING ubiquitinligases, cells were treated with 5 �M MLN4924 (R&D Systems)for 4 h before treatment with camptothecin.

Irradiation of cells

Cells were irradiated with 10 gray of gamma rays from acesium source (JL Shepherd and Associates). Cells were irradi-ated with 10 J/m2 of UV-C (254 nm) at a dose rate of 0.5 J/m2/s.

Cell cycle synchronization and flow cytometry

HEK-293 cells were synchronized in G1 by treatment with 2�g/ml aphidicolin (Sigma) for 16 h and then released into reg-ular media for either 5 h (S phase) or 10 h (G2 phase) asdescribed before (29). Cell cycle stage was determined by flowcytometry using a BD CYTOMICS FC500 Flow Cytometer (BDBiosciences).

Western blotting and antibodies

Nuclear extracts for Western blotting were prepared by lys-ing cells in hypotonic lysis buffer (10 mM Tris-HCl, pH 7.5, 1.5mM MgCl2, 5 mM KCl, and protease and phosphatase inhibi-tors) followed by extraction in nuclear extraction buffer (50 mM

Tris-HCl, pH 7.5, 0.5 M NaCl, 2 mM EDTA, 10% sucrose, 10%glycerol, and protease and phosphatase inhibitors) (29). Thefollowing primary antibodies were used: phospho-EXO1(Ser-714) (15); EXO1, phospho-KAP-1 (Ser-824), KAP-1,CtIP, BLM (Bethyl); phospho-p53 (Ser-15), CHK1, phospho-CHK1 (Ser-317), phospho-ATM/ATR substrate (Cell Sig-naling); p53, actin, 53BP1 (H-300), Cullin1, Cullin4 (SantaCruz); V5 (Invitrogen); RPA (Calbiochem); CyclinA (Abcam);BrdU (BD Biosciences); �H2AX (Millipore). The followingsecondary antibodies were used: horseradish peroxidase-conjugated secondary antibodies (Bio-Rad) and Alexa 488/568-conjugated secondary antibodies (Thermo FisherScientific).

RT-PCR

HEK-293 cells were treated with 1 �M camptothecin for 4 h.Cells were harvested and RNA was isolated using the RNeasykit (Qiagen). The RNA was then reverse-transcribed to makecDNA using the SuperScript III First-Strand Synthesis Systemfor RT-PCR (Invitrogen). qRT-PCR of the cDNA was per-formed using SYBR� Green PCR Master Mix (Applied Biosys-tems) and primers against EXO1, p21, and TFRC. Primers arelisted in supplemental Table 1.

Cloning and mutagenesis

EXO1b was subcloned from Flag2-EXO1 (15) intopDONR221 (Invitrogen) using the Gateway BP Clonase IIenzyme mix (Invitrogen). Site-directed mutagenesis of EXO1

was carried out using Pfx50 polymerase (Invitrogen) and Taqligase (New England BioLabs); see supplemental Table 2 forprimer sequences. EXO1 was then transferred to pLenti6.3/V5-DEST (Invitrogen) using the Gateway LR Clonase II enzymemix (Invitrogen) for C-terminal V5-tagged fusion proteinexpression, as described before (23). Serines 432, 652, 674, 676,694, and 714 of EXO1 were mutated to alanine to generate6A-EXO1.

Transfection of cells

Depletion of EXO1, ATR, CHK1, or CHK2 was carried out bytransfection with the appropriate siRNAs (supplemental Table3; Invitrogen) using electroporation (Lonza) for U2OS cells andLipofectamine2000 (Invitrogen) for HEK-293 cells; cells wereharvested 48 h later to verify knockdown by Western blotting.Cells were transfected with scrambled siRNA as control; seesupplemental Table 3 for siRNA sequences. For ectopic expres-sion of proteins, cells were transfected with the appropriateexpression plasmid and assayed after 24 – 48 h. pHAGE DNCullin1, pHAGE DN Cullin4A, and pHAGE DN Cullin4B weregifts from Stephen Elledge (Addgene plasmid #41911, 41914,41915). For ectopic expression of siRNA-resistant EXO1in cells with knockdown of endogenous EXO1, cells weretransfected with the expression plasmid 24 h after siRNAtransfection.

Ubiquitination assay

HeLa cells were transfected with His-Ub (WT or �GG) (49)and V5-EXO1 (WT or 6A) expression plasmids using Effectene(Qiagen). After 48 h, cells were treated with 10 �M MG-132 for4 h before the addition of 1 �M camptothecin. Cells were har-vested at the indicated times post-camptothecin treatment.Harvested cells were resuspended in denaturing lysis buffer (6 M

guanidine-HCl, 100 mM Na2HPO4/NaH2PO4, pH 8, 10 mM

Tris-HCl, pH 8, 5 mM imidazole, and 10 mM �-mercaptoetha-nol). Extracts were clarified and incubated with 100 �l of nickel-nitrilotriacetic acid beads for 4 h with rotation. Beads were seri-ally washed with lysis buffer without imidazole, wash buffer A(8 M urea, 100 mM Na2HPO4/NaH2PO4, pH 8, 10 mM Tris-HCl,pH 8, and 10 mM �-mercaptoethanol), wash buffer B (8 M urea,100 mM Na2HPO4/NaH2PO4, pH 6.8, 10 mM Tris-HCl, pH 6.8,0.2% Triton-X, and 10 mM �-mercaptoethanol) and wash bufferC (8 M urea, 100 mM Na2HPO4, NaH2PO4, pH 6.8, 10 mM Tris-HCl, pH 6.8, 0.1% Triton-X, and 10 mM �-mercaptoethanol).Bound proteins were eluted with elution buffer (200 mM imid-azole, 150 mM Tris-HCl, pH 6.8, 30% glycerol, 5% SDS, and 720mM �-mercaptoethanol) and analyzed by Western blotting.

Immunoprecipitation

Whole cell extracts of HEK293 cells expressing V5-taggedEXO1 were made in lysis buffer (20 mM Tris-HCl, pH 7.5, 80mM NaCl, 2 mM EDTA, 10% glycerol, and 0.2% Nonidet P-40)supplemented with protease and phosphatase inhibitors.Anti-V5 antibodies were bound to Dynabeads (Invitrogen), andbeads were incubated for 4 h with cell extracts following themanufacturer’s protocol. Beads were washed extensively in lysisbuffer and then boiled in 1� SDS loading buffer before Western

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blotting. Normal mouse IgG (Santa Cruz) was used for controlimmunoprecipitations.

Exonuclease assay

V5-tagged EXO1 proteins were immunoprecipitated fromHEK293 cells using Dynabeads as described previously (23).Nuclease activity of wild-type or mutant EXO1 was assessed byanalyzing the degradation of a linearized 3�- radioactivelylabeled 7.8-kb plasmid (pLVX-Tight Puro). Each nuclease reac-tion contained EXO1 protein equivalent to the immunopre-cipitate from 2 mg of cell lysate and 50 ng of substrate in a finalvolume of 35 �l of nuclease assay buffer (20 mM HEPES, pH 7.5,40 mM KCl, 5 mM MgCl2, 0.05% Triton X-100, 5% glycerol, 100�g/�l BSA, 0.5 mM DTT, and 1 mM ATP). Reactions were incu-bated at 37 °C, and samples were removed at the indicatedintervals. Samples were resolved on 0.8% agarose gels andtransferred onto a Hybond-XL nylon membrane (AmershamBiosciences). Images were acquired using phosphorimagingscreens and a Fujifilm Starion scanner. Quantitation was car-ried out using Multi Gauge software (Fujifilm). Gel lanes weremarked and used to generate line traces, and peaks weredetected automatically using default settings. Area under thecurve (AUC) for the 7.8-kb reagent peak was calculated for eachlane. The fraction of substrate remaining was calculated bydividing AUC of each sample by AUC of the starting material.Three independent time courses were quantified for eachprotein.

Immunofluorescence staining

To stain for RPA foci, cells were seeded onto glass chamberslides (Lab-Tek) and treated with 0.1 �M camptothecin for 4 h.The cells were fixed with 4% paraformaldehyde/PBS and per-meabilized with 0.5% Triton-X/PBS before incubation withantibodies (60). To obtain clear RPA foci, cells were subject toin situ fractionation (98). The average number of RPA foci pernucleus was determined after scoring at least 50 nuclei andsubtracting background (average numbers of foci in mock-treated cells). For quantifying DSB repair kinetics, cells weretreated with 0.1 �M camptothecin for 2 h after which the mediawere replaced with camptothecin-free media. Cells were co-im-munostained with anti-53BP1 or �H2AX antibody at differenttime points post-camptothecin treatment as described previ-ously (61). Images were captured using a Leica DH5500B fluo-rescence microscope (X40 objective lens) coupled to a LeicaDFC340 FX camera using Leica Application Suite v4 acquisi-tion software.

BrdU/ssDNA assay

Cells grown in the presence of 10 �M BrdU (Sigma) for 16 hwere treated with 0.1 �M camptothecin for 4 h and immunoflu-orescence stained with anti-BrdU antibody under non-dena-turing conditions (to detect BrdU incorporated into ssDNA)(60). For clarifying BrdU/ssDNA foci, cells were subject to insitu fractionation (98). The average number of BrdU/ssDNAfoci per nucleus was determined after scoring at least 50 nucleiand subtracting background (average numbers of foci in mock-treated cells).

Laser live cell imaging

Cells were transfected with GFP-RPA construct, laser micro-irradiated and time-lapse-imaged, and the fluorescence inten-sities of micro-irradiated areas were plotted after backgroundsubtraction (fluorescence intensities of un-irradiated areas) asdescribed before (16). Cells were irradiated, and live cell imageswere taken with a pulsed nitrogen laser (Spectra-Physics; 365nm, 10 Hz) coupled to a Carl Zeiss Axiovert 200M microscope(�63 oil immersion objective). Mean fluorescence intensitiesfor each time point were determined using Axiovision softwarev4 from at least 30 independent measurements, and totalincrease in fluorescence signal was plotted versus time.

NHEJ and HR assays

To measure HR, GFP expression in HEK-293 cells with anintegrated DR-GFP reporter was quantified by flow cytometry(62). To measure NHEJ, red fluorescent protein (RFP) expres-sion in HEK-293 cells with an integrated GFP-to-RFP reporterwas quantified by flow cytometry (61). For both assays, cellswere depleted of endogenous EXO1 using siRNA and trans-fected with the V5-EXO1 (WT or 6A) expression plasmid; after24 h, cells were transfected with an I-SceI expression vector,and GFP or RFP expression was quantified by flow cytometryafter an additional 72 h. GFP-positive or RFP-positive frequen-cies were corrected for transfection efficiencies (quantified byparallel transfection with a wild-type GFP expression vector).

Metaphase chromosome preparations

Cells were treated with 0.1 �M camptothecin for 2 h afterwhich the media was replaced with camptothecin-free media.Colcemid (Sigma), along with 1 mM caffeine (Sigma) to bypassG2/M arrest, was added to cells 24 h after camptothecin treat-ment. Metaphase chromosome spreads were prepared 16 hlater as described before (99).

Colony formation assay

Cells were treated with the indicated doses of camptothecinfor 4 h and plated in triplicate onto 60-mm dishes (1000 cells/dish). Surviving colonies were stained with crystal violet�10 –14 days later as described before (100).

Statistical analyses

Statistical analyses were performed using GraphPad Prism7software. Two-tailed Student’s t test was used to determinestatistical significance (ns, not significant; *, p � 0.01; **, p �0.005; ***, p � 0.001; ****, p � 0.0001).

Author contributions—N. T., B. M., M. C. H., F. L. B., J. L. H., andS. B. conducted the experiments. N. T., P. R. P., J. H., K. K. K., andS. B. designed the experiments and interpreted the results. B. M.and S. B. wrote the paper.

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BurmaSandeepBoffo, Molly Catherine Hardebeck, Patrick Ryan Potts, Kum Kum Khanna and

Nozomi Tomimatsu, Bipasha Mukherjee, Janelle Louise Harris, Francesca Ludovicato ensure accurate DNA repair

DNA-damage-induced degradation of EXO1 exonuclease limits DNA end resection

doi: 10.1074/jbc.M116.772475 originally published online May 17, 20172017, 292:10779-10790.J. Biol. Chem. 

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