DNA Repair 3 (2004) 1285–1295

Relative levels of the two mammalian Rad23 homologs determinecomposition and stability of the xeroderma pigmentosum

group C protein complex

Yuki Okudaa,b, Ryotaro Nishia,b,c, Jessica M.Y. Ngd,1, Wim Vermeulend,Gijsbertus T.J. van der Horstd, Toshio Morie, Jan H.J. Hoeijmakersd,

Fumio Hanaokaa,c,f , Kaoru Sugasawaa,c,∗a Cellular Physiology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

b Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japanc CREST, Japan Science and Technology Agency, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan

d MGC-Department of Cell Biology and Genetics,Center for Biomedical Genetics, Erasmus Medical Centre, P.O. Box 1738, 3000DR Rotterdam, The Netherlands

e Radioisotope Center, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8521, Japanf Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan

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is via theu tein that isi HR23Br protein. Inw d stabilitya as actually∼ nlyt te that thet r part thed©

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Received 27 January 2004; accepted 18 March 2004

Available online 8 July 2004

bstract

Mammalian cells express two Rad23 homologs, HR23A and HR23B, which have been implicated in regulation of proteolysbiquitin/proteasome pathway. Recently, the proteins have been shown to stabilize xeroderma pigmentosum group C (XPC) pro

nvolved in DNA damage recognition for nucleotide excision repair (NER). Because the vast majority of XPC forms a complex withather than HR23A, we investigated possible differences between the two Rad23 homologs in terms of their effects on the XPCild-type mouse embryonic fibroblasts (MEFs), endogenous XPC was found to be relatively stable, while its steady-state level anppeared significantly reduced by targeted disruption of themHR23Bgene, but not by that ofmHR23A. Loss of bothmHR23genes causedtrong further reduction of the XPC protein level. Quantification of the two mHR23 proteins revealed that in normal cells mHR23B is10 times more abundant than mHR23A. In addition, overexpression of mHR23A in themHR23A/Bdouble knock out cells restored not o

he steady-state level and stability of the XPC protein, but also cellular NER activity to near wild-type levels. These results indicawo Rad23 homologs are largely functionally equivalent in NER, and that the difference in expression levels explains for a majoifference in complex formation with as well as stabilization effects on XPC.2004 Elsevier B.V. All rights reserved.

eywords:Nucleotide excision repair; Xeroderma pigmentosum; XPC; HR23; Ubiquitin; Proteasome

∗ Corresponding author. Tel.: +81 48 467 9532; fax: +81 48 462 4673.E-mail address:sugasawa@riken.jp (K. Sugasawa).

1 Present address: Department of Developmental Neurobiology, St. Judehildren’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105,SA.

1. Introduction

Nucleotide excision repair (NER) is a major DNApair pathway, which eliminates a wide variety of hedistorting base lesions, such as ultraviolet light (UV)-indupyrimidine dimers as well as chemically-induced intrastrcrosslinks and bulky adducts[1]. Impaired NER activity habeen associated with several, UV-sensitive human gedisorders, including the highly cancer-prone syndrome

568-7864/$ – see front matter © 2004 Elsevier B.V. All rights reserved.oi:10.1016/j.dnarep.2004.06.010

1286 Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295

roderma pigmentosum (XP). Thus far seven NER-deficientgenetic complementation groups of XP (XP-A through G)have been identified, for all of which the responsible genes(XPA–XPG) were already cloned[2].

Two distinct subpathways are discerned in the mammalianNER system: i.e., global genome NER (GG-NER) that op-erates genome wide, and transcription-coupled NER (TC-NER) that is specialized to eliminate transcription-blockinglesions on the DNA strand of active genes[3,4]. A majordifference between these two subpathways resides in theirstrategies for lesion recognition. Blockage of the transloca-tion by RNA polymerase II is supposed to be responsible forprimary damage recognition in TC-NER[5]. In GG-NER, onthe other hand, a protein complex containing the XPC protein,one of the XP-responsible gene products[6–8], is involvedin this key step[9,10]. Although the purified XPC complexspecifically recognizes and binds to certain types of lesions[9,11], a series of biochemical analyses have demonstratedthat the complex is actually a structure-specific DNA bindingfactor, which shows a specific affinity for a junction of double-and single-stranded DNA rather than lesions per se[12,13].These biochemical properties provide a plausible explana-tion for how GG-NER can detect such a wide diversity ofstructurally-unrelated DNA lesions. After the subpathway-specific damage recognition, the following steps in the re-p aredb undt asea re-q ndX ite isn ture-s hichc con-t ult-i oly-m er,a in[

sen-t theX am-mw 23o -t pro-tu ractwlM pieso tingaX do-m genep to

XPC at least in vitro[29], most XPC is complexed in vivowith HR23B, and thus only a minor fraction of the complexcontains HR23A[15]. HR23B was found far more abun-dant in cells than XPC, and the absence of the latter pro-tein was shown not to affect the level of HR23B[30,31].In a cell-free system competent for NER, complex forma-tion with HR23B greatly enhances physical stability of theXPC protein, which is indispensable for inducing efficientNER in vitro [15,32]. This is in marked contrast to cen-trin 2, which has been shown to be dispensable for recon-stitution of the cell-free NER reaction with purified proteins[15,33,34].

The in vivo roles of Rad23 and its mammalian homologsin NER have been investigated in several genetic studies. InS. cerevisiae, Rad23 forms a complex with Rad4[35,36],the presumed counterpart of mammalian XPC[6], and dis-ruption of theRAD23gene caused a NER defect and mildUV-sensitivity [37,38]. Possible involvement of the protea-some in regulation of NER has been suggested in yeast([19,39–41], see also review in[42]), although the roles ofRad23 have not been definitely established. Nevertheless,when Rad4 was overexpressed, lack ofRAD23expressionpromoted multiubiquitylation and proteasomal degradationof the exogenous Rad4[43]. Targeted disruption was alsocarried out for the mouseHR23(mHR23) genes, and embry-o insw o-cg wild-t s arefc ein-fo relyr de-f blet -G singt able.H tabi-l torso res-s ss logsr pro-t gra-d hilet ver-e C (orR ex-p t oft hanH e be-h ic fi-b ek

air reaction are likely processed by a mechanism shetween both NER subpathways. The DNA duplex aro

he lesion is locally unwound by the XPB and XPD helicctivities of the transcription factor TFIIH, which alsouires the action of replication protein A (RPA), XPA aPG proteins. The opening of the helix at the damage secessary for subsequent dual incision by the two strucpecific NER endonucleases, XPF-ERCC1 and XPG, wauses the release of a stretch of 25–30 nucleotidesaining the lesion. DNA repair synthesis filling the resng single-stranded gap is then carried out by DNA p

erase (� or �) in a PCNA- and RFC-dependent mannnd the final nick is sealed by DNA ligase I (see review

14]).As described above, the XPC complex plays an es

ial role in the lesion recognition for GG-NER. BesidesPC protein, this heterotrimeric complex contains a malian homolog ofSaccharomyces cerevisiaeRad23[7,8] asell as centrin 2[15]. Mammalian cells express two Radrthologs, called HR23A and HR23B[7,16]. The amino

erminal part of the yeast Rad23 as well as both HR23eins exhibits a marked homology to ubiquitin[7,17]. Thisbiquitin-like (UbL) sequence has been shown to inteith the 19S proteasome regulatory complex[18–20]and be

ikely involved in controlling protein degradation[21–23].oreover, the Rad23 family of proteins shares two cof the ubiquitin-associated (UBA) domain, also suggeslink to the ubiquitin/proteasome pathway[24–27]. The

PC-interacting site was mapped between the two UBAains, and is also conserved between the two HR23roducts[28]. Although both HR23 proteins can bind

nic fibroblasts lacking either or both of the mHR23 proteere isolated[44,45]. As predicted from our previous bihemical studies, cells in which either one of themHR23enes was knocked out showed UV resistance in the

ype range, suggesting that the two Rad23 homologunctionally redundant at least for NER[29]. Analysis ofells in which both of the mHR23 proteins were absent rorced this interpretation:mHR23A/mHR23Bdouble knockut cells (hereafter referred to as “DKO”) exhibited a seveeduced level of the XPC protein as well as a specificect in GG-NER, resulting in a UV sensitivity comparao that ofXPC−/− cells [45]. Moreover, exogenous XPCFP fusion protein, when expressed in the DKO cells u

he strong SV40 promoter, was found to be very unstowever, the XPC-GFP protein became dramatically s

ized either by treatment of cells with proteasome inhibir by DNA damage in the presence of additional expion of human HR23B (hHR23B)[45]. These observationtrongly suggest that Rad23 and its mammalian homoegulate the GG-NER reaction, at least partly throughecting their partners (i.e., Rad4 and XPC) against deation by the ubiquitin/proteasome system. However, w

hese stabilization effects have been shown mainly with oxpressed proteins, the regulation of endogenous XPad4) under physiological conditions is still largely unlored. In addition, it also remains unknown why mos

he XPC protein forms a complex with HR23B, rather tR23A. To answer these questions, we investigated thavior of endogenous XPC protein in mouse embryonroblasts, in which either or both of themHR23genes wernocked out.

Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295 1287

2. Materials and methods

2.1. Cell lines and cultures

Targeted disruption of themHR23AandmHR23Bgenesas well as isolation of mouse embryonic fibroblasts (MEFs)of various genotypes were described previously[44,45].The XPC−/− MEF was isolated from a 13.5-day em-bryo by crossing heterozygous mice. TheXPC targetedmice were originally established by Sands et al.[46]and gratefully donated by Dr. Allan Bradley (Baylor Col-lege of Medicine). All MEF cells were cultured at 37◦Cwith 5% CO2 in a 1:1 mixture of Dulbecco’s modi-fied Eagle’s medium (Nissui) and Ham’s F-10 nutrientmixture (Gibco), supplemented with 10% fetal bovineserum.

2.2. Preparation of cell extracts

For immunoblot analyses of the mHR23 proteins, MEFcells (cultured in 60-mm dishes) were washed twice with coldphosphate-buffered saline, and lysed in situ with 0.25 ml ofSDS lysis buffer [50 mM Tris–HCl (pH 8.0), 0.6% sodiumdodecyl sulphate (SDS), 10 mMN-ethylmaleimide (NEM),0.25 mM phenylmethylsulphonyl fluoride (PMSF), a pro-t Thec ed at9 n-t A,5 M,0 ix-tm c-t

in6 ereds MT n-i in-h llsw cul-t uffer,w mix-tt edurer actsw iono theX oad-i her ysisb DSl ractsw andW as as

2.3. Preparation of recombinant proteins and antibodies

For overexpression of the mHR23 proteins inE. coli, thefull-length cDNAs were cloned into pET-24a vector (No-vagen), so that the carboxyl-terminus of each protein wasfused to the hexahistidine (His6) tag. TheE. colistrain, BL21(DE3), was transformed with the expression constructs, andthe protein expression was induced for 3 h at 37◦C by addi-tion of 1 mM isopropylthio-�-d-galactoside. Purification wascarried out essentially as described previously for the His6-tagged human HR23 proteins[28]. Protein concentrationsof the purified fractions were determined by the Bradfordmethod[48] using a reagent purchased from Bio-Rad andbovine serum albumin as a standard.

Anti-sera recognizing the mHR23 proteins were obtainedby immunizing rabbits with the corresponding purifiedproteins. By using unrelated His6-tagged proteins, it wasconfirmed that reactivity of the obtained anti-sera with theHis6-tag part was negligible when used for quantificationof the proteins by immunoblotting. Anti-XPC antibodywas raised also in rabbits against the synthetic 19-aminoacid peptide (KTKREKKAAASHLFPFEKL), which corre-sponds to the carboxyl-terminus of the human XPC protein.The antibody that was affinity-purified with the peptidewas found to recognize not only the human but also them dured n ofm verysB p21a ly.

2p

ain-t m toa rei tha -r ast ia-g wereq singt revi-o

2 3p

am-m ascD in-r ay,u en-

ease inhibitor cocktail (Complete, Roche Diagnostics)].ell lysates were scraped into microfuge tubes, heat5◦C for 10 min, and then diluted with 1 ml of buffer co

aining 10 mM sodium phosphate (pH 7.2), 2 mM EDT0 mM NaF, 0.25 M NaCl, 1% Nonidet P-40, 10 mM NE.25 mM PMSF, and the protease inhibitor cocktail. The m

ures were centrifuged for 90 min at 50,000× g (Beck-an TLA45 rotor, 4◦C) to obtain the supernatant fra

ions.For detection of the mXPC protein, the MEF cells

0-mm dishes, washed twice with cold phosphate-buffaline, were lysed with 0.5 ml of NP lysis buffer [25 mris–HCl (pH 8.0), 1 mM EDTA, 10% glycerol, 1% No

det P-40, 10 mM NEM, 0.25 mM PMSF, the proteaseibitor cocktail]. After incubation on ice for 30 min, the ceere scraped into microfuge tubes and the remaining

ure dishes were washed once with 0.5 ml of the same bhich were then combined with the cell suspension. The

ures were centrifuged at 20,000× g for 10 min at 4◦C, andhe supernatants were discarded. This extraction procemoved a cytoplasmic protein, which strongly crossreith our anti-XPC antibody and interferes with detectf the endogenous mXPC protein. We confirmed thatPC protein, as well as lamin B1 that was used as a l

ng control, were not solubilized during this extraction. Tesulting pellets were washed once with 0.5 ml of NP luffer, and then dispersed by sonication in 0.25 ml of S

ysis buffer. Protein concentrations of the prepared extere determined according to the method of Schaffnereissmann[47], where bovine serum albumin was used

tandard.

ouse XPC protein in immunoblot analyses. The proceescribed here allowed significantly improved detectioouse XPC protein, and enabled us to visualize the

mall quantities of mXPC in the DKO cells[45]. Anti-lamin1 antibody (Santa Cruz Biotechnology) and anti-ntibody (BD Biosciences) were purchased, respective

.4. Measurement of the in vivo repair rate of (6-4)hotoproducts

Each MEF line was cultured to reach confluence and mained in the medium containing 0.2% fetal bovine seruvoid dilution of lesions by DNA replication. The cells we

rradiated with UV (at 10 J/m2, under a germicidal lamp wi254-nm peak) and incubated at 37◦C for various time pe

iods to allow repair of the photolesions. Genomic DNA when purified by using the QIAamp DNA blood mini kit (Qen), and the amounts of remaining (6-4) photoproductsuantified by the enzyme-linked immunosorbent assay u

he lesion-specific monoclonal antibody as described pusly[49,50].

.5. Isolation of stable transformants expressing mHR2roteins

To achieve a high level of the protein expression in malian cells, cDNA encoding mHR23A or mHR23B w

loned into pCAGGS vector ([51], a generous gift fromr. Jun-ichi Miyazaki, Osaka University). The hygromyc

esistance gene (Hygr) was expressed in a bicistronic wsing the internal ribosome entry site (IRES) of the

1288 Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295

cephalomyocarditis virus. For this purpose, a DNA fragmentcontaining IRES and downstream Hygr was excised fromthe pIREShyg vector (Clontech) and inserted 3′ to the cod-ing region of the mHR23 proteins. The resulting expressionconstructs were linearized and introduced into themHR23double knock out cells by electroporation using Gene-PulserII (Bio-Rad). Stable transformants were selected by culturingin the presence of 200�g/ml of hygromycin B (Invitrogen).

2.6. Other methods

For immunoblotting, proteins resolved by SDS–poly-acrylamide gel electrophoresis (SDS–PAGE) were trans-ferred onto polyvinylidene fluoride membranes (Immobilon-P; Millipore). Detection was performed by chemilumines-cence using an appropriate, alkaline phosphatase-conjugatedsecondary antibody (Sigma) as well as CDP-Star (RocheDiagnostics) as a substrate. Quantification was carried outwith the luminescent imaging analyzer (LAS-1000 plus; Fu-jifilm) and the accompanying software (ImageGauge v3.45),whereas X-ray films were also exposed to the blots, whichare shown in the present paper. DNA content analysis wasperformed with the laser scanning cytometer (LSC2; Olym-pus), for which cells were fixed on chamber slide glass andstained with propidium iodide.

3

3e

andH teds ousee thef res-e cord-i im-mT cellsw pro-t -s inl XPCp t atl ysisr eemt nt ofm -b lsoS -s ntita-t blotw tt e

Fig. 1. Steady-state levels of the endogenous mXPC protein in themHR23single or double knock out cells. (A) Whole cell extracts (each 5�g pro-tein) prepared from MEF cells of indicated genotypes were analyzed byimmunoblotting using either anti-mHR23A or anti-mHR23B antibody. (B)Lysates from the same set of MEF cells were subjected to immunoblot anal-yses using anti-XPC antibody. Lamin B1 was detected on the same blot asa loading control.

mHR23B−/− MEFs (lane 3). As observed before[45], loss ofbothmHR23genes led to a further strong reduction of mXPCto ∼10% of the wild-type level (lane 4). We confirmed thatthe mXPC protein levels hardly fluctuate throughout the cellcycle and thus do not depend on growth rates of the MEFs(data not shown). Although both mHR23 proteins can affectthe steady-state level of mXPC, the contribution of mHR23Aseems to be much smaller than that of mHR23B under phys-iological conditions. This is apparently coincident with thefact that a majority of XPC is bound in vivo to HR23B ratherthan HR23A[15].

3.2. Absence of HR23B expression reduces cellular NERactivity

Although it has been shown that the UV-sensitivity ofmHR23B−/− MEFs is in the wild-type range[44], the mXPCprotein content appears to be significantly reduced in thosecells. Taken together with our previous observation that XPCmay be rate-limiting for GG-NER[45], these findings raisedthe possibility that the GG-NER activity is nevertheless de-tectably affected in themHR23B−/− cells. To accurately as-sess NER activity, cells were irradiated with UV and theamount of (6-4) photoproducts that remained in genomicDNA was measured at various time points using a specificm t di-l lls.A sa , them t ofm ho-

. Results

.1. Two Rad23 orthologs differently affect thendogenous level of XPC

To examine possible differences between HR23AR23B in terms of XPC stabilization, we first estimateady-state levels of the endogenous mXPC protein in mmbryo fibroblasts (MEFs), in which either or both of

unctionalmHR23genes were lost by gene targeting. Pnce or absence of mHR23A and mHR23B proteins ac

ng to the genotype of each MEF line was confirmed byunoblot analysis of the cell lysate, as shown inFig. 1A.he endogenous mXPC protein in the same set of MEFas then examined, showing marked differences in the

ein level depending on the genotypes (Fig. 1B). In the abence of two functionalmHR23Aalleles, the mXPC prote

evel was not reduced (lane 2). Instead, even more mrotein (∼1.6-fold of wild-type) appeared to be presen

east in this specific MEF line used. DNA content analevealed that 80–90% of the MEF cells in this culture so be tetraploid (data not shown). Therefore, the amouXPC that is present in themHR23A−/− MEFs is comparale to wild-type if normalized by the DNA content (see aection 4). In marked contrast, absence ofmHR23Bexpresion caused a clear reduction of the mXPC protein. Quaive analyses, in which the lamin B1 signals on the sameere used as controls (lower panel inFig. 1B), indicate tha

he mXPC protein was∼50% of the wild-type level in th

onoclonal antibody against this photolesion. To prevenution of DNA lesions by replication, we used confluent ces shown inFig. 2,mHR23A−/− MEFs removed the lesiont a similar rate as the wild-type cells. On the other handHR23B−/− cells, which contain about half the amounXPC, exhibited significantly slower removal of (6-4) p

Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295 1289

Fig. 2. Effects of themHR23gene disruption on the repair rate of (6-4)photoproducts. MEF cells of indicated genotypes were UV-irradiated at10 J/m2 and incubated for various time to allow repair. Amounts of (6-4)photoproducts remaining in the genomic DNA were quantified by using theenzyme-linked immunosorbent assay, and expressed as percentages of theinitial amounts of the lesion generated. The mean values and standard errorswere calculated from at least two independent experiments.

toproducts. Unexpectedly, MEFs lacking bothmHR23genesshowed a low, but still detectable level of residual repair ac-tivity, although it has been previously shown that the DKOcells are as sensitive to UV asXPC−/− MEFs[45]. This sug-gests that the residual XPC protein, devoid of HR23, can stillinduce NER in vivo, which may be nonetheless insufficientto confer significant UV-resistance to proliferating cells.

F he mHC e cells yls( dicated by ‘C’). Cell lysates were subjected to immunoblotting either with anti-XPCa e cells, the cyclin-dependent kinase inhibitor p21 was also visualized to verify effecto otypes were treated with 0.1 mM cycloheximide for various time periods. Endogenousmba

3.3. Stability of endogenous mXPC and effects of DNAdamage

When the human XPC protein, fused to the carboxyl-terminal GFP-tag, was stably expressed together withhHR23B in the DKO cells, the exogenously expressed XPC-GFP was found unstable. However, the XPC-GFP proteinwas dramatically stabilized not only by addition of a protea-some inhibitor N-Cbz-Leu-Leu-Leu-al (CBZ-LLL), but alsoby different types of helix-distorting DNA damage, substratesfor the NER reaction[45]. Transcription of the exogenousXPC-GFPand hHR23Bgenes was driven by strong viralpromoters, which may exaggerate the effect on XPC proteinstabilization[45]. Therefore, we investigated the effect of in-hibition of proteolysis on the endogenous mXPC protein inthe MEFs of various genotypes. In contrast to the overex-pressed XPC-GFP protein, treatment of wild-type cells with10�M CBZ-LLL did not cause significant accumulation ofthe endogenous mXPC protein within a timeframe of 9 h,while the cyclin-dependent kinase inhibitor p21 was clearlystabilized during this incubation (Fig. 3A). In addition, inhi-bition of de novo protein synthesis with cycloheximide didnot lead to a significant reduction of mXPC (Fig. 3B). Theseresults indicate that endogenous mXPC protein is quite sta-ble in wild-type cells, and metabolic turnover of this proteini ally

ig. 3. Stability of endogenous mXPC in the presence or absence of tBZ-LLL for various time periods as indicated. As a mock treatment, th

DMSO), which was used for solvent of CBZ-LLL (lanes 7 and 13; inntibody or with anti-lamin B1 antibody. For the lysates from wild-typf the CBZ-LLL treatment (lanes 2–7). (B) MEF cells of indicated gen

XPC as well as lamin B1 that were present in the cell lysates were detectey the lamin B1 signal for each lane in (B). The values obtained are expressend standard errors were calculated from two independent experiments.

R23 proteins. (A) Wild-type andmHR23A−/− MEF cells were treated with 10�Mwere incubated for 9 h with the corresponding concentration of dimethulfoxide

s relatively slow under physiological conditions. Essenti

d by immunoblotting. (C) The amounts of mXPC were quantified and normalizedd as percentages of the initial amount of mXPC for each cell line. The mean values

1290 Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295

the same results were obtained with themHR23A−/− MEFs(Fig. 3A and B). On the other hand, when themHR23B−/−and DKO cells were treated with cycloheximide, a notabledecrease in mXPC could be observed (Fig. 3B and C). Af-ter 6-h treatment with cycloheximide, a∼40% decrease inmXPC was observed with themHR23B−/− cells, while theDKO cells showed 75% reduction of mXPC (Fig. 3C). Inthese cells, endogenous mXPC protein thus appears indeeddestabilized. When themHR23B−/− as well as DKO cellswere treated with CBZ-LLL, these cells started to die anddetach from the culture dishes within several hours (data notshown), whereas the wild-type andmHR23A−/− cells wereapparently resistant to the same treatment. Loss ofmHR23Bexpression thus seems to cause overall hypersensitivity tothe proteasome inhibitor and, therefore, the presumed in-volvement of the proteasome in the destabilization of en-dogenous mXPC (i.e., accumulation of mXPC during thetreatment with the inhibitor) could not be demonstrated withthese cells. These observations point to additional functionsof the HR23B protein, consistent with the complex phenotypeof themHR23B−/− mouse mutant[44].

We further examined the effect of UV-irradiation on theendogenous mXPC level (Fig. 4). When wild-type cellsandmHR23A−/− cells were irradiated with UV at 10 J/m2,the mXPC level was found slightly elevated: at 9 h post-i4 pro-nii ts,t heser bilizat fora pro-t

3 A

nsi-b ly am 3B,

F ccumul 0 J/mi d to im heo ach lan nitial amounto were c

HR23A alone appears insufficient to fully stabilize XPC, re-sulting in subphysiological levels of the protein. A possibleexplanation for this is that HR23B is able to bind and sta-bilize the XPC protein more effectively than HR23A or, al-ternatively, that expression levels of the two HR23 proteinsare quite different. To discriminate between these possibili-ties, the amount of each mHR23 protein present in the MEFlysates was quantified by immunoblot analysis using the pu-rified recombinant proteins as standards. For this purpose,the His6-tagged, full-length mHR23A and mHR23B proteinswere overexpressed inE. coliand purified to near homogene-ity (Fig. 5A). Based on the protein concentrations (deter-mined by the Bradford method), known amounts of the puri-fied proteins were included in immunoblot analysis, as stan-dards for quantification (Fig. 5B). In this manner 3�g proteinof wild-type cell extracts was found to contain∼0.4 ng ofmHR23B and less than 0.1 ng of mHR23A. The molar ratiobetween the two proteins was further normalized by detec-tion of both recombinant proteins with antibody reacting withthe common His6-tag. This indicated that mHR23B is∼10times more abundant than mHR23A in wild-type MEF ex-tracts. In extracts from themHR23A/Bdouble heterozygousMEFs, about half amount of each protein was detected whencompared with wild-type cell extracts (Fig. 5B). Notably ab-sence of mHR23B protein hardly affected the protein level ofm −/−t d to∼ fort

3l

int ncei maye e fora oge-n tentt irusi

rradiation, a∼10% increase in mXPC was observed (Fig.B). On the other hand, the increase in mXPC was moreounced in themHR23B−/− and DKO cells. After 9 h, mXPC

ncreased∼1.6-fold in themHR23B−/− cells, and∼4-foldn the DKO cells (Fig. 4B), although, in absolute amounhe accumulation in these cells was nonetheless small. Tesults suggest that, besides the mHR23-dependent staion, UV-irradiation may trigger additional mechanismsccumulation of endogenous XPC, for which the HR23

eins are not required.

.4. HR23B protein is much more abundant than HR23

The above data indicate that HR23B is mostly respole for the stabilization of XPC, whereas HR23A has oninor contribution. Furthermore, in the absence of HR2

ig. 4. Effects of the mHR23 proteins on damage-induced mXPC ancubated for various time periods. The cell lysates were then subjectef mXPC were quantified and normalized by the lamin B1 signal for ef mXPC in the wild-type MEF. The mean values and standard errors

-

ation. (A) MEF cells of indicated genotypes were UV-irradiated at 12 andmunoblot analyses using anti-XPC and anti-lamin B1 antibodies. (B) Tamountse in (A). The values obtained are expressed as percentages of the ialculated from two independent experiments.

HR23A, as shown inFig. 1A. In themHR23B MEFs,herefore, the level of total mHR23 proteins is reduce10% of the wild-type cells, which can largely account

he low level of the mXPC protein observed (Fig. 1B).

.5. Overexpression of mHR23A can compensate forack of mHR23B

If the two HR23 proteins are functionally equivalenterms of the XPC stabilization and the apparent differes mainly due to their relative abundance in cells, onexpect that overexpression of HR23A can compensatbsence of HR23B. To test this idea, mHR23A was exously expressed in the DKO cells under control of a po

ranscriptional promoter, which consists of cytomegalovmmediate early enhancer and chicken�-actin promoter[51].

Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295 1291

Fig. 5. Quantification of the mHR23 proteins in MEF cell extracts. (A) Purified recombinant mHR23 proteins used as standards. Two-microgram of eachprotein fraction was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. M: molecular weight markers. (B) Various amounts of the purifiedmHR23 proteins as well as whole cell extracts (each 3�g protein) from the indicated MEF cells were subjected to immunoblot analyses with anti-mHR23Aand anti-mHR23B antibodies. The same blot was also probed with the antibody reacting with polyhistidine tag (indicated by ‘His’).

Fig. 6. Overexpression of mHR23A can restore stability and NER functions of mXPC in the DKO cells. (A) ThemHR23A/BDKO cells were transfected withconstructs for stable overexpression of either mHR23A or mHR23B protein. Whole cell extracts were prepared from each two independently isolated clonesexpressing mHR23A (lanes 3 and 4) or mHR23B (lanes 5 and 6), and expression of the mHR23 proteins was detected by immunoblotting with each 5�gprotein of the extracts. As controls, the wild-type (lane 1) and parental DKO (lane 2) cell extracts were analyzed in parallel. (B) Steady-state levels of mXPCin the transformed cell lines were examined by immunoblotting. Lamin B1 signals on the same blot are also shown as loading controls. (C) In vivo repairrate of (6-4) photoproducts in the isolated transformants were measured by the enzyme-linked immunosorbent assay, as done inFig. 2. The mean values andstandard errors were calculated from at least two independent experiments. (D and E) One of the two isolated DKO transformants that express either mHR23Aor mHR23B was incubated for various time periods in the presence of 0.1 mM cycloheximide (D), or after UV-irradiation at 10 J/m2 (E). Endogenous mXPCand lamin B1 in the cell lysates were visualized by imunoblotting.

1292 Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295

Hygromycin-resistance was conferred by a bicistronic ex-pression cassette using an internal ribosome entry site, whichenabled us to select for cells with a certain expression levelof the exogenous protein by varying concentrations of hy-gromycin B in the culture medium. When stable transfor-mants were selected with 200�g/ml hygromycin B, severalclones were found to overexpress the mHR23A protein to alevel about 10 times that of wild-type cells (Fig. 6A, lanes3 and 4). Under similar conditions, expression of exogenousmHR23B reached its own physiological level (lanes 5 and6). Given that mHR23B is∼10 times more abundant thanmHR23A in wild-type cells, the total amount of the mHR23proteins (i.e., as a sum of mHR23A and mHR23B) expressedin these DKO transformants was comparable to that in wild-type cells. These amounts of mHR23 proteins in DKO cells,regardless whether it was mHR23A or mHR23B, restoredsteady-state levels of endogenous mXPC (Fig. 6B) as wellas the repair rate of (6-4) photoproducts (Fig. 6C) to nearwild-type levels. Furthermore, the response of mXPC upontreatment with cycloheximide (Fig. 6D) and UV (Fig. 6E) wasalso indistinguishable from that in wild-type cells. These re-sults indicate that the overexpressed mHR23A protein cansubstitute for mHR23B in terms of the stabilization of en-dogenous mXPC.

4

4

d int sta entw hem oteini y thep rtingN toa dra-m sa C ho-m er-e icallyu wasa ld-c e me-d tedb hatt sta-b

, wes ild-t if-il toxice iting

direct determination of XPC half life. In pulse-labeling ex-periments, incorporation of [35S]methionine into endogenousmXPC was found to be very low (data not shown), also in-dicating that de novo synthesis of this protein is maintainedat a rather low level. Because the expression of XPC-GFPwas driven by a strong SV40 promoter, the rate of synthesisof the exogenous protein is far higher than that of endoge-nous mXPC. However, in this case its degradation shouldalso be accelerated in order to maintain appropriate proteinlevels. These findings establish the existence of a mecha-nism that regulates XPC concentrations over a wide dynamicrange, and the difference in the rate of metabolic turnover pro-vides an explanation for the differences in response to CBZ-LLL treatment between the overexpressed XPC-GFP and en-dogenous mXPC. Although HR23 proteins and the ubiqui-tin/proteasome system appeared to regulate dynamically theGG-NER activity in the cells overexpressing XPC[45], ourpresent data may raise a question whether such regulationis relevant under the physiological conditions. However, ourexperiments do not exclude the possibility that, besides thestable XPC pool in the XPC-HR23B complex, there mightbe a minor pool of newly-produced XPC, which is not com-plexed with HR23B and thus immediately degraded by theproteasome. It is possible that such a nascent XPC pool thathas a higher turnover rate may become stable upon inductiono usee iods(

e ofm re-dC odel,i tionodw ellsw e in-h lianh oly-s ablyto fut tiono ofc t bet

wasl FP( en-d of am om-p hatt ingo s, int NA

. Discussion

.1. Stability of endogenous XPC protein

When XPC-GFP fusion protein was stably expressehemHR23A/BDKO MEFs together with hHR23B, robuccumulation of XPC-GFP was observed upon treatmith a proteasome inhibitor[45]. These data indicate that tetabolic turnover of this exogenously overexpressed pr

s quite fast, and that its rapid degradation is mediated broteasome. In addition, treatments inducing helix-distoER-type lesions, such as UV-irradiation and exposureN-cetoxy-2-acetylaminofluorene, also resulted in similaratic accumulation of XPC-GFP[45]. These observationre consistent with studies on Rad4, the presumed XPolog inS. cerevisiae[43]. When HA-tagged Rad4 was ov

xpressed in yeast, the protein was shown to be intrinsnstable, so that metabolically pulse-labeled Rad4-HAlmost completely turned over within 60 min after a cohase. This rapid degradation of Rad4-HA appeared to biated by multi-ubiquitylation, which was further promoy loss ofRAD23expression. In addition, it was shown t

reatment with certain DNA damaging agents transientlyilized Rad4-HA.

Compared to the exogenously expressed proteinshow here that endogenous XPC is relatively stable. In wype MEFs, treatment with CBZ-LLL failed to induce signcant accumulation of mXPC within 9 h (Fig. 3A). However,onger periods could not be tested due to non-specificffects caused by the proteasome inhibitor thus prohib

f DNA damage and/or binding to HR23, which may canhancement of GG-NER after relatively long time persee below)[45].

Instability of mXPC became apparent in the absencHR23Bexpression: the protein level was significantlyuced during treatment with cycloheximide (Fig. 3B and). These observations fit to the previously proposed m

n which the HR23 proteins negatively regulate degradaf XPC by the 26S proteasome[45]. Although we tried toemonstrate this directly by incubating themHR23B−/− asell as DKO cells in the presence of CBZ-LLL, these cere unexpectedly found quite sensitive to the proteasomibitor. Besides functions in NER, Rad23 and its mammaomologs have been implicated in the regulation of proteis mediated by the ubiquitin/proteasome system, presumhrough inhibition of the proteasome activity[20,27], controlf multiubiquitin-chain elongation[25,52], or recruitment obiquitylated target proteins to the proteasome[21]. If the

otal mHR23 protein level is severely reduced by disrupf themHR23Bgene, it is conceivable that accumulationertain proteins caused by proteasome inhibitors mighoxic for cells.

The response of endogenous mXPC to DNA damageimited, if compared with that of overexpressed XPC-GFig. 4). As mentioned above, the observed increase inogenous mXPC may be due to induced stabilizationinor, newly-synthesized XPC pool, possibly through clex formation with HR23. However, it is not excluded t

he level of XPC protein accumulation might vary dependn cell types and culture conditions utilized. Nevertheles

he presence of a long lasting elevation of the level of D

Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295 1293

damage, this could still lead to a significant increase in XPClevel. Interestingly, modest accumulation of mXPC was alsoobserved in themHR23B−/− as well as DKO cells, indicatingthe existence of additional mechanisms for mXPC accumu-lation, for which the mHR23 proteins are not necessary. Notonly through the HR23-dependent stabilization, the XPC pro-tein level may be probably regulated via multiple pathways,particularly when DNA is damaged. For instance, synthe-sis of the mXPC protein per se might be up-regulated uponDNA damage at transcription and/or translation levels. Alter-natively, the XPC protein might be stabilized by binding todamaged DNA, or by other unknown mechanisms. It has beenreported that the p53 tumor suppressor protein and BRCA1may be involved in accumulation of XPC in response to DNAdamage[53–55]. Further studies with themHR23B−/− andDKO cells may shed light on the molecular mechanisms forthe XPC accumulation.

4.2. Difference between HR23A and HR23B

The human XPC protein was initially thought to exist asa heterodimeric complex involving hHR23B[7,8]. Althoughanother human Rad23 homolog, hHR23A, was identified andthe two hHR23 proteins appeared to be interchangeable interms of their cell-free NER functions[7,29], it was un-c B,b se-q hiche ficanta in 2[

f thet ex-t witht ex.B ber ofm celli e-v cell[ bero andq th eralh pro-t 23B( PCw otalm s,m C,wo andm earst nceo atedh reg-u the

two HR23 proteins has not been precisely compared, whichmay affect efficiency of the complex formation with newly-synthesized XPC. On the other hand, themHR23A−/− MEFsused in this study appeared to contain a slightly, but sig-nificantly higher amount of mXPC than the wild-type cells(Fig. 1). This is not due to down-regulation of mXPC bymHR23A, as overexpression of this protein rescued the lowlevel of mXPC in the DKO cells (Fig. 6). Instead, this partic-ular MEF line was found to be partly polyploid, suggestingthat the steady-state level of XPC protein may be regulatednot only by HR23 protein level but also by DNA content ofcells.

Striking differences have been observed between thephenotypes of themHR23A−/− and mHR23B−/− mice.While themHR23A−/− mice were apparently normal[45],mHR23B−/− animals showed pleiotropic features, includingincreased embryonal lethality, severe developmental defectsand pertinent male sterility[44]. Because most of these phe-notypes have been observed neither with theXPA−/− norXPC−/− mice, these results point to certain functions ofmHR23B outside the NER context. One of the key ques-tions arising from these findings is whether mHR23B hasspecific functions, which cannot be taken over by mHR23A.Our present results may provide an explanation for this. Evenif the two HR23 proteins are fully redundant in all aspectso lti ro-t velyh t bea tob uchh rth-ee letalm ar-en upli-c ordert nceo

A

bo-r ass ca-t ) ofJ andT archP archA andJ UR-1 icala 02),N

lear why XPC is specifically targeted in vivo to HR23ut not to HR23A. However, the XPC complex was subuently purified to a much more concentrated form, wnabled us to detect a small, but nonetheless signimount of hHR23A, as well as the third subunit, centr

15].In this report, we measured the absolute amounts o

wo HR23 proteins that are present in wild-type MEFracts, and their expression levels appeared to correlatehe ratio of HR23A and HR23B found in the XPC complased on our quantitative analyses, the estimated numHR23B molecules that are present in a wild-type MEF

s in the order of 105, which is largely consistent with the priously calculated number of hHR23B in a single HeLa30]. Although in the same study a roughly similar numf hHR23A molecules was estimated, more extensiveuantitative immunoblot analyses likeFig. 3 revealed thaHR23A is actually less abundant than hHR23B in sevuman cell lines tested (data not shown). The mHR23A

ein level was not up-regulated in the absence of mHRFig. 1A). In these cells, the steady-state level of mXas found to be reduced correlating with the lower tHR23 protein level (Fig. 1B). Compared to wild-type cellHR23B−/− MEFs contain about the half amount of mXPhereas the total amount of mHR23 is reduced to∼10%f wild-type level. Thus the correlation between mXPCHR23 protein levels is not linear, and mHR23A app

o contribute more to stabilization of mXPC in the absef mHR23B than in its presence. It remains to be elucidow the complex formation between XPC and HR23 islated in vivo. For instance, intracellular localization of

f their functions, loss ofmHR23Bexpression may resun severe reduction of the overall level of the mHR23 peins. Normal development of mice may require a relatiigh level of mHR23 protein expression, which may nochieved by mHR23A alone. However, it still remainse examined whether mHR23B is always expressed migher than mHR23A in all tissues and cell types. Norn blot experiments have shown thatmHR23AmRNA lev-ls are relatively high in specific tissues, such as skeuscle and testis[16]. Because of the lack of any appnt phenotype associated with themHR23A−/− mice, it isonetheless enigmatic why the HR23 gene had to be dated during evolution. Further studies are necessary ino fully understand the biological significance of the presef HR23A.

cknowledgements

We thank members of the Cellular Physiology Laatory in RIKEN for helpful discussions. This work wupported by Grants-in-aid from the Ministry of Eduion, Culture, Sports, Science and Technology (MEXTapan, by the CREST program of the Japan Scienceechnology Agency, and by the Chemical Biology Reseroject of RIKEN. Y.O. was supported by the Junior Resessociate Program of RIKEN. J.H.J.H., G.T.J.v.d.H.,.M.Y.N. are supported by the Dutch Cancer Society (E774) the Dutch Science Organization (Medical, Chemnd Life Sciences Divisions), the EC (QLRT-1999-020IH (AG17242-02) and a Spinoza award.

1294 Y. Okuda et al. / DNA Repair 3 (2004) 1285–1295

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