ProliferatingCellNuclearAntigen(PCNA)-bindingProtein … · ificationofPCNAdetecteduponexposureofreplicatingcellsto DNA damage induced by UV light or hydroxyurea (HU) (13), whereas

Proliferating Cell Nuclear Antigen (PCNA)-binding ProteinC1orf124 Is a Regulator of Translesion Synthesis*□SReceived for publication, July 11, 2012, and in revised form, August 7, 2012 Published, JBC Papers in Press, August 17, 2012, DOI 10.1074/jbc.M112.400135

Gargi Ghosal, Justin Wai-Chung Leung, Binoj C. Nair, Ka-Wing Fong, and Junjie Chen1

From the Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center,Houston, Texas 77030

Background:Translesion synthesis involves proliferating cell nuclear antigen (PCNA)monoubiquitination and polymeraseswitching.Results: C1orf124 is required for cell survival following UV damage. It binds to monoubiquitinated PCNA and participates inpolymerase switching.Conclusion: C1orf124 serves as a central platform that facilitates translesion synthesis.Significance: This study provides a mechanism for translesion synthesis.

DNA damage-induced proliferating cell nuclear antigen(PCNA) ubiquitination serves as the key event mediating post-replication repair. Post-replication repair involves eithertranslesion synthesis (TLS) or damage avoidance via templateswitching. In this study, we have identified and characterizedC1orf124 as a regulator of TLS. C1orf124 co-localizes and inter-acts with unmodified and mono-ubiquitinated PCNA at UVlight-induced damage sites, which require the PIP box and UBZdomain of C1orf124. C1orf124 also binds to the AAA-ATPasevalosin-containing protein via its SHP domain, and cellularresistance to UV radiation mediated by C1orf124 requires itsinteractions with valosin-containing protein and PCNA. Inter-estingly, C1orf124 binds to replicativeDNApolymerase POLD3and PDIP1 under normal conditions but preferentially associ-ates with TLS polymerase � (POLH) upon UV damage. Deple-tion of C1orf124 compromises PCNA monoubiquitination,RAD18 chromatin association, and RAD18 localization to UVdamage sites. Thus, C1orf124 acts at multiple steps in TLS, sta-bilizes RAD18 and ubiquitinated PCNA at damage sites, andfacilitates the switch from replicative to TLS polymerase tobypass DNA lesion.

During DNA replication, replication forks may stall whenthey encounter secondary DNA structures, repetitivesequences, certain protein-DNA complexes, or lesions gener-ated by DNA-damaging agents. Especially in response to UVlight-induced DNA lesions, replicative DNA polymerases stallbecause they are unable to accommodate altered DNA bases intheir active sites. Although stalled replication forks are nor-mally stabilized following the activation ofDNAdamage check-points, they may also collapse and thus result in double-strandbreak formation, gross chromosomal rearrangements, and

genomic instability (1). DNA damage tolerance pathways, alsoknown as post-replication repair (PRR)2 pathways, function inpreventing replication fork collapse in response to DNA dam-age by allowing stalled replication forks to progress throughlesions (1–3). Earlier studies in both yeast andmammalian cellssuggest two major pathways for PRR: translesion synthesis(TLS) and damage avoidance by template switching. DuringTLS, the stalled replicative polymerase is replaced by TLS poly-merases, which are a class of specialized polymerases with lowprocessivity that can replicate over distortions in DNA anddirectly bypass lesions (4, 5). Depending on theTLS polymerasethat is recruited, UV light-induced cyclobutane pyrimidinedimers can be bypassed either in a relatively error-free mode(for example, when using DNA polymerase (pol) �) or by anerror-prone mechanism using pol � and Rev1 (4, 5). The mech-anism of lesion bypass by damage avoidance is unclear but isthought to involve template switching with the undamaged sis-ter chromatid and/or the use of homologous recombination (6,7). Thus, both of these direct (TLS) and indirect (templateswitching) bypass pathways allow for resumption of DNA rep-lication and leave lesions for repair at a later time point.A critical step in the regulation of PRR is the post-transla-

tional modification of proliferating cell nuclear antigen(PCNA), the replicative sliding clamp that plays an essentialrole in DNA replication. Following DNA damage and/or repli-cation stress, PCNA is either mono- or polyubiquitinated onLys-164 (3, 8–10). Studies suggest that monoubiquitination ofPCNA promotes direct lesion bypass by recruiting TLS poly-merases to stalled replication forks (5, 11, 12), whereas polyu-biquitination of PCNA promotes damage avoidance through aprocess that is still unclear (8). In yeast, ubiquitination of PCNAismediated by the Rad6 epistasis group and two RINGdomain-containing E3 ligases, Rad18 and Rad5. Rad18 mediates themonoubiquitination of PCNA, whereas Rad5 facilitates the fur-ther addition of Lys-63-linked polyubiquitin chains (5, 11, 12).In humans, monoubiquitination on Lys-164 is the major mod-

* This work was supported, in whole or in part, by National Institutes of HealthGrants CA089239, CA092312, and CA100109 (to J. C.).

□S This article contains supplemental Fig. 1.1 Recipient of Era of Hope Scholar Award W81XWH-05-1-0470 from the

United States Department of Defense and member of the MD AndersonCancer Center (supported by National Institutes of Health GrantCA016672). To whom correspondence should be addressed. Tel.: 713-792-4863; Fax: 713-745-6141; E-mail: jchen8@mdanderson.org.

2 The abbreviations used are: PRR, post-replication repair; pol, polymerase;TLS, translesion synthesis; PCNA, proliferating cell nuclear antigen; HU,hydroxyurea; Ub-PCNA, monoubiquitinated PCNA; TAP, tandem affinitypurification; MMC, mitomycin C; VCP, valosin-containing protein.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 41, pp. 34225–34233, October 5, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34225

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/cgi/content/full/M112.400135/DC1http://www.jbc.org/

ification of PCNAdetected upon exposure of replicating cells toDNA damage induced by UV light or hydroxyurea (HU) (13),whereas polyubiquitination of PCNA has recently beendetected at much lower levels (14). Monoubiquitination ofPCNA increases its affinity for TLS pol� and pol � andRev1 (11,13, 15, 16). The increased affinity of Y-family polymerases formonoubiquitinated PCNA (Ub-PCNA) is mediated by ubiqui-tin-binding domains that have been identified in all of theY-family polymerases (11, 17, 18) and therefore provide amechanism for polymerase switching, whereby the blockedreplicative DNA polymerase is replaced by a TLS polymerasethat can bypass the lesion (19). PCNA ubiquitination is the keyevent regulating PRR; however, it is insufficient, by itself, toaccount for the specificity of PRR pathway choice, as severalTLS polymerases have ubiquitin-interacting motifs (5). Fur-thermore, the precise molecular mechanism and regulatoryevents underlying the switch from replicative to translesionpolymerase in response to DNA damage is largely unknown.In this study, we have identified a previously uncharacterized

protein (C1orf124) as a regulator of TLS. C1orf124 is a multi-domain protein and contains an SprT-like domain at its N ter-minus, an SHP box and a PIP box in the middle region, and aUBZ (ubiquitin-binding zinc finger) domain at the C terminus.We show that C1orf124 localizes to sites of UV light-inducedDNAdamage in the cells and is required for cell survival follow-ing UV radiation. On the basis of the results presented below,we propose that C1orf124 is a key mediator protein involved inTLS, which plays an important role in the switch from replica-tive DNA polymerase to TLS polymerase for efficient lesionbypass upon UV damage.

EXPERIMENTAL PROCEDURES

Antibodies—Anti-C1orf124 antibodieswere raised by immu-nizing rabbits with GST-C1orf124 fusion proteins containingresidues 1–250 and 1–489 of human C1orf124 protein. Anti-sera were affinity-purified using an AminoLink Plus immobili-zation and purification kit (Pierce). Anti-�-actin and anti-FLAG antibodies were obtained from Sigma. Anti-RPA2antibody was from Abcam. Anti-PCNA antibody (PC10) wasobtained from Santa Cruz Biotechnology. Anti-RAD18 anti-body was obtained from Novus Biologicals. Anti-cyclobutanepyrimidine dimer antibody was from Cosmo Bios.Constructs—All cDNAs were subcloned into pDONR201

(Invitrogen) as entry clones and were subsequently transferredto gateway-compatible destination vectors for the expression ofN-terminally tagged fusion proteins. All deletionmutants weregenerated using the QuikChange site-directed mutagenesis kit(Stratagene) and verified by DNA sequencing.Cell Culture, Transfection, siRNAs, and shRNAs—HeLa and

293T cells were cultured in DMEM supplemented with 10%fetal bovine serum and 1% penicillin/streptomycin. Non-si-lencing control shRNA and shRNA target sets were pur-chased from Sigma. The C1orf124 targeting sequences are asfollows: 1, 5�-CTATGTCAAACGAGCTACTAACTCGAG-TTAGTAGCTCGTTTGACATAG-3�; and 2, 5�-GTACAA-CCACAGCTCAGAATTCTCGAGAATTCTGAGCTGTG-GTTGTAC-3�. The shRNA-resistant wild-type and mutantC1orf124 constructs were generated by changing nucleo-

tides in the shRNA1 targeting region (5�-GCAACTCTGGC-ACACTCGATCCTAGCAGCGATCGCTATGAGCATTA-3�). The shRNAs were packaged into lentiviruses bycotransfection with packaging plasmids pMD2G andpSPAX2 into 293T cells. 48 h later, the supernatant was col-lected for infection of HeLa cells. Infection was repeatedtwice with an interval of 24 h to achieve maximal infectionefficiency. Infected cells were selected with medium con-taining puromycin (2 �g/ml).Recombinant Proteins—GST proteins were expressed in

Escherichia coli BL21(DE3) cells and purified as follows. Cellswere pelleted and lysed in NETN buffer A (150mMNaCl, 1 mMEDTA, 20 mM Tris (pH 8.0) and 0.5% Nonidet P-40) supple-mented with 1 mM PMSF, 1 mM DTT, and 50 �g/ml lysozyme.Cells were sonicated and clarified by centrifugation at 12,000rpm for 20 min at 4 °C. After clarification, the supernatant wasincubated with glutathione-Sepharose beads (Sigma) for 2 h at4 °C.After threewasheswithNETNbufferA, beads coatedwiththe indicated proteins were used for pulldown experiments.GST Pulldown Assays and Immunoprecipitations—293T

cells were transfected with constructs encoding Myc-taggedPCNA and incubated for 24 h. Cells were lysed with high-saltbuffer (50 mM HEPES (pH 7.5), 300 mM NaCl, 1 mM EDTA,0.6% Triton X-100, 8% glycerol, 1 mM DTT, 1 mM PMSF, and 1mM NaF). The supernatant was clarified and then incubatedwith GST-C1orf124, GST-C1orf124�PIP, or GST protein pre-bound to glutathione-Sepharose beads for 1 h at 4 °C. Afterthree washes with HEPES/Triton buffer, the beads were resus-pended in 1� SDS sample buffer and analyzed byWestern blot-ting using anti-Myc antibody. For co-immunoprecipitationexperiments followingUVradiation, cellswere treatedwith 100J/m2 UV-C light and allowed to recover for 4 h. Cells were thencollected, lysed in 600 mM NaCl/HEPES/Triton buffer, dilutedto 150 mM NaCl, sonicated, and clarified by centrifugationbefore performing co-immunoprecipitation experiments.TandemAffinity Purification (TAP)—TAPwas performed as

described previously (20). Briefly, 293T cells were transfectedwith plasmids encoding SFB (S-protein, FLAG, and streptavi-din-binding peptide)-tagged constructs. Cell lines stablyexpressing tagged proteins were selected, and the expression ofexogenous proteins was confirmed by immunoblotting andimmunostaining. For affinity purification, a total of 20 10-cmdishes of 293T cells stably expressing SFB-tagged protein werecollected and lysed inNETNbuffer B (20mMTris-HCl (pH8.0),100mMNaCl, 1mM EDTA, and 0.5%Nonidet P-40) containing1 �g/ml each pepstatin A and aprotinin for 25 min. Crudelysates were cleared by centrifugation, and the supernatantswere incubated with 300 �l of streptavidin-Sepharose beads(AmershamBiosciences) for 2 h at 4 °C. The beadswerewashedthree times with NETN buffer B and then eluted with 2 mg/mlbiotin (Sigma) for 2 h at 4 °C. The eluates were incubated with100 �l of S-protein-agarose beads (Novagen) for 2 h at 4 °C andthen washed three times with NETN buffer B. The proteinsbound to beads were eluted by boiling with SDS sample buffer,resolved by SDS-PAGE, visualized by Coomassie Blue staining,and subjected to mass spectrometry analysis for protein identi-fication performed by the Taplin BiologicalMass SpectrometryFacility at Harvard University.

C1orf124 Is a Regulator of Translesion Synthesis

34226 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 41 • OCTOBER 5, 2012

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Immunoblotting—Cells were lysed with NETN buffer B onice for 30min. Cleared cell lysates were then collected, boiled in2� Laemmli buffer, and separated by SDS-PAGE. Membraneswere blocked in 5%milk in TBS/Tween buffer and then probedwith antibodies as indicated.Immunostaining—Cells cultured on coverslips were washed

with PBS, pre-extracted with 0.5% Triton solution for 2 min,and fixed with 3% paraformaldehyde for 10 min. Coverslipswere washed with PBS and then immunostained with primaryantibodies in 5%goat serum for 60min.Coverslipswerewashedand incubated with secondary antibodies conjugated with rho-damine or FITC for 60 min. Cells were then stained with DAPIto visualize nuclear DNA. The coverslips were mounted ontoglass slides with anti-fade solution and visualized using aNikonECLIPSE E800 fluorescence microscope with a Nikon PlanFluor 60� oil objective lens (numerical aperture, 1.30) at roomtemperature. Cells were photographed using a SPOT camera(Diagnostic Instruments, Inc.) and analyzed using Photoshopsoftware (Adobe). For micro-irradiation experiments, cellswere seeded on 35-mm glass bottom dishes (MatTek Corp.),incubated overnight, and then visualized with a NikonECLIPSE TE2000-U inverted microscope. Cells were micro-irradiated with a Micropoint ablation system (PhotonicsInstruments, St. Charles, IL) with the laser output set to 35%.An average of 20 cells were micro-irradiated and further cul-tured for 6 h prior to immunostaining. To irradiate cells withUV light, 5-�mNucleopore membrane filters (Millipore) wereused. Cells were treated with 10 J/m2 UV-C light and incubatedfor 4 h prior to immunostaining.Cell Survival Assays—1 � 103 cells were seeded onto 60-mm

dishes in triplicates. 24 h after seeding, cells were treated withUV light, HU, ionizing radiation, ormitomycinC (MMC) at theindicated concentrations. The mediumwas replaced 24 h later,and cells were then incubated for 14 days. Colonies formedwere fixed and stained with Coomassie Blue. The numbers ofcolonies were counted using a Gel Doc system with QuantityOne software (Bio-Rad).

RESULTS

C1orf124 Is a DNA Damage Protein That Functions inResponse to Replication Stress—SLX4 (also known as FANCP)is a Fanconi anemia protein that functions in the repair of inter-strand DNA cross-links generated by agents such as MMC,cisplatin, and platinum-based drugs (21–23). The precisemolecular function of SLX4 in MMC-induced DNA damagerepair is unclear. To obtain a better understanding of howSLX4is recruited to DNAdamage sites and themolecular function ofSLX4 in DNA cross-link repair, we performed TAP using celllysates prepared from 293T cells stably expressing tripleepitope (S-protein, FLAG, and streptavidin-binding peptide)-tagged SLX4 (SFB-SLX4). Mass spectrometry analysis revealedmany known SLX4-associated proteins and also a previouslyuncharacterized protein, C1orf124 (Fig. 1A). C1orf124 is pre-dicted to encode a protein of 489 residues with an SprT-likedomain (residues 45–231) at the N terminus, an SHP box (firstidentified in Shp1, the yeast ortholog of p47; residues 253–261)and a PCNA-interacting PIP box (residues 325–332) in the

middle, and a RAD18-like UBZ domain (residues 453–476) atthe C terminus (Fig. 1B).Because C1orf124 contains a PCNA-interacting PIP box

motif and a RAD18-like UBZ domain, we speculated thatC1orf124 could function in the DNAdamage response. Indeed,exogenously expressed C1orf124 localized to DNA damagesites generated by laser-induced micro-irradiation (Fig. 1C,upper panel) or following treatment of cells with HU (middlepanel) or UV radiation (lower panel). Moreover, endogenousC1orf124 co-localized with �H2AX (Fig. 1D, upper panel) andcyclobutane pyrimidine dimers (lower panel) in cells followingHU treatment or UV radiation, respectively, indicating thatC1orf124 functions in the DNA damage response. Cells withstable knockdown of C1orf124 (Fig. 1E) showedmarked hyper-sensitivity to UV light (Fig. 1F) andHU treatment (Fig. 1G), butwith only mild or no increased sensitivity to ionizing radiation(Fig. 1H) and MMC treatment (Fig. 1I). Together, these datasuggest that C1orf124 is a DNA damage protein involvedmainly in promoting cell survival following replication stress.C1orf124 Interacts and Co-localizes with PCNA at UV Light-

induced Damage Sites—To gain insight in C1orf124 functionsin the replication stress pathway, we performed TAP and iden-tified PCNA as the major C1orf124-associated protein (Fig.2A). C1orf124 has a characteristic PCNA-interacting motif(PIP box), which was first identified in the cyclin-dependentkinase inhibitor also called p21 or Cip1 (24). The PIP box(QNVLSNYF, residues 325–332) of C1orf124 (Fig. 2B) agreeswell with the consensus PIP box sequence, QXX(L/V/I/M)XX(F/Y)(F/Y), deduced from those identified in FEN1, p21,andXPG (24–26).We generated a deletionmutant of C1orf124(C1orf124�PIP) that lacks the entire PIP box. As shown inFig. 2C, GST-fused wild-type C1orf124, but not GST-C1orf124�PIP or GST alone, could bind to PCNA, confirmingthat C1orf124 interacts with PCNA via this conserved PIP boxmotif.C1orf124 also contains a RAD18-like ubiquitin-bindingUBZ

domain at its C terminus (Fig. 1B). The interaction betweenC1orf124 and PCNA did not change markedly following UVtreatment (Fig. 2D), and C1orf124 bound to both unmodifiedand monoubiquitinated PCNA (Fig. 2E). However, when theUBZ domain of C1orf124 was deleted, this mutant(C1orf124�UBZ) lost its ability to bind to Ub-PCNA (Fig. 2E).Its association with unmodified PCNA was also slightlyreduced (Fig. 2E), indicating that the UBZ domain of C1orf124helps the binding of C1orf124 to Ub-PCNA. Indeed, althoughfull-length C1orf124 and the C1orf124�SHP mutant co-local-ized with replication protein A at laser-induced DNA damagesites (Fig. 2F) and with PCNA at damage foci following UVtreatment (Fig. 2G), this damage-induced localization ofC1orf124 was abolished in C1orf124�PIP and C1orf124�UBZmutants, which lack the PIP box and UBZ domain, respectively(Fig. 2, F and G). These data suggest that C1orf124 localizes toDNA damage sites via its association with Ub-PCNA.C1orf124 Participates in the PRRPathway—Because both the

PIP box motif and the UBZ domain of C1orf124 are importantfor its interaction with PCNA and its localization to DNA dam-age sites, we suspected that C1orf124 might function down-stream of PCNA ubiquitination. However, we found that

C1orf124 Is a Regulator of Translesion Synthesis

OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34227

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

C1orf124 knockdown cells also showed reduced UV light-in-duced PCNA monoubiquitination (Fig. 3A). This reduction inPCNA ubiquitination was rescued by reconstitution ofC1orf124 knockdown cells with full-length C1orf124, but notwith the C1orf124�PIP or C1orf124�UBZ mutant (Fig. 3A).These results indicate that although binding to PCNA isrequired for the recruitment of C1orf124 following UV radia-tion, C1orf124 is also required formaintaining the level of ubiq-uitinated PCNA at DNA damage sites.

The E3 ubiquitin ligase RAD18 is exclusively required forPCNA monoubiquitination, which is believed to be critical forthe switch from normal replicative DNA polymerase to Y-fam-ily polymerase following DNA damage and therefore allowslesion bypass (9, 10, 13). Consistent with a key role of RAD18 inTLS, RAD18 depletion causes hypersensitivity to DNA-damag-ing agents (27–29). The reduction in PCNA ubiquitinationobserved in C1orf124 knockdown cells suggests that C1orf124may also regulate RAD18 function. RAD18 is a putative

FIGURE 1. C1orf124 is involved in the cellular response to DNA damage. A, TAP was performed using 293T cells stably expressing SFB-tagged SLX4. Theresults from mass spectrometry analysis are shown. B, schematic representation of C1orf124 protein. C, HeLa cells were transfected with plasmid encodingSFB-C1orf124. Cells were treated with laser micro-irradiation (upper panel), HU (middle panel), or UV radiation (lower panel) and analyzed by immunostainingwith the indicated antibodies. RPA, replication protein A; CPD, cyclobutane pyrimidine dimer. D, HeLa cells were infected with control (shControl) and C1orf124(shC1orf124) shRNA lentiviral particles. Immunostaining experiments were performed using the indicated antibodies 6 h after treatment with 10 mM HU (upperpanel) or 10 J/m2 UV-C light (lower panel). E, knockdown efficiency of C1orf124-specific shRNAs was confirmed by immunoblotting using lysates prepared fromHeLa cells expressing the indicated shRNAs. WB, Western blot. F–I, survival curves for the indicated cell lines in response to increasing doses of UV light (F), HU(G), ionizing radiation (IR; H), or MMC (I). Cell survival assays were performed as described “Experimental Procedures.” Data are presented as means � S.D. fromthree different experiments.

C1orf124 Is a Regulator of Translesion Synthesis

34228 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 41 • OCTOBER 5, 2012

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

C1orf124-associated protein identified by our mass spectrom-etry analysis (Fig. 2A). We confirmed this association by co-immunoprecipitation experiments (Fig. 3B). Interestingly, wefound that the localization of RAD18 toUV light-induced dam-age foci was diminished in C1orf124 knockdown cells (Fig. 3C).Moreover, the chromatin association of RAD18 following UVradiation was also reduced in C1orf124 knockdown cells (Fig.3D). Cells with co-depletion of RAD18 and C1orf124 exhibitedsimilar UV sensitivity as RAD18�/� cells or C1orf124 knock-down cells (Fig. 3E), indicating that C1orf124 and RAD18 func-tion in the same PRR pathway.C1orf124 Regulates TLS—DNA damage inhibits replication

fork progression by blocking replicative DNA polymerases. Toovercome this blockade, cells recruit specialized TLS poly-merases, which can insert nucleotides opposite the damagedbases. In particular, TLS by DNA pol � (also called POLH/

RAD30/XPV) is the major pathway for bypassing UV photo-products (30). Recruitment of pol� and other TLS polymerasesto stalled replication forks is mediated by monoubiquitinationof PCNA (13). Y-family polymerases possess UBZ motifs, andthe direct binding of TLS polymerases to Ub-PCNA facilitatestheir recruitment to stalled replication forks (11). However,there are also othermechanisms that contribute to TLS polym-erase recruitment. For example, RAD18 has been shown toassociate directlywith pol� and to guide the polymerase to sitesof DNA damage (16).Whenwe compared the results of TAP of C1orf124-contain-

ing protein complexes obtained from untreated cells (Fig. 2A)with those from UV light-irradiated cells (Fig. 4A), we foundthat we obtained more peptides derived from POLD3 (polym-erase delta 3/p66) and PDIP1 (POLD3-interacting protein1/POLDIP1/KCTD13) in the untreated sample than in the UV

FIGURE 2. C1orf124 interacts and co-localizes with PCNA at UV light-induced damage sites. A, TAP was performed using 293T cells stably expressingSFB-tagged C1orf124. The results from mass spectrometry analysis are shown. B, sequence alignment of the PIP box motif of C1orf124 with conserved PIP boxmotifs of other PCNA-interacting proteins, namely FEN1, p21, and XPG. The consensus PIP box sequence (Qxx�xx��, where � � L/V/I/M and � � Y/F) isindicated. C, C1orf124 interacts with PCNA via the PIP box motif. 293T cells were transfected with plasmid encoding Myc-tagged PCNA. Cell lysates wereincubated with GST, GST-C1orf124�PIP, or GST-C1orf124, and immunoblotting was performed using the indicated antibodies. WB, Western blot. D, PCNAinteracts with C1orf124 in the absence and presence of UV damage. 293T cells were cotransfected with plasmids encoding SFB-tagged PCNA and Myc-taggedC1orf124. 24 h later, cells were left untreated or treated with 100 J/m2 UV-C light and collected 4 h later. Coprecipitation was carried out using S-protein beads,and immunoblotting was performed using the indicated antibodies. E, the UBZ domain of C1orf124 is required to bind to ubiquitinated PCNA. 293T cells weretransfected with plasmids encoding SFB-tagged C1orf124 and C1orf124�UBZ. 24 h later, cells were treated with 100 J/m2 UV-C light and collected 4 h later.Coprecipitation was carried out using S-protein beads, and immunoblotting was performed using the indicated antibodies. F and G, the PIP box motif and UBZdomain mediate the recruitment of C1orf124 to DNA damage sites. HeLa cells were transfected with constructs encoding SFB-tagged full-length (FL) C1orf124,C1orf124�SHP, C1orf124�PIP, or C1orf124�UBZ. Cells were treated with laser micro-irradiation (F) or 10 J/m2 UV-C light (G) and incubated for 6 h prior toimmunostaining with the indicated antibodies. RPA, replication protein A.

C1orf124 Is a Regulator of Translesion Synthesis

OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34229

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

light-irradiated sample. Interestingly, POLH (pol �) was iden-tified only as a C1orf124-associated protein in the UV light-treated sample (Figs. 2A and 4A), suggesting that C1orf124mayassociate with different polymerases before and after DNAdamage. Co-immunoprecipitation experiments showed thatthe binding of C1orf124 to POLD3 or PDIP1 was dramaticallyreduced followingUV irradiation (Fig. 4,B andC). On the otherhand, the association of C1orf124 with translesion polymerasePOLH was markedly increased in cells treated with UV radia-tion (Fig. 4D). These results suggest that C1orf124may directlyparticipate in the switching of replicative polymerase to transle-sion polymerase following UV damage and therefore mediatelesion bypass.C1orf124Coordinates with Valosin-containing Protein (VCP)

inMediating Cellular Resistance toUVDamage—BesidesDNAreplication and repair proteins, we also identified VCP andVCP-associated proteins (VCPIP and UBXD10) as C1orf124-

binding proteins (Figs. 2A and 4A). VCP (also known as p97 andCdc48) belongs to the hexameric AAA-ATPase family andfunctions in diverse cellular activities that include ubiquitin-de-pendent endoplasmic reticulum-associated protein degrada-tion protein quality control (31, 32), autophagy (32), endosomalsorting (33), and protein degradation at the outer mitochon-drial membrane (34). Recent studies imply that VCP acts byextracting protein complexes bound to chromatin rather thanpromoting protein degradation. For example, Aurora B hasbeen shown to be extracted from mitotic chromosomes byVCP/p97 and its cofactors (35). Furthermore, VCP/p97 regu-lates DNA replication by mediating the removal of replicationlicensing factor Cdt1 that is bound to PCNA (36). VCP/p97 hasalso been shown to function in repair of ionizing radiation-induced double-strand breaks. VCP/p97 is recruited to ionizingradiation-induced damage sites in an RNF8-dependent man-ner, where it catalyzes the removal of the Lys-48-conjugated

FIGURE 3. C1orf124 participates in the PRR pathway. A, C1orf124 is required for UV light-induced PCNA ubiquitination. HeLa cells with stable knockdown ofC1orf124 were transfected with a control vector or reconstituted with shRNA-resistant (shR) constructs encoding full-length (FL) C1orf124, C1orf124�PIP, orC1orf124�UBZ. Cells were treated with 100 J/m2 UV light and collected 4 h later. The levels of unmodified PCNA and Ub-PCNA were analyzed by immuno-blotting with anti-PCNA antibody. WB, Western blot. B, C1orf124 interacts with RAD18. 293T cells were transfected with plasmids encoding SFB-tagged RAD6,RAD18, and PCNA together with plasmids encoding Myc-tagged C1orf124. Coprecipitation was carried out using S-protein beads, and immunoblotting wasperformed using the indicated antibodies. C, C1orf124 regulates the chromatin association of RAD18. HeLa cells with stable expression of control (shControl)and C1orf124 (shC1orf124) shRNAs were left untreated or treated with 10 J/m2 UV-C light and incubated for 6 h prior to immunostaining with the indicatedantibodies. CPD, cyclobutane pyrimidine dimer. D, C1orf124 regulates the chromatin association of RAD18. HeLa cells with stable expression of control andC1orf124 shRNAs were left untreated or treated with 100 J/m2 UV-C light and incubated for 4 h. Soluble and chromatin-bound RAD18 levels were analyzed byimmunoblotting with anti-RAD18 antibody. E, C1orf124 and RAD18 function in the same PRR pathway. C1orf124-depleted cells, RAD18�/� cells, and cellsdepleted of both RAD18 and C1orf124 were treated with increasing doses of UV light. Survival curves are shown for the indicated cell lines. Data are presentedas means � S.D. from three different experiments.

C1orf124 Is a Regulator of Translesion Synthesis

34230 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 41 • OCTOBER 5, 2012

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

protein substrates to allow for proper assembly of downstreamsignaling effectors, including RAD51, BRCA1, and 53BP1 (37).In addition, VCP/p97 also mediates the removal of the RNApolymerase II complex when it is stalled at UV light-inducedDNA lesions (38). These studies indicate that VCP plays a rolein the DNA damage response. Thus, it is possible that the asso-ciation between C1orf124 and VCP may be important forC1orf124 function following UV irradiation.We found that C1orf124 binds to VCP via the SHP box

(Fig. 5A). Deletion of the PIP box or UBZ domain ofC1orf124 did not influence its interaction with VCP(Fig. 5A). To assess the function of C1orf124-VCP interac-tion following UV irradiation, we generated constructs en-coding shRNA-resistant SFB-tagged wild-type C1orf124,C1orf124�PIP, C1orf124�SHP, or C1orf124�UBZ. Only theexpression of shRNA-resistant wild-type C1orf124, but notany of these C1orf124 deletion mutants, could rescue the UVhypersensitivity in C1orf124-depleted cells (Fig. 5B). Thesedata indicate that the binding of C1orf124 to VCP and toubiquitinated PCNA is required for its in vivo functionmedi-ating cell survival following UV damage.

DISCUSSION

In this study, we have identified and characterized thePCNA-binding protein C1orf124. Cells depleted of C1orf124showed marked increases in cellular sensitivity to UV andHU treatment (Fig. 1, F and G), suggesting a function ofC1orf124 in the DNA damage response. C1orf124 interactedwith unmodified and ubiquitinated PCNA (Fig. 2, C–E) andlocalized to UV light-induced DNA damage sites. Depletionof C1orf124 resulted in a marked decrease in PCNA monou-

biquitination, which was accompanied by a reduction inRAD18 chromatin association and RAD18 localization toDNA damage sites (Fig. 3, A–C). Theses results suggest thatC1orf124 is required to stabilize RAD18 and Ub-PCNA atthe sites of DNA damage.Our findings agree with the observations in a recent study

characterizing the function of C1orf124 in the UV light-in-duced damage response (39). However, the precise mecha-nism by which C1orf124 confers cellular resistance to UVdamage is still unclear. In this study, we have shown thatC1orf124 binds VCP via the SHP box and that the binding ofC1orf124 to VCP/p97 is crucial for cellular resistance to UVdamage (Fig. 5, A and B). Studies have shown that VCP/p97functions in regulating DNA metabolic processes by medi-ating proteasomal degradation or catalyzing the extractionof proteins or protein complexes from the chromatin (35,38). On the basis of this information and the data presentedin our study, we propose that C1orf124 may function to sta-bilize Ub-PCNA and RAD18 on DNA damage sites by pre-venting their removal or extraction from the chromatin byVCP/p97 during TLS (Fig. 5C). C1orf124 may carry out itsregulatory function by sequestering the substrates (RAD18and PCNA) away from the enzymatic action of VCP/p97either by directly inhibiting VCP activity or by physicallydisrupting VCP-substrate interaction via steric hindrance.Thus, in C1orf124-depleted cells, VCP can gain access to itssubstrates and catalyzes the removal of RAD18 andUb-PCNA from the chromatin (Fig. 5C, right panel) and thusresults in the reduction of RAD18 and Ub-PCNA at damagesites. Our hypothesis is supported by the observation that

FIGURE 4. C1orf124 displays preferential binding to replicative and translesion DNA polymerases before and after UV damage, respectively. A, TAPwas performed using 293T cells stably expressing SFB-tagged C1orf124 following UV treatment. The results from mass spectrometry analysis are shown. 293Tcells were transfected with plasmids encoding SFB-tagged POLD3 (B), SFB-tagged PDIP1 (C), and SFB-tagged POLH (D) together with plasmid encodingMyc-C1orf124. Cells were left untreated or treated with 100 J/m2 UV-C light and collected 4 h later. Coprecipitation was carried out using S-protein beads, andimmunoblotting was performed using the indicated antibodies. WB, Western blot.

C1orf124 Is a Regulator of Translesion Synthesis

OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34231

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

overexpression of a catalytically inactive dominant-negativemutant of VCP (E305Q/E578Q) restored RAD18 chromatinassociation in C1orf124 stable knockdown cells (supplemen-tal Fig. 1, A and B). Furthermore, the reduction of PCNAubiquitination observed in C1orf124 knockdown cells wasalso rescued by the expression of this catalytically inactivemutant of VCP (supplemental Fig. 1C).Interestingly, we also found that C1orf124 preferentially

bound to replicative polymerase POLD3 or TLS polymerasePOLH before or after DNA damage (Fig. 4, B–D). Thus, wepropose that C1orf124 may directly regulate the switch fromreplicative polymerase to translesion polymerase followingDNAdamage (Fig. 5C). It is likely that this function of C1orf124may involve its regulated associations with these polymerasesand potentially also the function of VCP/p97. Further studiesare needed to elucidate precisely how this switch occurs at themolecular level.

Acknowledgments—We thank all members of the Chen laboratory foradvice and technical assistance. We thank Henry Adams and theGenetics Department Microscopy CORE facility at The University ofTexas MD Anderson Cancer Center.

REFERENCES1. Branzei, D., and Foiani, M. (2010) Maintaining genome stability at the

replication fork. Nat. Rev. Mol. Cell Biol. 11, 208–2192. Chang, D. J., and Cimprich, K. A. (2009) DNA damage tolerance: when it’s

OK to make mistakes. Nat. Chem. Biol. 5, 82–903. Lee, K. Y., and Myung, K. (2008) PCNA modifications for regulation of

post-replication repair pathways.Mol. Cells 26, 5–114. Prakash, S., Johnson, R. E., and Prakash, L. (2005) Eukaryotic translesion

synthesis DNA polymerases: specificity of structure and function. Annu.Rev. Biochem. 74, 317–353

5. Waters, L. S., Minesinger, B. K., Wiltrout, M. E., D’Souza, S., Woodruff,R. V., and Walker, G. C. (2009) Eukaryotic translesion polymerases andtheir roles and regulation in DNA damage tolerance.Microbiol. Mol. Biol.

FIGURE 5. C1orf124 acts with VCP in promoting cell survival following UV damage. A, C1orf124 interacts with VCP via the SHP box. 293T cells weretransfected with plasmids encoding Myc-tagged full-length C1orf124, C1orf124�SHP, C1orf124�PIP, and C1orf124�UBZ together with plasmid encodingSFB-tagged VCP. Coprecipitation was carried out using S-protein beads, and immunoblotting was performed using the indicated antibodies. WB, Western blot.B, interaction of C1orf124 with VCP and PCNA is crucial for the in vivo function of C1orf124. C1orf124-depleted (C1orf124 shRNA1) HeLa cells stably expressingshRNA-resistant (shR) wild-type C1orf124, C1orf124�SHP, C1orf124�PIP, or C1orf124�UBZ were generated. Cells were treated with the indicated doses of UVlight and incubated for 14 days. Survival curves are shown for the indicated cell lines. Data are presented as means � S.D. from three different experiments. C,model depicting the molecular function of C1orf124 in regulating TLS as described under “Discussion.” RPA, replication protein A.

C1orf124 Is a Regulator of Translesion Synthesis

34232 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 41 • OCTOBER 5, 2012

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/cgi/content/full/M112.400135/DC1http://www.jbc.org/cgi/content/full/M112.400135/DC1http://www.jbc.org/cgi/content/full/M112.400135/DC1http://www.jbc.org/

Rev. 73, 134–1546. Broomfield, S., Hryciw, T., and Xiao, W. (2001) DNA post-replication

repair and mutagenesis in Saccharomyces cerevisiae. Mutat. Res. 486,167–184

7. Unk, I., Hajdú, I., Blastyák, A., and Haracska, L. (2010) Role of yeast Rad5and its human orthologs, HLTF and SHPRH, in DNA damage tolerance.DNA Repair 9, 257–267

8. Ulrich, H. D. (2009) DNA Repair (Amst) 8, 461–4699. Hoege, C., Pfander, B., Moldovan, G. L., Pyrowolakis, G., and Jentsch, S.

(2002) RAD6-dependent DNA repair is linked to modification of PCNAby ubiquitin and SUMO. Nature 419, 135–141

10. Stelter, P., and Ulrich, H. D. (2003) Control of spontaneous and damage-induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425,188–191

11. Bienko, M., Green, C. M., Crosetto, N., Rudolf, F., Zapart, G., Coull, B.,Kannouche, P., Wider, G., Peter, M., Lehmann, A. R., Hofmann, K., andDikic, I. (2005) Ubiquitin-binding domains in Y-family polymerases reg-ulate translesion synthesis. Science 310, 1821–1824

12. Kannouche, P. L., and Lehmann, A. R. (2004) Ubiquitination of PCNA andthe polymerase switch in human cells. Cell Cycle 3, 1011–1013

13. Kannouche, P. L., Wing, J., and Lehmann, A. R. (2004) Interaction ofhuman DNA polymerase � with monoubiquitinated PCNA: a possiblemechanism for the polymerase switch in response to DNA damage.Mol.Cell 14, 491–500

14. Motegi, A., Liaw, H. J., Lee, K. Y., Roest, H. P., Maas, A., Wu, X., Moinova,H., Markowitz, S. D., Ding, H., Hoeijmakers, J. H., and Myung, K. (2008)Polyubiquitination of proliferating cell nuclear antigen by HLTF andSHPRH prevents genomic instability from stalled replication forks. Proc.Natl. Acad. Sci. U.S.A. 105, 12411–12416

15. Guo, C., Sonoda, E., Tang, T. S., Parker, J. L., Bielen, A. B., Takeda, S.,Ulrich, H. D., and Friedberg, E. C. (2006) REV1 protein interacts withPCNA: significance of the REV1 BRCT domain in vitro and in vivo. Mol.Cell 23, 265–271

16. Watanabe, K., Tateishi, S., Kawasuji, M., Tsurimoto, T., Inoue, H., andYamaizumi, M. (2004) Rad18 guides pol � to replication stalling sitesthrough physical interaction and PCNA monoubiquitination. EMBO J.23, 3886–3896

17. Plosky, B. S., and Woodgate, R. (2004) Switching from high-fidelity repli-cases to low-fidelity lesion-bypass polymerases. Curr. Opin. Genet. Dev.14, 113–119

18. Guo, C., Tang, T. S., Bienko, M., Parker, J. L., Bielen, A. B., Sonoda, E.,Takeda, S., Ulrich, H. D., Dikic, I., and Friedberg, E. C. (2006) Ubiquitin-binding motifs in REV1 protein are required for its role in the tolerance ofDNA damage.Mol. Cell. Biol. 26, 8892–8900

19. Friedberg, E. C., Lehmann, A. R., and Fuchs, R. P. (2005) Trading places:how doDNApolymerases switch during translesionDNA synthesis?Mol.Cell 18, 499–505

20. Yuan, J., Ghosal, G., and Chen, J. (2009) The annealing helicase HARPprotects stalled replication forks. Genes Dev. 23, 2394–2399

21. Gari, K., and Constantinou, A. (2009) The role of the Fanconi anemianetwork in the response to DNA replication stress. Crit. Rev. Biochem.Mol. Biol. 44, 292–325

22. Svendsen, J. M., Smogorzewska, A., Sowa, M. E., O’Connell, B. C., Gygi,S. P., Elledge, S. J., and Harper, J. W. (2009) Mammalian BTBD12/SLX4assembles a Holliday junction resolvase and is required for DNA repair.Cell 138, 63–77

23. Kim, Y., Lach, F. P., Desetty, R., Hanenberg, H., Auerbach, A. D., andSmogorzewska, A. (2011) Mutations of the SLX4 gene in Fanconi anemia.Nat. Genet. 43, 142–146

24. Gulbis, J.M., Kelman, Z., Hurwitz, J., O’Donnell,M., andKuriyan, J. (1996)Structure of the C-terminal region of p21WAF1/CIP1 complexed with hu-man PCNA. Cell 87, 297–306

25. Querol-Audí, J., Yan, C., Xu, X., Tsutakawa, S. E., Tsai, M. S., Tainer, J. A.,Cooper, P. K., Nogales, E., and Ivanov, I. (2012) Repair complexes of FEN1endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from theirPCNA counterparts by functionally important stability. Proc. Natl. Acad.Sci. U.S.A. 109, 8528–8533

26. Maga, G., and Hubscher, U. (2003) Proliferating cell nuclear antigen(PCNA): a dancer with many partners. J. Cell Sci. 116, 3051–3060

27. Nakajima, S., Lan, L., Kanno, S., Usami, N., Kobayashi, K., Mori, M.,Shiomi, T., and Yasui, A. (2006) Replication-dependent and -independentresponses of RAD18 to DNA damage in human cells. J. Biol. Chem. 281,34687–34695

28. Yamashita, Y.M., Okada, T.,Matsusaka, T., Sonoda, E., Zhao, G. Y., Araki,K., Tateishi, S., Yamaizumi, M., and Takeda, S. (2002) RAD18 and RAD54cooperatively contribute tomaintenance of genomic stability in vertebratecells. EMBO J. 21, 5558–5566

29. Tateishi, S., Niwa,H.,Miyazaki, J., Fujimoto, S., Inoue,H., andYamaizumi,M. (2003) Enhanced genomic instability and defective post-replicationrepair in RAD18 knockout mouse embryonic stem cells. Mol. Cell. Biol.23, 474–481

30. Day, T. A., Palle, K., Barkley, L. R., Kakusho, N., Zou, Y., Tateishi, S.,Verreault, A., Masai, H., and Vaziri, C. (2010) Phosphorylated Rad18 di-rects DNA polymerase � to sites of stalled replication. J. Cell Biol. 191,953–966

31. Meusser, B., Hirsch, C., Jarosch, E., and Sommer, T. (2005) ERAD: the longroad to destruction. Nat. Cell Biol. 7, 766–772

32. Tresse, E., Salomons, F. A., Vesa, J., Bott, L. C., Kimonis, V., Yao, T. P.,Dantuma, N. P., and Taylor, J. P. (2010) VCP/p97 is essential for matura-tion of ubiquitin-containing autophagosomes, and this function is im-paired by mutations that cause IBMPFD. Autophagy 6, 217–227

33. Ramanathan, H. N., and Ye, Y. (2012) The p97 ATPase associates withEEA1 to regulate the size of early endosomes. Cell Res. 22, 346–359

34. Meyer, H., Bug, M., and Bremer, S. (2012) Emerging functions of theVCP/p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 14,117–123

35. Dobrynin, G., Popp, O., Romer, T., Bremer, S., Schmitz, M. H., Gerlich,D.W., andMeyer, H. (2011) Cdc48/p97-Ufd1-Npl4 antagonizes Aurora Bduring chromosome segregation in HeLa cells. J. Cell Sci. 124, 1571–1580

36. Havens, C. G., andWalter, J. C. (2011) Mechanism of CRL4Cdt2, a PCNA-dependent E3 ubiquitin ligase. Genes Dev. 25, 1568–1582

37. Meerang, M., Ritz, D., Paliwal, S., Garajova, Z., Bosshard, M., Mailand, N.,Janscak, P., Hübscher, U., Meyer, H., and Ramadan, K. (2011) The ubiq-uitin-selective segregaseVCP/p97 orchestrates the response toDNAdou-ble-strand breaks. Nat. Cell Biol. 13, 1376–1382

38. Verma, R., Oania, R., Fang, R., Smith, G. T., and Deshaies, R. J. (2011)Cdc48/p97mediates UV-dependent turnover of RNA Pol II.Mol. Cell 41,82–92

39. Centore, R. C., Yazinski, S. A., Tse, A., and Zou, L. (2012) Spartan/C1orf124, a reader of PCNA ubiquitylation and a regulator of UV light-induced DNA damage response.Mol. Cell 46, 625–635

C1orf124 Is a Regulator of Translesion Synthesis

OCTOBER 5, 2012 • VOLUME 287 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34233

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Gargi Ghosal, Justin Wai-Chung Leung, Binoj C. Nair, Ka-Wing Fong and Junjie ChenRegulator of Translesion Synthesis

Proliferating Cell Nuclear Antigen (PCNA)-binding Protein C1orf124 Is a

doi: 10.1074/jbc.M112.400135 originally published online August 17, 20122012, 287:34225-34233.J. Biol. Chem.

10.1074/jbc.M112.400135Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2012/08/17/M112.400135.DC1

http://www.jbc.org/content/287/41/34225.full.html#ref-list-1

This article cites 39 references, 14 of which can be accessed free at

by guest on Decem

ber 12, 2020http://w

ww

.jbc.org/D

ownloaded from