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d n a r e p a i r 7 ( 2 0 0 8 ) 751–761

avai lab le at www.sc iencedi rec t .com

journa l homepage: www.e lsev ier .com/ locate /dnarepai r

irect involvement of the tumor suppressor p53 inucleotide excision repair

u-Ching Changa, Kun-Yan Janb, Chun-An Chenga, Chu-Bin Liaoc, Yin-Chang Liua,c,∗

Institute of Molecular Medicine, National Tsing-Hua University, Hsin-Chu 30043, TaiwanInstitute of Cellular and Organismic Biology, Academia Sinica, Taipei 11529, TaiwanDepartment of Life Science, National Tsing-Hua University, Hsin-Chu 30043, Taiwan

r t i c l e i n f o

rticle history:

eceived 18 September 2007

eceived in revised form

7 January 2008

ccepted 28 January 2008

ublished on line 14 March 2008

eywords:

53

VC

ucleotide excision repair

a b s t r a c t

The tumor suppressor p53 enhances repair of UVC-induced DNA damage. The comet-NE

assay, a conventional alkaline comet assay which includes a nuclear digestion step, was

used to examine the effects of p53 on the excision activity of nuclear extracts (NEs). In

contrast with untreated NEs, NEs immunodepleted of p53 or NEs of p53-null cells were

unable to excise UVC-induced DNA adducts. Introduction of p53 by transfection restored

the excision activity to NEs of p53-null cells. Deletion of the N-terminal 99 amino acids

and/or the C-terminal 85 amino acids of p53 barely affected the excision activity, whereas

further deletion of the C-terminus of p53 by another 10 amino acids completely abolished

the excision activity of NEs. Immunostaining following localized UV irradiation was used

to examine the effects of p53 on the recruitment of repair proteins for nucleotide excision

repair (NER). Although recruitment of XPC occurred regardless of the presence of p53, the

omet-NE assay recruitment of XPB was p53-dependent. However, p53 with the 95 amino acid deletion at

its C-terminus was unable to support this recruitment of XPB. Consistently, intact p53 (but

not the C-terminal 95 residue truncated version) was detected in co-immunoprecipitation

assays with an anti-XPB antibody. These results support the hypothesis that p53 facilitates

NER through direct involvement by protein–protein interactions.

(XPG and ERCC1-XPF). The oligonucleotide excision is followed

. Introduction

n responding to DNA damage incurred by UV radiation orany organic compounds, cells may trigger the nucleotide

xcision repair (NER) mechanism to repair the lesions [1,2].he NER mechanism is conserved in all organisms, and isital to their survival. Defects in NER usually have deleteriousffects such as in Xeroderma Pigmentosum (XP), Trichoth-

odystrophy and Cockayne syndrome in humans [3]. The NER

echanism involves several biochemical steps which includeamage recognition, damage site unwinding, dual incision

∗ Corresponding author at: Institute of Molecular Medicine, National Tsax: +886 3 571 5934.

E-mail address: ycliu@life.nthu.edu.tw (Y.-C. Liu).568-7864/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2008.01.019

© 2008 Elsevier B.V. All rights reserved.

and gap-filling DNA synthesis [2–4]. The damage recognitionstep may involve RNA polymerase II or XPC-hHR23B if thedamage is or is not in the transcribing region, respectively.Once the damage has been identified and verified, the dam-age site, including its 3′- and 5′-end adjacent nucleotides, isunwound by helicases (XPB and XPD of the TFIIH complex)before the damaged DNA is excised by two endonucleases

ing-Hua University, Hsin-Chu 30043, Taiwan.

by repair synthesis which is mediated by DNA polymerases �

or �, PCNA, RPA and replication factor C. The remaining gap isthen sealed by ligase I.

7 ( 2

752 d n a r e p a i r

The tumor suppressor p53 is known as the guardian ofgenome integrity and mutations of p53 are often detected inhuman cancers [5,6]. Disruption of p53 function may com-promise the cellular capacity to repair the UV-induced DNAdamage [7,8]. This effect may be via the transactivation of p53,which may induce the expression of genes whose encodedproteins are involved in NER, e.g. p48 and XPC [9–11]. How-ever, the direct involvement of p53 in NER has been suspectedsince p53 is able to bind repair proteins such as XPB andXPD [12,13]. A previous study [12] found that recombinant p53failed to enhance DNA repair synthesis in vitro, in which aUV-irradiated plasmid was used as a substrate for the repairproteins in whole cell extracts.

In this study, we re-examined the possibility of the directinvolvement of p53 in NER, using the newly developed comet-NE assay [14], a modified version of the conventional alkalinecomet assay. This technique includes a nuclear extract (NE)digestion step following the preparation of nucleoids inagarose gels. DNA adducts, if any, in the nucleoids would berecognized and excised by repair proteins in the NE. Gapsdue to the excision of oligonucleotides would not be closed inthese experimental conditions and would be detected by thecomet assay. We have used the comet-NE assay in our recentstudies [15,16]. Our present study indicates that p53 is vitalto the excision activity of NEs. NEs immunodepleted of p53or NEs of p53-null cells are unable to excise the UV-inducedDNA adducts. Deletion of the N-terminal 99 residues or the C-terminal 85 residues of p53 did not affect the excision activityof the NEs. Immunostaining following localized UV irradia-tion indicates that the recruitment of XPB but not of XPC isp53-dependent.

2. Materials and methods

2.1. Cell cultures and p53 expression plasmids

NB4 (human acute promyelocytic leukemia) and H1299(human lung adenocarcinoma) cells were maintained in 1×RPMI -1640 medium (Gibico, Grand Island, NY, USA). TTD10VI(skin biopsies of patients with homozygous of XPD genedefect) cells were maintained in 1× Dulbecco’s Modified EagleMedium (Gibico, Grand Island), and XPC cells (human skinfibroblast cells and XPC gene lost 2 nucleotides cause frameshift) were maintained in 1× Minimum Essential Medium(Gibico, Grand Island). The culture medium was supple-mented with 10% fetal bovine serum (FBS), 100 U/ml penicillin,100 �g/ml streptomycin and 0.03% glutamine and cells werecultured at 37 ◦C in a water-saturated atmosphere contain-ing 5% CO2. The expression plasmids of truncated p53 (�C30,�C55, �C75 and �C95) were provided by Dr. Y.-S. Lin (Devel-opment Center for Biotechnology, Taipei). The expressionplasmids of truncated p53 (�N99, �C85) were constructedinto the same empty vector. The expression plasmids of trun-cated p53 with NLS tag (i.e. NLS-�C75 and NLS-�C95) wereconstructed with the complementary DNA strands 5′-AAA

GGT ACC ATG CCC AAG AAG AAG CGC AAG GTG CCC AAGAAG AAG CGC AAG GTG AAG CTT AAA-3′ (the nucleotidesunderline are NLS sequence) at the N-terminus of p53. Thegene cloning was performed according to standard protocols.

0 0 8 ) 751–761

2.2. UV irradiation

Micropore UV irradiation was performed as described previ-ously [17]. Cells were cultured in 35-mm glass bottom dishes(MatTek, Ashland, MA, USA) for 2 days. After being rinsed withPBS, cells were carefully covered with a polycarbonate isoporemembrane (Millipore, Bedford, MA, USA). The filter-maskedcells were then UV irradiated at 200 J/m2 with a germicidallamp (254 nm, Sankyo Denki Co., Tokyo, Japan) in a UV box. Forwhole cell irradiation, cells were exposed to UV without anyfilter at the indicated doses. Doses of UV-irradiation were cal-ibrated with a UV radiometer (UVP Inc., San Gabriel, CA, USA).

2.3. Preparation of NEs

NEs were prepared as described previously [16]. Cells werefirst treated with 2.5 mM hydroxyurea and 25 �M cytosine-�-d-arabinofuranoside for 16 h. The cells were washed withhypotonic buffer (20 mM Hepes, pH 7.5, 5 mM KCl, 0.5 mMMgCl2, 0.5 mM dithiothreitol and 0.2 M sucrose), and wereallowed to swell in the hypotonic buffer without sucrose for10 min on ice. The swollen cells were then ruptured with10 strokes of a Dounce homogenizer and homogenates wereforced through a 22G needle 10 times. Each mixture wascentrifuged at 2000 × g for 5 min and nuclear pellets wereresuspended in buffer (20 mM Hepes, pH 7.5, 5 mM KCl, 0.5 mMMgCl2, 0.5 mM dithiothreitol and 10% sucrose) and stored at−70 ◦C. The nuclei were thawed on ice and allowed to swellin 100 mM NaCl on ice for 1 h. The ruptured nuclei were cen-trifuged at 15,000 × g for 20 min at 4 ◦C, and the supernatantswere filtered through a YM-10 Microcon filter (Millipore, Bed-ford, MA, USA). Protein concentrations were determined witha BCA Protein Assay Kit (Pierce Chemical Co., Chester, UK);bovine serum albumin was used as a standard.

2.4. Comet-NE assay

The comet-NE assay was performed as described previously[16]. For preparing the gels on each slide, 100 �l 1.4% agarosein phosphate-buffered saline (PBS) at 65 ◦C was placed ontoglass microscope slides pre-warmed at 60 ◦C. Coverslips wereapplied immediately, and the slides were chilled on ice. Cover-slips were then removed and 100 �l aliquots of cells containingagarose were then added. This was done by mixing equal vol-umes of 1.2% low-melting agarose and the cell suspensions(1 × 106 cells/ml in PBS); the mixtures were kept at 40 ◦C. Cov-erslips were added and removed as described above. Next,100 �l 1.2% low-melting agarose was applied as the third layerof agarose. After the third layer of the gel was made, the slideswere immersed in ice-cold cell lysis solution and were storedat 4 ◦C for at least 2 h. The cell lysis solution contained 2.5 MNaCl, 100 mM EDTA, 10 mM Tris (pH adjusted to 10 with NaOH),1% N-laurylsarcosine, 1% Triton X-100 and 10% DMSO (addedimmediately before use).

After cell lysis, the slides were washed three times withdeionized water. Digestion of NEs was performed by adding

a total of 20 �l excision mixture containing 0.6 �g NE, 50 mMHepes-KOH (pH 7.9), 70 mM KCl, 5 mM MgCl2, 0.4 mM EDTA,2 mM ATP, 40 mM phosphocreatine and 2.5 mM creatine phos-phokinase on each slide. A coverslip was applied, and the

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d n a r e p a i r 7

lides were incubated at 37 ◦C for 2 h in a sealed box con-aining a piece of wet tissue paper. After the incubation, thelides were denatured in 0.3N NaOH, 1 mM EDTA for 20 min.lectrophoresis was carried out in the same denaturationolution at 25 V, 300 mA for 25 min. Each slide was washedriefly in deionized water, blotted and then transferred to 0.4 Mris–HCl, pH 7.5. DNA was stained by adding 40 �l propid-um iodide (50 �g/ml) onto the slide. A coverslip was appliednd slides were examined using a fluorescence microscopeAxioplan 2, Zeiss Co., Thornwood, NY, USA). Images of 50ells per treatment were recorded with a close-circuit dis-lay camera (CoolSNAP). The migration of DNA from theucleus of each cell was measured with a computer program

http://tritekcorp.com) using the parameter of tail moment,hich is defined as the product of the tail length and the

raction of total DNA in the tail.

.5. Immunodepletion

Es were incubated with specific antibodies (1:10, w/w), andhe mixtures were gently shaken in a rotator for more than2 h at 4 ◦C. The mixtures were centrifuged at 8000 × g for0 min to remove precipitates and the supernatants were useds immunodepleted NEs in the comet-NE assays. Antibodiessed for this experiment were p53 (sc-126; Santa Cruz, CA,SA), XPA (sc-853; Santa Cruz), OGG1 (sc-12075; Santa Cruz),PG (sc-12558; Santa Cruz) and actin (sc-1616; Santa Cruz).

.6. DNA transfection

53 expression plasmids of various lengths were transfectednto H1299 cells with the LipofectamineTM 2000 transfectioneagent (Invitrogen, Inc., Carlsbad, CA, USA) according to the

anufacturer’s instructions. Subsequent analyses followingransient transfection were performed 24 h following trans-ection.

.7. Western blotting

estern blot analysis was performed according to a stan-ard protocol. Proteins in whole cell lysates were separatedy electrophoresis and were transferred to PVDF membranesAmersham Pharmacia Biotech, Amersham, UK) after whichhey were probed with p53 (sc-6243; Santa Cruz) and PCNANA03, Oncogene, Cambridge, MA) antibodies. To detect theb:Ag complexes, an ECL detection kit (Pierce Chemicalo., Chester, UK) was used according to the manufacturer’s

nstructions.

.8. Assay of p53 transactivation capacity

he plasmid WWP-Luc, a luciferase reporter containing the53 binding site derived from the promoter of p21Waf1/Cip1

as provided by Dr. B. Vogelstein (Johns Hopkins University,altimore, MD). The EGFP-N3 control plasmid was purchasedrom Clontech (Clontech, Heidelberg, Germany). Exponen-

ially growing cells (4 × 105) were plated in 35-mm dishesefore transfection. Cells were transfected with different trun-ated fragments of the p53 plasmid plus WWP-Luc and theGFP-N3 plasmid using the LipofectamineTM 2000 transfection

8 ) 751–761 753

reagent (Invitrogen, Inc.) according to the protocols providedby the manufacturer. Twenty-four hour after transfection,the cells were washed twice with PBS and were lysed in200 �l luciferase cell culture lysis reagent (Promega, Madi-son, WI, USA). Luciferase activities from 40 �l aliquots of celllysates were measured using the Wallac Victor 1420 MultilabelCounter (PerkinElmer, Monza, Italy). Results are expressed asrelative p53 activity compared with the empty vector control(Neo) after normalizing against EGFP expression.

2.9. Host cell reactivation

The plasmid CMV-Luc is a luciferase reporter containing theCMV promoter which can stably express luciferase in cells.To induce damage to the plasmid, the CMV-Luc plasmid wasUV-irradiated at a dose of 100 J/m2. H1299/Neo or H1299/p53cells were transfected with CMV-Luc (damaged or undam-aged) using the LipofectamineTM 2000 transfection reagent(Invitrogen, Inc.) according to the protocols provided by themanufacturer. The cells were harvested at the indicated times(1–3 days after CMV-Luc transfection) for determination ofluciferase activity as described above. Similarly, cells were alsotransfected with the plasmid EGFP-N3 to measure transfec-tion efficiency as a control. Results are expressed as the ratiobetween the level of cells transfected with damaged plasmidsand that of cells transfected with undamaged plasmids afternormalization for EGFP expression.

2.10. Immunofluorescence staining of CPD, XPC, XPBand p53 after micropore UV irradiation

This method was performed as described previously [17] withsome modification. In brief, 1 h after UV irradiation, cells werewashed with PBS and treated with an ice-cold solution of 2%formaldehyde and 0.5% Triton X-100 in PBS for 15 min. Cellswere incubated with PBS containing 20% FBS and 0.1% TritonX-100 at 37 ◦C for 30 min. Cells were incubated with the pri-mary antibody (XPB, XPC or p53 antibody) and then with thesecondary antibody (Alexa Fluor 488) at 37 ◦C for 30 min. Forthe previous incubation steps, primary antibody (1:100 dilu-tion) or secondary antibody (1:2000 dilution) solutions werefreshly made. For detection of CPD, DNA was denatured bytreating cells with 2N HCl at 37 ◦C for 30 min. The cells, afterwashing with PBS and blocking with 20% FBS, were incubatedwith anti-CPD antibody [18,19] and then with the secondaryantibody (Alexa Fluor 594) at 37 ◦C for 30 min. For thesesteps, the anti-CPD antibody solution (1:1500 dilution) andthe secondary antibody solution (1:2000 dilution) were freshlymade. Nuclear DNA was counterstained with 0.1 �g/ml 4′,6-diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene,OR, USA). Fluorescence images were captured using a digitalcamera mounted on an Olympus IX71 microscope (OlympusOptical Co., Tokyo, Japan).

2.11. Immunoprecipitation analysis

Immunoprecipitation of XPB-interacting protein was per-formed as described previously [20] with some modification.H1299 cells were harvested 4 h after UV irradiation at 25 J/m2.Cells were lysed in ice-cold IP lysis buffer (50 mM Tris–HCl, pH

7 ( 2 0 0 8 ) 751–761

Fig. 1 – The effect of p53 on the repair of UV-induced DNAdamage. DNA repair was examined by (A) host cellreactivation assay or (B) comet-NE assay. (A) H1299 cells(stably transfected with either p53 expression vector orempty vector, i.e. H1299/p53 or H1299/Neo, respectively)were transfected with UV-damaged or undamagedCMV-Luc plasmid and were harvested at the indicatedtimes (1–3 days) for luciferase activity assay. Activity isexpressed as the relative level to cells transfected with theundamaged plasmid. (B) H1299/p53 and H1299/Neo cellswere UV irradiated (3 J/m2) and were harvested at theindicated times (0–12 h) after irradiation. DNA lesions wereanalyzed by comet-NE assay using a NE from NB4 cells.Percentage of damage remaining is reported relative to the

754 d n a r e p a i r

7.4, 100 mM NaCl, 0.5% NP-40, 0.5% Triton X-100, 0.02% sodiumazide, 100 mM sodium fluoride, 1 mM PMSF, 1 mM EDTA, pH8.0, 1 mM benzamidine, 40 �g/ml aprotinin and 40 �g/ml leu-peptin). Mixtures were centrifuged (12,000 × g, 30 min) and thesupernatants (cell extracts) were collected. To reduce non-specific binding, cell extracts were pre-incubated with proteinA-Separose beads (pre-swollen in lysis buffer) before beingmixed with the anti-XPB antibody. Ag-Ab complexes werepulled down with protein A-Sepharose beads. The presenceof p53 in the immunoprecipitates was analyzed by Westernblotting.

3. Results

3.1. p53 enhances DNA repair

The reported stimulatory effect of p53 on DNA repair was con-firmed in this study using a host cell reactivation assay. TheDNA repair rate of human H1299 cells transfected with p53was faster than that of isogenic p53-null H1299 cells (Fig. 1A).In addition, the enhancement of the excision of UV-inducedDNA lesions by p53 was recapitulated with the comet-NE assaysince p53-transfected cells had a faster excision rate comparedto p53-null cells (Fig. 1B). In the latter experiment, the DNAlesions at specific times after UV irradiation were detected bythe comet-NE assay. To validate this relative novel method forthe following analyses, we performed a complementation testusing NE from two NER defective cell lines (XPC and TTD).As the results shown in Fig. 2A, no NE excision activity wasdetected unless the two different NEs were mixed together.Complementation was clearly seen when the NEs were com-bined.

3.2. p53 is necessary for the excision activity of the NE

To determine if p53 in the NE is essential to the exci-sion activity, we incubated the NE with a p53 antibody toremove p53 before the NEs were used in the comet –NE assay(Supplementary Fig. S1). The tail moments with such NEs weregreatly reduced (compared to columns 2 and 3, Fig. 2B). Simi-larly, NEs pre-incubated with antibodies to XPA or XPG lost theexcision activity (columns 4 and 5). In contrast, pre-incubationwith antibodies to OGG1 or actin did not reduce the excisionactivity of the NEs (columns 6 and 7). Thus, these results sug-gest that p53, like XPA or XPG, is essential for the excision ofUV-induced DNA adducts.

The above conclusion was further supported by an exper-iment with NEs of p53 null H1299 cells and isogenicp53-transfected cells. The NEs of H1299 cells had little exci-sion activity while the excision activity was restored in NEsof p53-transfected H1299 cells (columns 2 and 3, respectively,Fig. 2C).

3.3. The C-terminal 85 amino acids and theN-terminal 99 amino acids of p53 are dispensable for the

excision activity of the NEs

To characterize the structural moiety of p53 that is essen-tial to the excision activity, the NEs from cells expressing

initial level at 0 h after irradiation.

various truncated versions of p53 were analyzed by the comet-NE assay. As shown in Fig. 3A, truncation of the N-terminal99 amino acids, which includes the transactivation domain(residues 1–42) and the proline-rich region (residues 61–94),did not significantly affect the excision activity. Similarly,deletion of the C-terminal end up to 75 amino acids, whichincludes the negative regulatory domain (residues 363–393)and the oligomerization domain (residues 324–355) [21,22],

barely affected the excision activity (column 5 of Fig. 3A andcolumns 4–6 of Fig. 3B). However, if the C-terminal 95 aminoacid residues were deleted, the excision activity was com-pletely abolished (column 7, Fig. 3B). Since nuclear localization

d n a r e p a i r 7 ( 2 0 0 8 ) 751–761 755

Fig. 2 – p53 is an essential component of NEs necessary to excise the UV-induced DNA adducts. NB4 cells were UVirradiated (3 J/m2) and were harvested immediately for comet-NE assay with the indicated NEs or buffer only (column 1). (A)Use of the comet-NE assay to show complementation between NE of XPC cells and NE of TTD cells. Comet-NE assay with NEof XPC, TTD or NB4 cells (column 2, 3 and 5, respectively) or the mixed NEs between NE of XPC cells and NE of TTD cells(column 4). (B) NEs of NB4 cells pre-incubated with nothing (column 2) or anti-p53, XPA, XPG, OGG1 or Actin antibodies(columns 3–7), respectively. (C) NEs of H1299, H1299/p53 and H1299/Neo cells, respectively. Insert: Western blotting analysisof p53 protein in extracts of the indicated cells.

smwntNpppNt

ignal (NLS) of p53 was completely absent in �C95 mutant, itay be suspected if the loss of NE excision activity with �C95as because of the failure of the truncated protein to reside inuclei. Thus, we appended a consensus sequence of NLS [23]o �C95 (at N-terminus). We found that though the addition ofLS greatly increased the �C95 protein level in nuclei (bottomanel, Fig. 3C), it did not affect the NE excision activity (top

anel, Fig. 3C). The NE excision activity of p53 null cells com-lemented with NLS-�C95 remained inactive as those withoutLS. As a control experiment, the NLS was also added to �C75,

he NLS did not affect the NE excision activity, i.e. the NE exci-

sion activity of p53 null cells complemented with NLS-�C75was active as those without NLS (Fig. 3C). Thus, the loss of NEexcision activity with �C95 is not attributed to the absenceof NLS. Meanwhile, we also found that further deletion at theC-terminus to remove 85 amino acids had little effect on theexcision activity (�C85 of Fig. 3C).

On the other hand, the transactivation activity assay shows

that transactivation activity, unlike the NE excision activity, isvery sensitive to the deletion at either C- or N-termini (Fig. 4),indicating that the effect of p53 on NE excision activity is inde-pendent on its transactivation activity.

756 d n a r e p a i r 7 ( 2 0 0 8 ) 751–761

Fig. 3 – Effects of deleting the N-terminal or C-terminal ends of p53 on excision activity of NEs. NB4 cells were UV irradiated(3 J/m2) and were harvested immediately for comet-NE assay with the indicated NEs or buffer only (column 1). (A) NEs ofH1299 cells transfected with vector only (column 2) or expression plasmids of p53 of various lengths (columns 3–5):full-length (column 3), truncation of N-terminal 99 residues (column 4); truncation of both N-terminal 99 and C-terminal 75residues (column 5). (B) NEs of H1299 cells transfected with vector only (column 2) or expression plasmids of p53 of variouslengths (columns 3–7). C-terminal truncation for 30, 55, 75 or 95 residues (columns 4–7), respectively. Inserts: Western

d n a r e p a i r 7 ( 2 0 0

Fig. 4 – Transactivation capacity of p53 of various lengths.H1299 cells expressing p53 of indicated lengths (asdescribed for Fig. 3) were transfected with a p21Waf1/Cip1

promoter containing plasmid for the p53 transactivationaa

3s

TpnCtteCp(pmeacSHen�vammt

bcnNtp

ssay. Data are expressed as levels relative to the basalctivity (column 1, Neo i.e. cells not expressing p53).

.4. p53 is essential for the recruitment of XPB to CPDites

o study the effect of p53 on the recruitment of DNA repairroteins, we adopted the UV micropore local irradiation tech-ique combined with immuno-staining. As shown in Fig. 5,PDs were induced locally by UV; XPC co-localized with

he CPD sites regardless of the expression of p53. In con-rast, XPB only co-localized with the CPD sites if the cellsxpressed wild-type p53 (Fig. 6A). Co-localization of XPB andPD did not occur in cells expressing the �C95 mutant of53 (Fig. 6B) or in cells transfected with the empty vector

H1299/Neo) (Supplementary data, Fig. S2). Thus, wild-type53 but not its �C95 mutant appears necessary for the recruit-ent of XPB to CPD sites. Our data suggest that p53 is

ssential to recruit XPB but not XPC. Consistent with thebove findings, wild-type p53 but not the �C95 mutant waso-immunoprecipitated with an anti-XPB antibody (Fig. 7).imilar XPB levels were detected in both H1299/p53 and1299/�C95 cells (data not shown). Thus, the difference inffect between the full-length p53 and its �C95 mutant wasot related to the XPB level. In addition, truncation mutantC75 was recognized by XPB, in agreement with the pre-ious conclusion that deletion of C-terminal 75 but not 95

mino acids did not affect the NE excision activity. In sum-ary, these data suggest that wild-type p53, but not its �C95utant, may associate with XPB via protein–protein interac-

ions.

lotting analysis of p53 protein in NEs of the indicated cells. PCNontrol. (C) The effect of nuclear localization signal of p53 on NEuclear localization signal; Tet, tetramerization domain; Reg, regEs of H1299 cells transfected with expression plasmids of full-l

ag. The activity was determined with comet-NE assay as describrotein in cytosolic (Cyto) or nuclear (NE) fractions of the indicate

8 ) 751–761 757

4. Discussion

Our study suggests that p53 is necessary for the excision ofUVC- induced DNA adducts. This effect of p53 may be viaprotein–protein interactions, which facilitate the recruitmentof NER proteins (e.g. XPB) to the DNA damage sites. This is con-sistent with previous observations that the loss of p53 functionin mammalian cells results in reduced repair of UV-inducedDNA damage [7].

4.1. The use of comet-NE assay

Although a number of previous studies suggested that p53may be directly involved in NER [8,13,24], an approachsimilar to ours has not been performed. In the presentstudy, a recently developed comet-NE assay combined withimmunodepletion was used to test the role of p53 in NER.A similar approach was used in our recent work includ-ing the role of p53 in DNA repair in hamster cells [16].The success of complementation with NE from XPC andTTD cells shown in Fig. 2A suggests a future comple-mentation test with NE of p53-null cells and purified p53protein.

4.2. The role of p53 in the recruitment of XPB

The results of our immunostaining experiment (Fig. 6) agreewith a previous study [24] that p53 is essential for the recruit-ment of XPB to CPD sites (or 6-4 PP sites, data not shown).However, our study disagrees that p53 is necessary for therecruitment of XPC (Fig. 5). Our data indicate that XPC canbe recruited to CPD sites regardless of the expression ofp53. Moreover, anti-XPC antibodies, in contrast to anti-XPBantibodies, were unable to co-immunoprecipitate p53 (datanot shown). The reason for this discrepancy is unclear andwhether it is due to differences in the cell types used in thesestudies remains to be clarified. Our data indicate that deletionof the C-terminal 95 amino acids (but not 85) of p53 abolishthe excision activity of the NE (Fig. 3B and 3C). Further, p53with a deletion of the C-terminal 95 amino acids (i.e. residues1–298) was not co-immunoprecipitated by the anti-XPB anti-body (Fig. 7). We suspect that the sequence of residues 299–308of p53 may be crucial for interactions between p53 and XPB,and we are testing that possibility with site-specific mutants.The sequence of concern is LPPGSTKRAL where STKR are

hydrophilic and the rest residues are hydrophobic. We arecurrently manipulating the region to see if the electrostaticeffect or Van der Waals force play role in the NE excisionactivity.

A (proliferating cell nuclear antigen) was used as a loadingexcision activity. Top panel: the structures of p53 (NLS,ulatory domain). Middle-panel: relative excision activity ofength p53 or its truncation versions with or without NLSed above. Bottom-panel: Western blotting analysis of p53d cells.

758 d n a r e p a i r 7 ( 2 0 0 8 ) 751–761

Fig. 5 – Recruitment of XPC to CPD sites is p53-independent. H1299 cells expressing (H1299/p53) or not expressing(H1299/Neo) p53 were UV irradiated at the indicated dose through micropore filters; the cells were fixed 30 min after theirradiation for DNA staining (DAPI) or immunostaining for the indicated antigens.

4.2.1. Involvement of the interactions of p53 with othercomponents of TFIIH complexA previous study [12] suggested that p53 may have multipleinteractions with XPB, XPD and p62 of the TFIIH complex.Although the C-terminal 73 amino acids of p53 are notrequired for p53–p62 interactions, similar information aboutp53–XPB (p53–XPD) interactions is unavailable. Due to the lackof a good antibody against XPD, we were unable to characterizethe possibility of p53–XPD interactions.

4.3. Involvement of the nuclear localization sequence(NLS) of p53

Residues 298–318 of p53 may contain the signal sequence fornuclear localization [23,25]. To test the possibility whetherthe loss of excision activity with �C95 p53 was due to theabsence of the truncated p53 in nuclei, we have added a NLSback to �C95 p53 and found NLS did not improve the exci-sion activity despite its stimulatory effect on �C95 p53 proteinin nuclear fraction (Fig. 3C). The NLS, derived from consen-sus sequence, had no inhibitory effect on NE excision activityas the NE excision activities of �C75 and NLS-�C75 weresimilar.

4.4. Involvement of histone acetylation in the excision

activity of NEs

The effect of p53 on histone acetylation has been linked to itsinvolvement in NER [26–28]. Through interactions with p300, a

histone acetyltransferase, p53 may mediate acetylation of his-tone H3 which in turn relaxes the condensed chromatin andthus increases the accessibility of DNA damage sites [26]. Ourpresent study does not support the model of chromatin acces-sibility because of the following considerations. First, it hasbeen shown that the N-terminal domain of p53 is essential forits interaction with p300. However, our data indicate that trun-cation of the p53 N-terminal did not affect the excision activity(Fig. 3A). Second, if p53 played a role in chromatin accessibilityin our study, it would have affected the recruitment of XPC tothe CPD sites. However, our study shows that the recruitmentof XPC to CPD sites is p53-independent (Fig. 5). Although itremains possible that alteration of chromatin structures mayplay role in the subsequent steps of NER, our data with comet-NE indicates that p53 enhanced excision of UV-induced lesionin an environment that nucleosome structures are largelydestroyed. Thus, the effect of p53 on NE excision activity isindependent on the function of p53 in regulating chromatinstructure.

4.5. Remaining questions and conclusions

It remains to be known how p53 is recruited to the CPD sites.Although it appears that XPC does not interact with p53,whether the damage-bound XPC is able to recruit p53 remains

to be determined. Further, we did not monitor the kinetics ofp53 association with CPD and apparently, it should be dissoci-ated from the complex at a certain point. It will be of interestto determine when p53 leaves the repair complex.

d n a r e p a i r 7 ( 2 0 0 8 ) 751–761 759

Fig. 6 – Recruitment of XPB to CPD sites is p53-dependent. Images are similar to those shown in Fig. 5 except for theimmunostaining of XPB instead of XPC.

760 d n a r e p a i r 7 ( 2 0 0 8 ) 751–761

Fig. 7 – The C-terminal 95 amino acids of p53 are essential for co-immunoprecipitating of p53 with an anti-XPB antibody.H1299 cells expressing (H1299/p53, H1299/�C75, H1299/�C95) p53 were UV irradiated (25 J/m2); cells were harvested 4 hafter the irradiation for preparation of cell extracts. The cell extracts were used for Western blot analysis of p53 either

ates

r

directly (left 3 lanes, indicated as input) or in immuoprecipit(right 3 lanes).

In conclusion, our study indicates that the tumor suppres-sor p53 protein may facilitate NER via its direct involvementin the repair process by assisting repair proteins such as XPBto verify the damage sites.

Acknowledgements

We thank Dr. Toshio Mori (Nara Medical University, Japan) forproviding the monoclonal antibody to CPD and TTD10VI cellsand technical guidance in performing the local UV irradiation.We thank Dr. Hai-Mei Huang (National Tsing-Hua University,Hsin-Chu) for providing the H1299 cells, Dr. Wen-Ya Huang(National Cheng Kung University, Tainan) for XPC cells, Dr.B. Vogelstein (Johns Hopkins University, Baltimore, MD) forplasmid WWP-Luc and Dr. Young-Sun Lin (Development Cen-ter for Biotechnology, Taipei) for the expression plasmids oftruncated p53 (�C30, �C55, �C75 and �C95). This work wassupported by grants from the National Science Council, Tai-wan (NSC 95-2311-B-007-020; NSC 96-2311-B-007-011).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.dnarep.2008.01.019.

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