dna-pkcs and parp1 bind to unresected stalled ... · replication forkprogression andprocessing,...

Molecular and Cellular Pathobiology

DNA-PKcs and PARP1 Bind to Unresected StalledDNAReplication ForksWhere TheyRecruit XRCC1to Mediate RepairSongminYing1,2, Zhihui Chen3, Annette L.Medhurst4, JessicaA.Neal5,6, ZhengqiangBao2,Oliver Mortusewicz7, Joanna McGouran8, Xinming Song3, Huahao Shen1,9,Freddie C. Hamdy10, Benedikt M. Kessler8, Katheryn Meek5,6, and Thomas Helleday7

Abstract

A series of critical pathways are responsible for the detection,signaling, and restart of replication forks that encounter blocksduring S-phase progression. Small base lesions may obstructreplication fork progression and processing, but the link betweenrepair of small lesions and replication forks is unclear. In thisstudy, we investigated a hypothesized role for DNA-PK, animportant enzyme in DNA repair, in cellular responses to DNAreplication stress. The enzyme catalytic subunit DNA-PKcs wasphosphorylated on S2056 at sites of stalled replication forks inresponse to short hydroxyurea treatment. UsingDNA fiber experi-ments, we found that catalytically activeDNA-PKwas required for

efficient replication restart of stalled forks. Furthermore, enzy-matically active DNA-PK was also required for PARP-dependentrecruitment of XRCC1 to stalled replication forks. This activitywasenhanced by preventing Mre11-dependent DNA end resection,suggesting that XRCC1 must be recruited early to an unresectedstalled fork. We also found that XRCC1 was required for effectiverestart of a subset of stalled replication forks. Overall, our worksuggested that DNA-PK and PARP-dependent recruitment ofXRCC1 is necessary to effectively protect, repair, and restart stalledreplication forks, providing new insight into how genomicstability is preserved. Cancer Res; 76(5); 1078–88. �2015 AACR.

IntroductionThe integrity of DNA replication forks is essential for the

maintenance of genomic stability. It is becoming increasinglyclear that a variety of lesions can be produced at stalled replication

forks, and that these may trigger various responses. Multiplecellular pathways are required to ensure faithful duplication ofthe genome in an intricate network of cellular events that detect,signal, and repair any lesions that will impede the process of DNAreplication. First, cells can activate pathways that will utilize lowfidelity polymerases that allow replication to bypass the genotoxicstress in a process termed translesion synthesis, for subsequentpostreplication repair (1, 2). Second, when the blocking lesion istoo great, forks collapse forming a single DNA double-strand endthat is generally repaired by homologous recombination (HR;refs. 3–5), which can also be inappropriately joined to a singleDNA end from another collapsed fork, resulting in replicationstress–induced copy number variation as demonstrated byGloverand colleagues (6). Finally, the cell can pause replicationwhile thelesion is repaired and then restart replication from the same place.The molecular mechanisms behind translesion synthesis andreplication-induced DNA double-strand break repair have beenrelatively well studied but the processes behind replication restartremain ill-defined (2, 7).

Small base lesions are very common spontaneous lesions andmayobstruct replication fork progression (8) aswell as processingof stalled replication forks. However, to date, there is no estab-lished link between repair of small lesions and replication forks.In repair of small lesions, PARP1 has been demonstrated tointeract with and recruit the DNA repair protein, XRCC1, to sitesof DNA single-strand breaks (SSB; refs. 9, 10). XRCC1 has beenshown to be a scaffold protein that participates in both the SSBrepair and the base excision repair (BER) pathways (11, 12), whileinformation on its potential role in S-phase cells is lacking.

DNA-PKcs is an early component of the NHEJ pathway thatrepairsDNAdouble-strand breaks by a relatively rapid and simple

1Department of Respiratory and Critical Care Medicine of SecondAffiliatedHospital, ZhejiangUniversity School ofMedicine, Hangzhou,China. 2Department of Pharmacology, Zhejiang University School ofMedicine, Hangzhou,China. 3Department of Gastrointestinal and Pan-creatic Surgery, First Affiliated Hospital, Sun Yat-sen University,Guangzhou, China. 4Department of Oncology, CR-UK/MRC OxfordInstitute for Radiation Oncology, University of Oxford, Oxford, UnitedKingdom. 5Department ofMicrobiology&MolecularGenetics,Collegeof VeterinaryMedicine, Michigan StateUniversity, East Lansing, Michi-gan. 6DepartmentofPathobiology&Diagnostic Investigation,Collegeof VeterinaryMedicine, Michigan StateUniversity, East Lansing, Michi-gan. 7Science for Life Laboratory, Division of Translational Medicineand Chemical Biology, Department of Medical Biochemistry and Bio-physics, Karolinska Institutet, Stockholm, Sweden. 8Nuffield Depart-ment of Medicine, HenryWellcome Building for Molecular Physiology,University of Oxford, Oxford, United Kingdom. 9State Key Laboratoryfor Respiratory Diseases,Guangzhou,China. 10Nuffield Department ofSurgical Sciences, John Radcliffe Hospital, University of Oxford,Oxford, United Kingdom.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S. Ying and Z. Chen contributed equally to this article.

CorrespondingAuthors: ThomasHelleday, Karolinska Institutet, Science for LifeLaboratory, Stockholm, S-171 21, Sweden. Phone: 4608-5248-0000; Fax: 4420-8661-3107; E-mail: [email protected]; and Songmin Ying, E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-0608

�2015 American Association for Cancer Research.

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mechanism that essentially ligates severed DNA ends with com-paratively low fidelity (13, 14). Although cells deficient in com-ponents of the NHEJ pathway display a marked hypersensitivityto agents that cause DNA double-strand breaks, it has also beenreported sensitive to fork stalling agents in hamster cell lines(3, 5, 15). To examine a potential role for DNA-PKcs in mechan-isms that alleviate genotoxic stress caused by fork stalling, weassessed the functional role of DNA-PKcs and its phosphorylationin living cells treated with a replication inhibitor, hydroxyurea(HU; ref. 16).

Here, we demonstrate that (i) DNA-PKcs is phosphorylated atS2056 as a consequence of fork stalling; (ii) XRCC1 is recruited byPARP1 and DNA-PKcs at stalled replication forks; and (iii)XRCC1's recruitment to stalled forks is important for effectivereplication restart. This establishes one of the first link betweenXRCC1-mediated repair and replication repair.

Materials and MethodsCell lines and reagents

V-C8 and V-C8þB2 cell lines were kindly provided by Mal-gorzata Z. Zdzienicka (N. Copernicus University, Bydgoszcz,Poland; ref. 17). EM9-V, EM9-XH, and EM9-CKM cell lineswere kindly provided by Keith Caldecott (University of Sussex,Falmer, Brighton, United Kingdom). V3-derived cells weregrown in a-MEM (Gibco) supplemented with 10% FCS, pen-icillin-streptomycin, G418, and puromycin. All these Chinesehamster ovary cell lines and a human osteosarcoma cell lineU2OS were grown in DMEM with 10% FCS at 37�C under anatmosphere containing 5% CO2. All cell lines were authenti-cated by single tandem repeat analysis and grown up to amaximum of 20 passages and for less than 4 months followingresuscitation. All cell lines were routinely tested for morphol-ogy, doubling times, as well as being mycoplasma free byverification using the Mycoprobe Mycoplasma Detection Kit(R&D Systems). The GFP-XRCC1 construct was from Val�erieSchreiber (Universit�e de Strasbourg, Illkirch, France). Olaparib(10 mmol/L, otherwise indicated) was purchased from SelleckChemicals. Mre11 nuclease activity inhibitor mirin (100 mmol/L,otherwise indicated), CK2 inhibitor TBB (10 mmol/L, otherwiseindicated), and DNA-PKcs inhibitor NU7026 (10 mmol/L, other-wise indicated) were purchased from Sigma.

ImmunofluorescenceFollowing the indicated treatment, cells were fixed and stained

as previously described (18). The primary antibodies used wereXRCC1 (Abcam), phospho-specific DNA-PKcs S2056 (Abcam),cyclin A (Santa Cruz Biotechnology), gH2AX (Millipore), RPA(Neomarkers), and 53BP1 (Bethyl Laboratories, Inc.). The secon-dary antibodieswereAlexa Fluor 555–conjugated goat anti-mouseIgG (Molecular Probes). DNA was counterstained with DAPI.

High-throughput microscopyFor high-throughput microscopy, cells were seeded in 96-well

plates (10,000 cells/well), fixed in 4% formaldehyde þ 0.5%Triton X-100 for 20minutes, and permeabilized with 0.5% TritonX-100 for 10 minutes. For quantitative microscopy, images weretakenwith an ImageXpress (MolecularDevices)microscopeusinga 20� lens. Foci numbers per cell were counted using Cell Profiler(Broad Institute, Cambridge, MA) software and plotted usingMicrosoft Excel.

Western blottingCells were lysed in RIPA buffer in the presence of 1 � protease

inhibitor cocktail (Sigma). An aliquot of 50 mg total protein wasrun on an SDS-PAGE gel and transferred to Hybond ECL mem-brane (Amersham Pharmacia). This membrane was immuno-blotted with XRCC1 (Abcam), RAD51 (Santa Cruz Biotechnolo-gy), Ku70 (NeoMarkers), Ku80 (NeoMarkers), tubulin (Sigma),DNA-PK (Calbiochem), and GRB2 (BD Transduction Laborato-ries) antibodies in 5% milk overnight. Immunoreactive proteinswere visualized using ECL reagents (Roche) following the man-ufacturer's instructions.

Toxicity assayTwo hundred cells were plated in duplicate into 6-well plates

overnight before the addition of indicated treatment. Seven to tendays later, when colonies could be observed, cells were fixed andstained with methylene blue in methanol (4 g/L). Coloniesconsisting of >50 cells were subsequently counted.

Pull-down of GFP-XRCC1 protein and its interaction partnersExtracts from approximately 1 � 108 cells were prepared by

resuspension and incubation of the cell pellet in 2-mL lysis buffer(20 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 0.5 mmol/LEDTA, 2 mmol/L PMSF, 0.5% NP40, 1� mammalian proteaseinhibitor mix) for 30 minutes on ice. After centrifugation, super-natants were diluted to 5 mL with immunoprecipitation buffer(20 mmol/L Tris–HCl pH 7.5, 150 mmol/L NaCl, 0.5 mmol/LEDTA). Extracts were incubated with 5 mg of a GFP-Trap_A beads(GFP nanotrap; Chromotek) for 2 hours at 4�C with constantmixing (19). GFP-XRCC1 and its interacting proteins were pulleddownby centrifugation at 2,700� g. The beadswerewashed twicewith 5 mL of wash buffer (20 mmol/L Tris–HCl pH 7.5, 300mmol/L NaCl, 0.5 mmol/L EDTA) and were resuspended in 2�SDS sample buffer and boiled at 95�C for 10minutes. Beads werecollected by centrifugation and the supernatant was run on anSDS-PAGE gel.

Mass spectrometry analysisGel slices were subjected to in-gel trypsinolysis as described

(20). The analysis of digested material was performed by liquidchromatography/tandem mass spectrometry (MS/MS) using anWaters Q-TOF Premier mass spectrometer (Waters) coupled to anano-UPLC system (NanoAcquity,Waters) using a reversed phase75 mm� 250mm column as described (21). MS/MS spectra weresearched against a UniProt SwissProt Human database(v2011.11.18, 20,326 sequences) in Mascot v2.3.01, allowingtwomissed cleavage and 50 ppm/0.1 kDamass deviations inMS/MS-MS, respectively. Carbamidomethylation of cysteine was afixed modification. Oxidation of methionine, deamidation ofasparagines and glutamine, and phosphorylation of serine andthreonine were used as variable modifications.

siRNA treatmentsiRNA against DNA-PKcs was purchased from Qiagen

(Hs_PRKDC _5). A nontargeting control "All stars negative con-trol"wasused fromQiagen.Cellswere transfected intoU2OS cellsusing Oligofectamine (Invitrogen) according to the manufac-turer's instructions. In brief, cells were double transfected 24hours apart. Cells were incubated for 72 hours after the initialtransfection before further analysis.

DNA-PKcs, PARP1, and XRCC1 Mediate Replication Repair

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DNA fiber assayCells were pulse-labeled with 25 mmol/L chloro-deoxyuridine

(CldU) and 250 mmol/L 5—iododeoxyuridine (IdUrd) as indi-cated. Labeled cells were harvested and DNA fiber spreadsprepared as described earlier (22). For immunodetection ofCldU-labeled tracts, acid-treated fiber spreads were incubatedwith rat anti-BrdUrd monoclonal antibody (AbD Serotec)that recognizes CldU, but not IdUrd for 1 hour at room tem-perature. Slides were fixedwith 4% formaldehyde and incubatedwith an Alexa Fluor 555–conjugated goat anti-rat IgG (Molec-ular Probes) for 1.5 hours at room temperature. IdUrd-labeledpatches were detected using a mouse anti-BrdUrd monoclonalantibody (Becton Dickinson) that recognizes IdUrd, but notCldU overnight at 4�C, followed by an Alexa Fluor 488–conju-gated goat anti-mouse F(ab0)2 fragment (Molecular Probes) for1.5 hours at room temperature. Fibers were examined using ausing a Bio-Rad Radiance confocal microscope using a 60 �(1.3NA) lens. The lengths of CldU (AF 555, red) and IdUrd (AF488, green) labeled patches were measured using the ImageJsoftware, and mm values were converted into kb using theconversion factor 1 mm ¼ 2.59 kb (22). Replication structureswere quantified using the Cell Counter Plug-in for ImageJ (KurtDe Vos, University of Sheffield, Sheffield, United Kingdom).

Statistical analysisThe paired one-tailed Student t test was used for statistical

analyses (P� 0.05, nonsignificant; �, P < 0.05; ��, P < 0.01; ��, P <0.001).

ResultsXRCC1 is recruited by PARP to unresected replication forks toprotect fork stability and to promote survival

Wehave shownpreviously that PARPplays an important role inrepairing replication fork damage by mediating restart and pro-tecting stalled DNA replication forks (18, 23). Here, we investi-gated whether PARP can also recruit XRCC1 to stalled DNAreplication forks. As can be seen, XRCC1 nuclear foci are inducedfollowing replication stress that results from exposure to HU (Fig.1A and B). Next, cells were labeled with 5-ethynyl-20-deoxyur-idine (EdU) 20minutes beforeHU treatment tomark the locationof stalled DNA replication forks, and then stained for XRCC1.More than 80% of XRCC1 foci colocalized with sites of DNAreplication, as defined by EdU incorporation (Fig. 1B). These dataindicate that XRCC1 is recruited to sites of stalled replicationforks. HU-treated cells were also costained for the cyclin A proteinto specifically identify S–G2 phase cells; XRCC1 foci were presentonly in the cyclin A–positive cells (Fig. 1C), confirming thatXRCC1 foci only form in replicating cells after HU treatment.

GFP-XRCC1 focus formation was analyzed following a com-bined treatment with HU and the PARP inhibitor olaparib (24).We found that PARP activity was required forHU-induced XRCC1focus formation (Fig. 1D). Furthermore, clonogenic assays wereperformedwith the XRCC1-deficient CHOcell strain EM9. EM9-Vcells (vector only transfected) are hypersensitive to HU as com-pared with the EM9 cells complemented with an XRCC1 expres-sion construct (Fig. 1E). We conclude that XRCC1 is required forthe survival of cells in response to replication stalling.

Next, we asked whether the Mre11 nuclease affectsXRCC1's accumulation or retention at stalled replication forks.When Mre11 nuclease activity was inhibited by the Mre11

inhibitor, mirin (25), increased numbers of XRCC1 foci wereobserved (Fig. 2A); moreover, induction of HU-induced XRCC1foci was potentiated by the inhibition of Mre11's nucleaseactivity (Fig. 2B). These data suggest that Mre11-induced proces-sing of stalled forks can suppress XRCC1 relocalization toreplication lesions. The induction of XRCC1 foci followingloss of Mre11 activity was fully dependent on PARP activity(Fig. 2C).

It has been reported that Mre11-mediated resection of stalledreplication forks was upregulated in the absence of BRCA2 (23).Here, the localization of XRCC1 in BRCA2-defective V-C8 andBRCA2 complemented V-C8þB2 cell lines was analyzed. Inaccordance with the previously reported hyperactivation of PARPactivity, XRCC1 foci were observed in BRCA2-defective cellswithout exposure toDNA-damaging agents (Fig. 2D).Noobviousdifference in the level of XRCC1 foci was found in V-C8 and V-C8þB2 cells following HU treatment for 24 hours (Supplemen-tary Fig. S1A), suggesting that BRCA2 does not play a direct role inthe recruitment of XRCC1 protein to damage in response toreplication stalling. However, the induction of XRCC1 by inhi-bition of Mre11 was dramatically enhanced in the absence offunctional BRCA2 (Fig. 2D). These data suggest that in the absenceof BRCA2 and Mre11's nuclease activity, there is an increase inunresected stalled forks; PARP targets XRCC1 to these unresectedstalled replication forks. This notion was further proved bydepletion of CtIP in U2OS cells, where increased levels of XRCC1foci formation were observed (Supplementary Fig. S1B and S1C),suggesting that CtIP-mediated resectionmay suppress the recruit-ment of XRCC1 protein to stalled forks.

Given that XRCC1 foci are increased in Mre11-inhibited cells,we reasoned that these two proteins may have compensatoryfunctions in repairing replication damage, and hence be goodcandidates to have a synthetic lethal relationship (as is the casewith PARP and BRCA2). To test this hypothesis, we investigatedwhether Mre11 activity is required for survival of XRCC1-defective cells. Consistent with this notion, XRCC1-deficientcells (EM9-V) are remarkably sensitive to Mre11 inhibition ascompared with isogenic, XRCC1 complemented cells (EM9-XH; Fig. 2E).

CK2 activity is required to recruit XRCC1 to stalled DNAreplication forks

Next, we tested whether XRCC1 protein levels are altered inresponse to replication damage. We found that XRCC1 proteinlevels increased, especially at early time points following HUtreatment, whereas RAD51 protein levels peaked after 24 hoursof treatment (Fig. 2F and Supplementary Fig. S2A). These dataare consistent with the findings that XRCC1 foci form early afterHU treatment, whereas RAD51 form later (26). This is alsoconsistent with the notion that XRCC1 may be involved inrepairing more simple DNA lesions than RAD51, which isrequired for repair of late forming DSBs following HU treat-ment (26).

Casein kinase-2 (CK2) phosphorylates and stabilizes XRCC1 toenable the assembly of DNA SSB repair protein complexes at sitesof DNA damage (27–29). Hence, we tested whether HU-inducedstabilization of XRCC1 protein at DNA damage sites is also CK2dependent. XRCC1 is stabilized following HU exposure in cellsexpressing wild-type XRCC1 but not in cells expressing the CKM-mutant form of XRCC1 that cannot be phosphorylated by CK2(Fig. 2F). To confirm that CK2 activity is needed, cells were treated

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with the CK2 inhibitor TBB, which also prevented accumulationof XRCC1 (Supplementary Fig. S2B). Furthermore, CK2 inhibi-tion prevents HU-induced GFP-XRCC1 focus formation (Supple-mentary Fig. S2C). Taken together, these data reveal a CK2-dependent stabilization of XRCC1 in response to replicationstalling.

XRCC1 binds to DNA-PK subunits in response to replicationstalling

To better understand how XRCC1 functions at stalled DNAreplication forks, a GFP-nanotrap strategy was utilized to "pulldown" GFP-XRCC1 and its interacting proteins. Mass spectrom-

etry analysis was performed to analyze components of thecomplex. As expected, a number of known XRCC1-interactingpartners were identified, including PARP-1, DNA ligase-3,PARP-2, bifunctional polynucleotide phosphatase/kinase(PNKP), DNA polymerase b, and aprataxin (Fig. 3A). Interest-ingly, DNA-PK subunits, KU70 and KU80, were identified asXRCC1-binding partners (Fig. 3A). To quantify the interactionbetween GFP-XRCC1 and the KU70/KU80 complex, Westernblot analyses were performed. Although an interaction betweenXRCC1 and KU70/KU80 was evident even in unperturbed cells,the amount of KU70/KU80 that was pulled down togetherwith GFP-XRCC1 following HU treatment was significantly

Figure 1.PARP recruits XRCC1 to stalled replication forks. A, quantification of GFP-XRCC1 foci following 2 mmol/L HU treatment for 2 or 6 hours in U2OS cells. B,immunofluorescence staining of endogenous XRCC1 and EdU following treatment of 2 mmol/L HU for 6 hours in U2OS cells. C, immunofluorescence stainingof G FP-XRCC1 and cyclin A following treatment of 2 mmol/L HU for 6 hours in U2OS cells. D, left, quantification of GFP-XRCC1 foci following indicated treatmentin U2OS cells for 6 hours. Right, Western blot analysis of PAR and GRB2 following indicated treatment in U2OS cells for 6 hours. E, clonogenic survivalfollowing 24 hours treatment with increasing concentrations of HU EM9-V or EM9-XH cells.






greater (Fig. 3B). The interaction was not DNA/RNA mediatedas shown in the presence of benzonase, a broad-spectrumnuclease (Supplementary Fig. S3A). This suggests that bindingof KU70/KU80 to XRCC1 was induced following replicationdamage.

HU-induced DNA-PKcs foci are apparent using a phospho-specific DNA-PKcs antibody (anti-pS2056). Moreover, about50% colocalization of these foci with XRCC1 foci was observed(Fig. 3C). Neither depletion of PARP-1 (Supplementary Fig. S3Band S3C) nor loss of XRCC1 (Supplementary Fig. S3D) affectedthe formationof phosphorylatedDNA-PKcs foci.However, reduc-ing the expression of DNA-PKcs by siRNA resulted in a decrease inthe percentage of cells that displayed HU-induced phosphorylat-ed DNA-PKcs foci, suggesting that this was DNA-PKcs dependent(Supplementary Fig. S4A and S4B).

DNA-PKcs is phosphorylated in response to replication forkstalling and its kinase activity is required for cellular resistanceto HU

DNA-PK autophosphorylates itself onnumerous residues (like-ly more than 40) in response to DNA damage (30, 31). Phos-phorylation within two major clusters of sites (ABCDE and PQRclusters) induces conformational changes that allow DNA-PK toregulate DNA end access; these phosphorylations clearly promoteNHEJ. Here, we examined HU-induced DNA-PKcs phosphoryla-tion of serine 2056, within the "PQR" phosphorylation sitecluster, in a panel of V3 transfectants expressing wild-type andmutant forms of DNA-PKcs. S2056 is strongly phosphorylated inresponse to2mmol/LHU in cells expressingwild-typeDNA-PKcs;no phosphorylation is observed in cells lacking DNA-PKcs, or incells expressing the PQR mutant (alanine ablation of 2056 site)

Figure 2.Recruitment of XRCC1 to unresected forks promotes cell survival. A, quantification of endogenous XRCC1 foci following treatment with 100 mmol/L mirin for 6 or24 hours in U2OS cells. B and C, quantification of GFP-XRCC1 foci following indicated treatment for 6 hours in U2OS cells. D, quantification of endogenousXRCC1 foci following treatment with 100 mmol/L mirin for 6 or 24 hours in V-C8 or V-C8þB2 cells. E, clonogenic survival following continuous treatmentwith increasing concentrations of mirin in EM9-V or EM9-XH cells. F, Western blot analysis of XRCC1 and GRB2 following treatment of 2 mmol/L HU in EM9-V,EM9-XH, or EM9-CKM cells.

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confirming the specificity of the phospho-specific reagent. Inter-estingly, in cells expressing a catalytically inactive DNA-PKcsmutant K-R: mutated at K3752R, this phosphorylation event isalso observed, albeit at lower levels than in wild-type counterpart(Fig. 4A and Supplementary Fig. S4C). It is important to note thatHU-induced 2056 phosphorylation of catalytically inactive DNA-PKcs is more robust than observed in cells (expressing K3752R)treated with either zeocin (a DSB-inducing agent) or UV (22),suggesting that another protein kinase can target DNA-PKcs inresponse to HU.

Here, we exploited a panel of DNA-PKcs mutants to assesswhether DNA-PK's catalytic activity and phosphorylation affectscellular resistance to HU exposure. Consistent with previousreports, cells lacking DNA-PKcs (vector only) display modestsensitivity to HU compared with wild-type controls (Fig. 4B).Cells expressing a catalytically inactive mutant are similarly sen-sitive toHU as are cells that express noDNA-PKcs; cells expressingcatalytically inactive DNA-PKcs are also similarly sensitive toagents that induce DSBs as cells that express no DNA-PKcs. Incontrast, cells expressing the PQR>ala mutant that are also sim-ilarly sensitive toHUas the K>Ror vector only expressing cells, areonly slightly sensitive to agents that induce DSBs as comparedwith cells lacking DNA-PKcs or expressing kinase inactive DNA-PKcs. The ABCDEmutant cannot phosphorylate any of six sites inthe ABCDE cluster, which are important in initiating NHEJ and inpromoting end processing; the ABCDE mutant is markedly sen-sitive,muchmore so than cells completely lackingDNA-PKcs (Fig.4B). Emerging data suggest that the ABCDE mutant can initiateNHEJ and proceed slowly through the process; prolonged occu-pancy of ABCDEblockedDNA-PK at aDNAend, and the resultingslow repair is clearly more detrimental to cell survival than inother mutants, for example, catalytically inactive mutants, thatcan bind DNA ends, but do not initiate NHEJ (15). These data

suggest that DNA-PK's functional role at stalled replication forksrequires a similar series of phosphorylation events to facilitaterepair, perhaps by regulating access to DNA ends.

DNA-PKcs S2056 foci are present in S-phase and represent sitesof unresected replication stress

To investigate the role of DNA-PKcs in the cellular response toreplication stress, we examined the induction of phosphorylationat residue S2056 in response to HU. Asynchronous U2OS cellsdisplay punctate p2056 nuclear foci in response to a 2-hourexposure to 2 mmol/L HU presumably reflecting stalled replica-tion forks (Fig. 4C). Reducing the expression of DNA-PKcs bysiRNA resulted in a decrease in the percentage of cells thatdisplayed HU-inducible foci (Supplementary Fig. S4A andS4B). These suggest that DNA-PKcs is phosphorylated at S2056in response to HU-induced genotoxic stress.

Next, we costained U2OS cells for cyclin A. Cells that formHU-induced DNA-PKcs S2056 foci also display moderate levels ofcyclin A staining, confirming that these cells are in S-phase (Fig.4D). We also sought to investigate whether these foci representedsites of replication stress. We determined this by costaining cellswith EdU and gH2AX. We observed that approximately 80% ofS2056 foci colocalized with EdU confirming that phosphorylatedDNA-PKcs responds to regions of DNA that have been recentlyreplicated and undergone subsequent fork stalling (Fig. 4E).Furthermore S2056 phosphorylation was also found to stronglycolocalize with g-H2AX confirming its association with replica-tion stress (Fig. 4F). Although virtually all p2056 sites colocalizewith gH2AX and EdU, this represents only a subset (approximate-ly 50%) of all gH2AX and EdU foci. HU clearly induces gH2AXand EdU that lack p2056. We also examined whether DNA-PKcswas present at ssDNA regions by costaining for phosphorylatedDNA-PKcswithRPA. Toour surprise, therewasminimal (less than

Figure 3.Interaction of XRCC1 with DNA–PK complex under replication stress. A, Coomassie staining of GFP-nanotrap pulled down GFP-XRCC1 complexes with treatmentor 2 mmol/L HU for 6 hours in U2OS cells. Specific GFP-XRCC1–interacting protein bands were analyzed by mass spectrometry, with the identified proteinsnamed. B, Western blot analysis of XRCC1, KU80, and KU70 from A. C, immunofluorescence staining of GFP-XRCC1 and phospho-DNA-PKcs following2 mmol/L HU treatment for 6 hours in U2OS cells.






20%) colocalization of these proteins, suggesting that at least aproportion of these two proteins localize to different DNA struc-tures created by HU-induced fork stalling (Fig. 4G). These datasuggest DNA-PK is phosphorylated at unresected DNA endsduring replication stress.

Previously, we observed that HU-induced g-H2AX foci arequickly recovered after release from a 2-hour HU-induced repli-cation stalling (26). Here, we determined g-H2AX foci and theirsubsequent recovery in cells treated with an inhibitor of DNA-PKcs kinase activity (NU7026). Interestingly, we found only aslight defect (but not significantly different) in recovery of g-H2AXfoci in the presence of NU7026 (Supplementary Fig. S5A). Sim-ilarly, the formation of RPA foci, and their subsequent recovery,after HU-induced fork stalling, is normal in the presence ofNU7026 (Supplementary Fig. S5B). In contrast, 53BP1 foci, amarker for DNA double-strand ends, which have been suggestedto form at reversed replication forks (chicken foot 4-way junc-tions) in response to short exposure to HU treatment, persist inthe absence of DNA-PKcs kinase activity (Supplementary Fig.S5C). Thus, it seems most likely that DNA-PK binds the dou-ble-strand end structure (chicken foot) at reversed forks, or incases of complete fork collapse, the resulting single DNA end.These data are consistent with a previous report showing a

requirement for DNA-PK in the repair of 53BP1 foci inducedafter a 15-hour HU treatment (32).

DNA-PK is required for XRCC1 recruitment to stalled forks andfor replication restart

Functional association between DNA-PKcs and XRCC1 waspreviously shown following ionizing irradiation (33). Next, wetested whether DNA-PKcs was required for XRCC1 recruitment tostalled forks. XRCC1 foci were measured in DNA-PKcs–defectiveV3-3 and wild-type AA8 cells following treatment with HU for 6hours. A dramatic decrease in the level of XRCC1 foci was found inthe absence of DNA-PKcs (Fig. 5A). Next, we tested various DNA-PKcs–mutant cell lines, and found that KR or PQR mutation wasalso able to reduce the frequency of XRCC1 foci (SupplementaryFig. S6). Furthermore, the HU-induced stabilization of XRCC1protein was also lost by the inhibition of DNA-PKcs activity (Fig.5B). These results collectively suggest that DNA-PKcs activity isrequired for XRCC1 function and likely acts upstreamof XRCC1atstalled forks. To test whether these activities were also involved inthe maintenance of the integrity of stalled forks, we analyzedXRCC1-defective EM9-V and the complemented EM9-XH cells,with or without DNA-PKcs inhibitor, for their ability to restartstalled replication forks (Fig. 5C–E). We found that, either in the

Figure 4.DNA-PKcs is phosphorylated in response to replication fork stalling and is required for cellular resistance to HU. A, cells were treated with 2 mmol/L HU for2 hours before harvesting for polyacrylamide gel electrophoresis. Western blotting of whole-cell extracts from the indicated mutant strains was performedand membranes probed with the specified antibodies. B, clonogenic survival assays to assess cellular survival of various DNA-PKcs mutant strains afterexposure to HU. The indicated cell lines were exposed to increasing doses of HU for 24 hours and then allowed to recover for 7 to 10 days before stainingsurviving colonies. Error bars, SEM from at least three independent experiments. C, representative images of HU-induced DNA-PKcs S2056 foci in U2OS cells.D, representative images of U2OS cells depicting HU-induced phospho-DNA-PKcs costaining with cyclin A. E–G, representative images of U2OS cellsdepicting phospho-DNA-PKcs costaining with EdU, gH2AX, and RPA in response to a 2-hour treatment of HU (2 mmol/L).

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absence of functional XRCC1 or following inhibition of DNA-PKcs activity, more forks failed to resume replication after releasefroma2-hourHUblock (Fig. 5C andD).However, a combinationof these two deficiencies did not further decrease the frequency offork restart (Fig. 5C and D). Similar trends were found for neworigin firing after release from 2 hours HU block (Fig. 5E).

DNA-PKcs kinase activity is requisite but not sufficient forreplication restart

Our data clearly demonstrate that DNA-PK promotes cellularsurvival after exposure toHU. To explain its role in promoting cellsurvival after HU exposure, we suggest two possibilities. DNA-PKmight either (i) promote inappropriate NHEJ joining of DNAdouble-strand ends at two adjacent collapsed forks, resulting inincreased cellular survival, but at the expense of genomic stability;or (ii) promote replication restart after binding at the reversed fork(chicken foot structure). To investigate potential effects of DNA-PKon replication restart, theDNAfiber assaywas used.When cellsare treated with NU7026, a significant increase in HU-inducedstalled forks is observed (Fig. 6A and B), implying that DNA-PKpromotes replication restart after HU-induced fork arrest. Fur-thermore, a small but significant increase in the number of neworigins firing after HU treatment in NU7026-treated cells wasobserved, inferring that blocking DNA-PK activity results inincreased numbers of complete fork collapse.

To test whether DNA-PK's kinase activity is important forreplication restart, DNA fiber analyses were also performed onV3 cells lacking DNA-PKcs, or expressing a kinase deadmutant ofDNA-PKcs K-R (K3752R), as well as the two phosphorylation

clusters mutants, PQR and ABCDE (Fig. 6C and D). Comparedwith cells expressing wild-type DNA-PKcs, both V3 cells expres-sing no DNA-PKcs, or catalytically inactive DNA-PKcs displayedelevated levels of replication fork stalling in the presence of HU.These data demonstrate a clear requirement for DNA-PK's cata-lytic activity in replication restart. However, increased fork stallingwas also observed in cells expressing the ABCDE and PQRmutants that both retain full catalytic activity. Thus, we concludethat although DNA-PK's catalytic activity is required for replica-tion restart, it is not sufficient.

Interestingly, cells expressing the catalytically inactive mutantdisplayed heightened levels of neworigin firing, both in untreatedcells and in response to HU. In addition, cells expressing theABCDE mutant had increased sensitivity in response to HU ascompared with cells lacking DNA-PK. These data demonstrate adominant negative function of the ABCDE-mutant DNA-PK. Wesuggest that this can be explained by DNA-PK being trapped ontoreplication fork intermediates andmayprevent resolution of forksfor instance via PARP-mediated pathways.

DiscussionHere, we demonstrate that both PARP and DNA-PK recruit

XRCC1 to sites of stalled replication forks upon HU treatment. Insupport of a novel function for XRCC1 at stalled replication forks,we demonstrate that XRCC1 is required for effective restart andrepair of stalled forks.

Because we only observed a small defect in the restart of stalledforks, and no absolute requirement for XRCC1 to mediate repair

Figure 5.DNA-PK is required for stabilization and recruitment of XRCC1 to stalled forks to promote restart. A, quantification of XRCC1 foci following 2mmol/L HU treatment for6 hours in V3-3 or AA8 cells. B,Western blot analysis of XRCC1 and GRB2 following indicated treatment in U2OS cells. C–E, representative images of replication tract(C), quantification of fork restart (D), and quantification of new origin firing after release from 2 hours HU treatment with or without DNA-PKcs inhibitor (E).






at stalled replication forks, we suggest that the function of XRCC1is to mediate repair of a subset of stalled forks. There is apossibility that small lesions block a subset of stalled replicationforks that otherwise impede restart of the forks. Indeed, when theDNA double helix is opened up during replication, the unpairedbases in single-stranded DNA (ssDNA) are more prone to bedamaged and are not easily repaired when the complementarystrand is not base-paired. Furthermore, ligases and topoisome-rases are heavily involved in DNA replication, and failed topo-isomerase or ligase reactions may trap proteins at sites of stalledreplication, whichmay require XRCC1-mediated repair alongsideTDP1 or aprataxin. Alternatively, XRCC1 may also have a moredirect role in replication restart and repair. In conclusions, ourdata support a model where the scaffolding protein XRCC1 isinvolved in repair of stalled replication forks occurring on unre-sected DNA ends (Fig. 7).

It is interesting that an increased amount of XRCC1 recruitmentto replication forks was observed when Mre11 exonuclease activ-ity is impaired, suggesting that XRCC1 is recruited to either smallgaps at replication forks or DNA double-strand ends and not toextended ssDNA regions generated by end resection. This notionis also strengthenedby thefinding that PARP1preferentially bindsto replication fork structures containing only small ssDNA gaps(18) and that the Ku70/80 subunits of DNA-PK bind to DNAdouble-strand ends (34).

The HU hypersensitivity of cells expressing DNA-PK that can-not phosphorylate the ABCDE sites is in agreement with ourprevious observations that cells expressing the ABCDEmutant are

more sensitive to IR, zeocin, and mitomycin C than cellscompletely lacking DNA-PKcs (15, 35), in agreement with adominant negative effect of DNA-PK being trapped to replicationintermediates. The HU hypersensitivity of V3 cells expressing theABCDE mutant are consistent with the hypothesis that thisphenotype is the result of an inability of cells with mutations atthis phosphorylation cluster to correctly respond to replicationstress.

Combining the data presented in this article, we propose amodel where DNA-PKcs and PARP1 associate with DNA endsexposed at unresected stalled forks (Fig. 7). Early studiessuggest that DNA-PK activation occurs in trans (i.e., by inter-action with the DNA molecule bound by a second, synapsedDNA–PK complex; ref. 36). However, more recent data suggestthat DNA dependent, DNA-PK activation can occur at a singleisolated, DNA–PK complex, in cis (37). Previously, we dem-onstrated that Mre11 is recruited to only a portion of stalledreplication forks through PARP activity (18). It is very clear thatMre11 is recruited also in the absence of PARP activity and thedependence of PARP may vary between different conditionsused to stall replication forks. PARP was also demonstrated toprotect stalled replication forks fromMre11-mediated resection(23). We suggest that Mre11 is recruited by PARP to stalledforks that are not easily resected and that may require XRCC1-mediated repair. Under conditions where all stalled replicationforks are easily resected, we envision that PARP is not requiredfor Mre11 recruitment, but play a distinctive role in stabilizingstalled replication forks.

Figure 6.DNA-PKcs kinase activity is requiredfor replication restart. A and C,representative images of fibersobtained with the indicatedtreatment. B and D, quantification ofstained fiber with the indicatedtreatment. Error bars, SEM from atleast three independent experiments.Values marked with asterisks aresignificantly different (Student t test,� , P < 0.05; �� , P < 0.01).

Ying et al.





In conclusion, here we report that PARP and DNA-PKcs arerelocating and DNA-PK rapidly phosphorylated at S2056 atunresected stalled replication forks. We demonstrate that PARPand DNA-PK are required for relocation of XRCC1 to stalledreplication forks, and we uncovered a novel role for XRCC1,proposed to be for removal of small lesions, to enable effectiverepair and restart of stalled replication forks.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: S. Ying, A.L. Medhurst, F.C. Hamdy, K. Meek,T. HelledayDevelopment of methodology: S. Ying, Z. Chen, F.C. Hamdy, T. HelledayAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Ying, Z. Chen, A.L. Medhurst, J.A. Neal, O. Mortu-sewicz, J. McGouranAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Ying, A.L. Medhurst, Z. Bao, O. Mortusewicz,J. McGouran, H. Shen, B.M. Kessler, T. HelledayWriting, review, and/or revision of the manuscript: S. Ying, A.L. Medhurst,Z. Bao, O. Mortusewicz, F.C. Hamdy, B.M. Kessler, K. Meek, T. Helleday

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Chen, J. McGouran, X. SongStudy supervision: T. Helleday

AcknowledgmentsThe authors thank Drs. Penny Jeggo and Susan Lees-Miller for discussions.

Grant SupportThis work was supported financially by the National Natural Science Foun-

dation of China (31370901, 81422031), National 1000 Talents Program forYoung Scholars, and Zhejiang Provincial Natural Science Foundation of China(LR14H160001), the Swedish Cancer Society, the Swedish Children's CancerFoundation, the Swedish Research Council, the Swedish Pain Relief Founda-tion, the Medical Research Council, and the Torsten and Ragnar S€oderbergFoundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received March 3, 2015; revised August 18, 2015; accepted September 12,2015; published OnlineFirst November 24, 2015.

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Ying et al.




2016;76:1078-1088. Published OnlineFirst November 24, 2015.Cancer Res Songmin Ying, Zhihui Chen, Annette L. Medhurst, et al. Forks Where They Recruit XRCC1 to Mediate RepairDNA-PKcs and PARP1 Bind to Unresected Stalled DNA Replication

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