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M. El-mahdy, Qianzheng Zhu, Qi-En Wang, G. Wani, M. Prætorius-Ibba, A. Wani (2006)
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5338–5350 Nucleic Acids Research, 2007, Vol. 35, No. 16 Published online 9 August 2007 doi:10.1093/nar/gkm550 Ubiquitylation-independent degradation of Xeroderma pigmentosum group C protein is required for efficient nucleotide excision repair 1, 1 1 1 Qi-En Wang *, Mette Prætorius-Ibba , Qianzheng Zhu , Mohamed A. El-Mahdy , 1 1 1 1 1,2,3, Gulzar Wani , Qun Zhao , Song Qin , Srinivas Patnaik and Altaf A. Wani * 1 2 3 Department of Radiology, Department of Molecular and Cellular Biochemistry and Comprehensive Cancer Center, The Ohio State University, 460 W. 12th Ave, Columbus, OH 43210, USA Received March 13, 2007; Revised June 27, 2007; Accepted July 5, 2007 impaired NER activity. XP patients are classified into ABSTRACT seven groups (XP-A to -G), and the defects of correspond- The Xeroderma Pigmentosum group C (XPC) protein ing seven genes (XPA to XPG) are responsible for the is indispensable to global genomic repair (GGR), missing NER activity in these XP patients. It is becoming a subpathway of nucleotide excision repair (NER), increasingly clear and accepted that NER in mammalian and plays an important role in the initial damage cells is mediated by the sequential assembly of repair recognition. XPC can be modified by both ubiquitin proteins at the site of the DNA lesion, rather than and SUMO in response to UV irradiation of cells. by the action of a pre-assembled repairosome (3–5). XPC–hHR23B complex is most likely the initial damage Here, we show that XPC undergoes degradation recognition factor when the lesions are situated in the upon UV irradiation, and this is independent of transcriptionally inactive genome or non-transcribed protein ubiquitylation. The subunits of DDB-Cul4A strand of actively transcribed genes (5,6), whereas E3 ligase differentially regulate UV-induced XPC XPA–RPA serves an equally critical function in verifying degradation, e.g DDB2 is required and promotes, the presence of the DNA lesion (7). In addition, due to its whereas DDB1 and Cul4A protect the protein high affinity for UV-damaged DNA, the damaged DNA degradation. Mutation of XPC K655 to alanine binding protein (DDB) complex has also been implicated abolishes both UV-induced XPC modification in the damage recognition step of GGR. DDB is a and degradation. XPC degradation is necessary heterodimer of DDB1 and DDB2 components. Studies on for recruiting XPG and efficient NER. The overall the role of DDB2 in NER have raised some concerns (8). results provide crucial insights regarding the fate Nevertheless, accumulating evidence has confirmed that and role of XPC protein in the initiation of excision DDB2 is undeniably involved in GGR. For example, several studies have shown that the cells from some XP-E repair. patients or DDB2-deficient Chinese hamster V79 cells have a partial deficiency in NER (9–11). Microinjection of the purified DDB complex into XP-E cells reversed the INTRODUCTION NER defect (12–14). Since NER can be reconstituted with Living cells could, at any moment, suffer DNA damage. If purified components and damaged DNA in the absence of damage is left unrepaired, consequent genomic instability DDB (15,16), DDB is believed to be relevant only to the can compromise cell survival. Nucleotide excision repair NER within the chromatin context. Our previous studies (NER) is a versatile repair pathway that can eliminate a as well as work of other laboratories have clearly shown wide variety of lesions, e.g. UV-induced photolesions that DDB2 is a key factor in regulating GGR of CPD, including cyclobutane pyrimidine dimers (CPD) and 6-4 most likely through the recruitment of XPC to the DNA photoproducts (6-4PP), from the genome of UV exposed damage sites (17–19). cells (1). NER includes two distinct subpathways, global XPC is a 940-amino acid protein, and harbors domains genomic repair (GGR) which removes lesions from the that can bind to damaged DNA and repair factors, entire genome, whereas transcription coupled repair e.g. hHR23B, XPB and Centrin 2 (20–22). XPC always (TCR) eliminates the DNA damage in the transcribed exists in a bound form with hHR23B and Centrin 2 in strand of actively transcribing genes (2). An autosomal cells. This protein complex actively participates in the recessive disorder, Xeroderma Pigmentosum (XP) exhibits process of NER (21,23,24). Although hHR23B contains *To whom correspondence should be addressed. Tel: +1 614 292 9015; Fax: +1 614 293 0802; Email: wang.771@osu.edu; wani.2@osu.edu 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2007, Vol. 35, No. 16 5339 two ubiquitin-associated domains and one ubiquitin-like (GM04312) and XPA complemented cells (XP-A+XPA, domain, it can stabilize XPC and enhance the binding GM15876), XP-F (GM08437) and XPF comple- between XPC and damaged DNA (25). XPC protein mented cells (XP-F+XPF, kindly provided by Dr Gan can be modified upon UV irradiation, the modifica- Wang, Wayne State University, Detroit, MI, USA), tions include ubiquitylation and sumoylation (26,27). XP-G (XP3BR-SV) and XPG complemented cells Interestingly, the ubiquitylation of XPC does not lead to (XP-G+XPG, kindly provided by Dr Karlene Cimprich, its degradation, but increases the binding of XPC to Stanford University, Stanford, CA, USA) and XP-E damaged DNA (26). While the role of sumoylated XPC is (GM01389) cells were grown in MEM supplemented still unclear, it was speculated to protect XPC from with 10% FCS and antibiotics. Mouse embryo fibroblast degradation (27). DDB2 is required for the UV-induced ts20 (thermosensitive for E1 ubiquitin-activating enzyme) XPC modifications. Among the modifications, the UV- and its parental cell line A31N (Kindly provided by induced XPC ubiquitylation is regulated by DDB-Cul4A Dr Harvey L. Ozer, UMDNJ-New Jersey Medical School) E3 ubiquitin ligase complex comprised of DDB1, DDB2, were cultured in 50% F-10 + 50% DMEM medium Cul4A, Roc1 and COP9 signalosome (28). DDB–Cul4A containing 10% FCS and antibiotics. HeLa cells with complex can ubiquitylate both DDB2 and XPC, but the over-expressed FLAG-HA-DDB2 and V5-His-XPC fates of ubiquitylated DDB2 and XPC appear to be quite (HeLa-DDB2-XPC cells) were generated in our lab and different, ubiquitylated DDB2, but not XPC, is subjected cultured in DMEM containing 500mg/ml G418 (36). All to proteasomal degradation (26). cells were cultured at 378C in a humidified atmosphere XPC expression can be induced following UV irradia- of 5% CO except A31N and ts20 cells, which are tion through transcriptional activation (29). Furthermore, maintained at 328C. For overall UV exposure, the cells overexpressed exogenous XPC has been found to be were washed with PBS, irradiated with varying UV doses intrinsically unstable and is degraded by the proteasome and incubated in suitable medium for the desired time (30). Nevertheless, the association with hHR23B protein period. The irradiation was performed with a germicidal partly stabilizes these XPC in vivo. Similarly, tagged Rad4, lamp at a dose rate of 0.8 J/m /s as measured by a the homolog of XPC in yeast, is found to be actively Kettering model 65 radiometer (Cole Palmer Instrument degraded by the 26S proteasome, and the turnover is Co., Vernon Hill, IL, USA). protected by Rad23 protein (31,32). However, the endo- genous mouse XPC protein is shown to be stable, with Site-directed mutagenesis, plasmid construction and transfection a half life of over 6 h (33), and the endogenous Rad4 in yeast is also stable in the absence of UV light, but is XPC-V5-His and DDB1-V5-His plasmids were generated degraded following UV irradiation (34). Our previous in our lab. pXPC3 plasmid containing XPC with study suggested that human XPC undergo degradation N-terminal 1–117 amino acids deletion (1–117) was following UV irradiation (27), and this degradation kindly provided by Dr Randy Legerski (The University of precedes the XPC induction observed later in the process. Texas MD Anderson Cancer Center, Houston, TX, USA). In this report, we have demonstrated that XPC is indeed Cul4A-c-Myc plasmid was kindly provided by Dr Yue degraded by 26S proteasome upon UV irradiation, Xiong (University of North Carolina, Chapel Hill, NC, and this degradation is independent of ubiquitylation. USA). DDB2-FLAG plasmid (kindly provided by Dr Furthermore, we provide evidence showing that the Gilbert Chu, Stanford University, Stanford, CA, USA) subunits of DDB–Cul4A complex differentially affect the was used to generate point mutants R273H and K244E, UV-induced XPC degradation. Additionally, we have and XPC-V5-His plasmid was used to generate point found that K655 residue of XPC protein is intimately mutants K655A and K917A by QuikChange Site-Directed involved in the UV-induced modifications as well Mutagenesis kit (Stratagene, La Jolla, CA, USA). The as degradation of XPC. Elimination of UV-induced plasmids were transfected into cells either by FuGene 6 XPC degradation impairs the efficient NER of CPD (Roche, Indianapolis, IN, USA) or Lipofectamine 2000 through an effect on the recruitment of XPG to damaged (Invitrogen, Carlsbad, CA, USA) according to the DNA sites. manufacture’s instruction. To generate stably transfected cell lines, G418 (500mg/ml) was added to the medium for selection and resistant colonies confirmed by western MATERIALS AND METHODS blotting. Cell culture and UV irradiation Western blot analysis Normal human fibroblasts OSU-2 cells, established and maintained in culture as described earlier (35), The cells were trypsinized and washed once with PBS. Li-Fraumeni Syndrome fibroblast strain designated 041 The cell pellets were lysed by boiling for 10 min in a cells (kindly provided by Dr Michael Tainsky, MD sample buffer (2% SDS, 10% glycerol, 10 mM DTT, Anderson Cancer center, Houston, TX, USA), XP-C 62 mM Tris–HCl pH 6.8, protease inhibitor cocktail). (GM15983), HeLa cells with over-expressed FLAG Protein samples were loaded on 8–16% Tris–Glycine gels and HA-tagged DDB2 (HeLa-DDB2 cells) (a gift of (Invitrogen) and separated by PAGE. The proteins Dr Yoshihiro Nakatani, Dana-Farber Cancer Institute, were then transferred to nitrocellulose membrane, Boston, MA, USA) were grown in DMEM supplemented blocked by 5% milk and immunoanalyzed. The antibodies with 10% fetal calf serum (FCS) and antibiotics. XP-A used were, rabbit anti-XPC and rabbit anti-DDB2 5340 Nucleic Acids Research, 2007, Vol. 35, No. 16 (generated in our lab) (27), rabbit anti-Cul4A and rabbit Immuno-slot blot analysis anti-DDB1 (a gift from Dr Yue Xiong), goat anti-Lamin The amount of CPD in DNA was quantified with non- B (Santa Cruz Biotechnology, Santa Cruz, CA, USA), competitive immuno-slot blot assay. Briefly, XP-C cells in mouse anti-hHR23B (BD Bioscience, San Jose, CA, 100 mm plates were transiently co-transfected with DDB2 USA), rabbit anti-V5 (Bethyl Laboratories, Montgomery, and either empty vector, wild type or K655A XPC TX, USA) and mouse anti-c-Myc (Invitrogen). mutant. Twenty-four hours post-transfection, cells were split into 60 mm plates and grown for an additional 24 h. Local UV irradiation and immunofluorescence After UV exposure (10 J/m ) and desired incubation periods, cells were recovered by trypsinization and XP-C cells growing on glass coverslips were transfected immediately lysed for DNA isolation. The identical with XPC-V5-His plasmid for 48 h. The cells were then amounts of DNA samples were loaded on nitrocellulose washed with PBS and UV irradiated through an isopore membranes and the amount of CPD was detected with polycarbonate filter (Millipore, Bedford, MA, USA), monoclonal anti-CPD antibody (TDM-2). The intensity containing pores of a 5 mm in diameter, as described of each band was determined by laser densitometric previously (37). The cells were then double stained with scanning and the amount of damage remaining, compared rabbit anti-XPC and mouse anti-CPD (TDM-2, MBL with the initially induced DNA damage, was used to International, Woburn, MA, USA), or rabbit anti-XPC calculate the relative repair rates. and mouse anti-XPA (Lab Vision, Fremont, CA, USA), or rabbit anti-XPC and mouse anti-XPG (Lab Vision) or mouse anti-V5 (to visualize XPC, Invitrogen) and RESULTS rabbit anti-XPB (Santa Cruz). Fluorescence images were obtained with a Nikon Fluorescence Microscope E80i XPC is degraded following UV irradiation (Nikon, Tokyo, Japan) fitted with appropriate filters for Our previous studies have indicated that the level of XPC FITC and Texas Red. The digital images were then in cells decreases upon UV irradiation (27). To further captured with a cooled CCD camera and processed with confirm this phenomenon, we compared the decay rates of the help of its SPOT software (Diagnostic Instruments, XPC in UV- or mock-irradiated normal human fibroblast, Sterling Heights, MI, USA). OSU-2 cells pre-treated with cycloheximide (CHX), an inhibitor of de novo protein synthesis. Figure 1A shows GST pull down assay that in the absence of new XPC synthesis, XPC protein exhibits a high decay rate following UV irradiation, and GST and the fusion protein GST-hSug1 were expressed the distinct pattern of XPC degradation could be observed in Escherichia coli strain DH5a transformed with as early as 30 min after UV treatment (lanes 5–8). To rule either pGEX4T-1 or pGEX-hSug1 (kindly provided by out the possibility that the decreased XPC level at Dr Andrew Paterson, The University of Alabama at 125 kDa is due to the conversion of XPC to slower Birmingham, Birminghan, AL, USA). After purification, migrating modified forms, we over exposed the film to GST and GST-hSug1 were separately incubated show the corresponding levels of various XPC bands. As with glutathione Sepharose 4B beads (Amersham shown in Figure 1A, the modification of XPC is not fully Bioscience, Uppsala, Sweden) at 48C for 2 h in PBS. The obvious at 2 h time point and yet the level of XPC at nuclear extract from OSU-2 cells were prepared by 125 kDa is lower than that seen at 1 h time point. We then incubating OSU-2 cells in nuclear extract (NE) buffer scanned all bands of XPC and quantified the total XPC (20 mM HEPES, pH 7.9, 25% glycerol, 0.42 M NaCl, amount and normalized by Lamin B level. As shown in 1.5 mM MgCl , 0.2 mM EDTA, protease inhibitor cock- Figure 1A, total XPC amount did decrease with the tail) for 20 min and NE was collected by centrifugation. elongation of incubation time following UV irradiation. GST or GST-hSug1 bound beads were incubated with This result indicates that the decrease of XPC observed either NE, or purified recombinant XPC (a gift of Dr Yue after UV irradiation is most likely due to the protein Zou, East Tennessee State University, Johnson City, TN, degradation. Moreover, we treated the cells with protea- USA) at 48C for 2 h in NE buffer. After washing five times some inhibitor MG132 prior to UV irradiation to test the with NE buffer, the beads were boiled in 2SDS loading involvement of 26S proteasome in this UV-induced XPC buffer for 5 min and the supernatant was subjected to degradation. We found that the UV-induced decrease of western blot analysis. XPC levels is promptly inhibited in the presence of MG132 (Figure 1B, lanes 1 and 2 versus 3 and 4). siRNA transfection A similar result regarding the effect of MG132 on the Cul4A and DDB1 siRNA oligonucleotides were syn- immediate fate of XPC was also seen with repair-deficient thesized by Dharmacon (Lafayette, CO, USA) in a XP-A cells (Supplementary Figure 1). These combined purified and annealed duplex form. The sequences data indicate that UV irradiation causes XPC protein targeting Cul4A and DDB1 were 5 -GAACAGCGAUC degradation via proteasome-mediated proteolysis. 0 0 GUAAUCAAUU-3 and 5 -UAACAUGAGAACUCU Moreover, Figure 1B showed that when protein degrada- UGUC-3 , respectively. Specific and control siRNA tion is inhibited by treatment with MG132, more XPC transfections were performed with Lipofectamine 2000 was detected in UV-treated cells than that in mock-treated (Invitrogen) according to manufacture’s instruction. cells (lane 3 versus 4). However, when we further inhibited Nucleic Acids Research, 2007, Vol. 35, No. 16 5341 A UV-induced XPC degradation is independent of CHX treatment time (h) 0.5 1.0 1.5 2.5 0.5 1.0 1.5 2.5 2 ubiquitylation UV (20 J/m ) ++ + −− − − − Repair time (h) 0 0.5 1.0 2.0 Previous studies have implied that UV-induced ubiquity- lation of XPC is reversible and does not serve as a signal 125 kDa XPC for degradation (26). Therefore, we reasoned that 1 0.98 0.85 0.87 1 0.75 0.57 0.21 Relative amount UV-induced XPC degradation observed in our experi- XPC modified (exposed XPC ments might be independent of ubiquitylation. To test this longer) 125 kDa hypothesis, we analyzed the UV-induced changes of in vivo XPC levels in mammalian cells capable of conditional Lamin B inactivation of E1 enzyme. Ts20 cells growing at 12 3 4 5 6 7 8 permissive 328C, thus harboring normal E1 activity, exhibit a small extent of XPC degradation upon irradia- CHX (100 µg/ml) −− − − + + + + tion (Figure 1C, lane 5 versus 6). However, when E1 is MG132 (10 µM) −− ++ − − + + inactivated by transferring cultures to non-permissive UV (20 J/m , 1 h) − + − + − + − + 398C (38,39), these cells showed considerable XPC XPC degradation following irradiation (lane 7 versus 8). Relative amount 1 0.52 1 1.71 1 0.33 1 1.15 Exactly the same XPC degradation response is seen in Lamin B parent control A31N cells at both permissive and non- 12 3 4 5 6 7 8 permissive temperatures (Figure 1C, lanes 1–4). In essence, the ubiquitylation defect failed to impinge on A31N ts20 the protein degradation. These in vivo data clearly indicate 32°C 39°C 32°C Temperature 39°C that UV-induced XPC degradation is independent of 2 ubiquitylation and suggest a direct interaction of XPC UV (20 J/m , 2 h) − + − + − + − + with proteasome. To substantiate this idea of direct XPC ubiquitylation-independent interaction, the GST pull Relative amount 1 0.80 1 0.44 1 0.83 1 0.39 down assay was conducted with the whole cell lysates Lamin B prepared from OSU-2 fibroblasts. We found that recom- binant hSug1, a subunit of 19S proteasome, physically binds to XPC protein (Figure 1D). The interaction was DE further tested with purified recombinant XPC and hSug1, and the result shows that XPC protein can bind to hSug1 directly (Figure 1E). Taken together, we believe that XPC UV-induced XPC degradation is independent of ubiqui- XPC 1 2 3 tylation and that XPC can bind to 26S proteasome through direct interaction with hSug1. Figure 1. XPC is degraded independent of ubiquitylation upon UV irradiation. (A) OSU-2 cells were treated with 100 mg/ml of CHX 0.5 h NER process does not affect UV-induced XPC degradation prior to UV irradiation at 20 J/m or mock treatment. Cells were further incubated in the medium containing CHX for different time To explore the relationship between XPC degradation and periods. The whole cell lysates were subjected to western blot the NER process, we examined the kinetics of UV-induced analysis using anti-XPC antibody. The same membrane was also XPC degradation in normal human fibroblast as well as immunoblotted for Lamin B as a loading control. (B) OSU-2 cells were treated with either MG132 (10mM) or CHX (100ug/ml) or both human cell lines belonging to different XP complementa- for 1 h prior to UV irradiation at 20 J/m or mock treatment and the tion groups and the corresponding cell lines corrected for cells were incubated in the same medium for another 1 h. The cell the cognate repair deficiency. As shown in Figure 2A, lysates were subjected to immunoblotting as described above. (C) A31N normal human fibroblast, OSU-2 cells, showed a sig- and ts20 cells were cultured for 16 h at 328Cor398C, UV irradiated at 20 J/m , and cultured for another 1 h at the same temperatures. The nificant decrease in XPC levels at 1 h, followed by an whole cell lysates were subjected to immunoblotting and the level of increase until it again reached the control levels at 8h XPC was detected with anti-XPC antibody. (D and E) The cell lysates after UV irradiation. Meanwhile, all three XP-A, XP-F from OSU-2 cells (D) or purified recombinant XPC protein (E) were and XP-G cell lines exhibit the typical XPC degradation incubated with recombinant GST or GST-hSug1 proteins bound to GST beads. The protein bound to the beads was subjected to upon UV irradiation (Figure 2B–D), indicating that UV- immunoblotting using anti-XPC antibody. Relative amount of total induced XPC degradation is not affected by the absence of XPC at various times post-UV or mock-treatment were quantified any of these essential repair factors and is independent relative to the respective unirradiated levels and normalized by Lamin of the productive cellular excision repair process. In B controls. addition, XP-A and XP-F cells exhibit a similar XPC dynamics as that of repair-proficient OSU-2 cells, characterized by a prompt decrease at 1 and 2 h followed protein synthesis by treatment with CHX, this by restoration beginning at 4 h following UV irradiation UV-induced XPC increase did not occur (lane 7 versus (Figure 2B and D, lanes 1–5). On the contrary, XP-G cells 8). These results indicate that both degradation and demonstrated continued XPC degradation without any induction of XPC protein occurs in tandem within UV detectable recovery of XPC at later intervals (Figure 2C, irradiated cells. lanes 1–5). Nevertheless, ectopic expression of XPG 10% Input GST GST-hSug1 Input (5%) GST GST-hSug1 5342 Nucleic Acids Research, 2007, Vol. 35, No. 16 OSU-2 1.2 UV (20 J/m ) − ++++ 1.0 Repair time (h) 01248 0.8 0.6 XPC 0.4 XPC 0.2 (exposed longer) 0.0 02468 10 Lamin B Repair time (h) XP-A XP-A+XPA 1.2 UV (20 J/m ) − ++++ − ++ + + 1.0 Repair time (h) 01248 01 24 8 0.8 0.6 XPC XP-A 0.4 XPC XP-A+XPA 0.2 (exposed longer) 0.0 02468 10 Lamin B Repair time (h) 1 2 34 5 6 789 10 XP-G XP-G+XPG 1.2 2 XP-G UV (20 J/m ) − ++++ − +++ + 1.0 XP-G+XPG Repair time (h) 01248 0 124 8 0.8 0.6 XPC 0.4 XPC 0.2 (exposed longer) 0.0 02468 10 Lamin B Repair time (h) 1 2 34 5 6 789 10 XP-F XP-F+XPF 1.6 1.4 UV (20 J/m ) − ++++ − +++ + 1.2 Repair time (h) 01248 0 124 8 1.0 0.8 XPC 0.6 XP-F 0.4 XPC XP-F+XPF (exposed 0.2 longer) 0.0 02468 10 Lamin B Repair time (h) 1 2 34 5 6 789 10 Figure 2. The dynamic of UV-induced XPC degradation. NER-proficient OSU-2 cells (A), various NER-deficient XP cells, like XP-A (B), XP-F (C), XP-G (D) as well as their corresponding repair factor-complemented cell lines were UV irradiated at 20 J/m and then incubated for the indicated times. Whole cell lysates were subjected to immunoblotting as described in Figure 1A. The levels of total XPC in each lane were quantified and normalized by the initial amount of XPC and Lamin B and plotted on the right. in XP-G cells was able to restore the normal XPC dynamics the ectopic expression of XPF in XP-F cells prevented the (Figure 2C, lanes 6–10), indicating that XPG is required for expected XPC decrease observed upon UV treatment the recovery of XPC protein following repair of (Figure 2D, lanes 6–10). Since the UV-induced XPC UV damage in fully repair-competent cells. Interestingly, degradation is easily seen in CHX-treated XPF-corrected Relative XPC level Relative XPC level Relative XPC level Relative XPC level Nucleic Acids Research, 2007, Vol. 35, No. 16 5343 repair-proficient XP-F cells (Supplementary Figure 2), A XP-E we conclude that the XPF protein does not interfere with UV (20 J/m ) − + + the XPC degradation. Nonetheless, XPF protein could Repair time (h) 01 4 additionally be stimulating the new synthesis of XPC which is also inducible upon UV irradiation. XPC Relative amount 1 1.05 1.15 DDB2 is essential for promoting UV-induced XPC Lamin B degradation 12 3 The requirement of DDB2 protein for the UV-induced XPC modifications (ubiquitylation and sumoylation) has V-79 previously been reported by our laboratory and others DDB2 transfection −− ++ (26,27). Here, we extend this work by investigating the UV (20 J/m , 1h) − + − + role of DDB2 in UV-induced XPC degradation. We approached this question by first following the post- XPC irradiation fate of XPC protein in experiments with Relative amount 1 1.03 1 0.64 DDB2-deficient XP-E cells. The results clearly show that DDB2 UV-induced XPC degradation fails to occur in cells lacking DDB2 (Figure 3A). Since XP-E cells posed Lamin B difficulty in transfecting cDNA constructs, we used 12 3 4 another DDB2-deficient Chinese Hamster V79 cell line to further observe the effect of restoring DDB2 into these cells on UV-induced XPC degradation. As expected, DDB2 (mg) 0 0 0.5 1.0 2.0 4.0 8.0 DDB2 expression restored the XPC degradation following UV (20 J/m , 1 h) − + + + + + + UV irradiation (Figure 3B). This DDB2-mediated response was more clearly demonstrable in another cell DDB2 line, 041, that lacks the DDB2 because of the absence of p53 inducer. As shown in Figure 3C, XPC remains fully Lamin B intact upon UV irradiation of these cells. However, XPC transient transfection of DDB2 cDNA into these cells restored the normal UV-induced XPC degradation with a Relative amount 1 1.17 0.87 0.82 0.76 0.65 0.51 distinct dose-response relationship, i.e. greater XPC Lamin B degradation with higher DDB2 expression. Finally, we 17 2 3 4 5 6 tested whether the expression of mutated DDB2 can functionally substitute for the wild-type DDB2. Two XP-E mutations are single amino acid substitutions Transfection Vector Wt R273H K244E (K244E and R273H) corresponding to XP-E patients UV (20 J/m , 1 h) − + − + − + − + XP82TO and the related individuals XP2RO and XP3RO, respectively (40). Extracts from cells of these lines are XPC defective in the ability to bind UV-irradiated DNA Relative amount 1 0.92 1 0.19 1 1.07 1 0.90 fragments (9). These two naturally occurring mutants of DDB2 DDB2, R273H and K244E, along with wild-type DDB2, were separately and stably transfected into 041 cells and Lamin B evaluated for the fate of XPC. As expected, only the wild- 12 3 4 5 6 7 8 type DDB2 promotes UV-induced XPC degradation (Figure 3D), which unambiguously indicates that the Figure 3. DDB2 is required and promotes UV-induced XPC degrada- tion. (A) XP-E cells were UV irradiated at 20 J/m and incubated for damaged DNA binding activity of DDB2 is a strict the indicated times. The whole cell lysates were subjected to requirement for it to participate in the XPC degradation. immunoblotting using anti-XPC antibody. (B) V79 cells were tran- siently transfected with DDB2-FLAG and UV irradiated at 20 J/m . DDB1 and Cul4A protect XPC from degradation After incubation for another 1 h, the whole cell lysates were prepared and subjected to immunoblotting using anti-XPC antibody. (C) 041 upon UV irradiation cells were transiently transfected with various amounts of DDB2- DDB-Cul4A E3 ligase is believed to be functionally FLAG. Twelve hours post-transfection, cells were split into two 60 mm plates and grown for another 24 h. The cells were mock or UV essential for the XPC ubiquitylation upon UV irradiation irradiated at 20 J/m and incubated for another 1 h.The whole cell (26). Furthermore, our results indicate that DDB2, as one lysates from mock-irradiated cells were subjected to immunoblotting of the subunits of this same E3 ligase, is also required for using anti-DDB2 antibody, and those from UV-treated cells were UV-induced XPC degradation. However, the role of other subjected to immunoblotting using anti-XPC antibody. (D) 041 cells stably expressing wild-type or mutant (R273H and K244E) DDB2- subunits of this E3 ligase in this important FLAG were UV irradiated at 20 J/m and incubated for another 1 h. cellular process is not known. In order to address this The whole cell lysates were subjected to immunoblotting using anti- question, we utilized a siRNA-based gene silencing DDB2 and anti-XPC antibodies. Relative amount of total XPC at strategy to squelch the activity of individual complex various times post-UV were quantified relative to the respective components within cells. As shown in Figure 4A and B, unirradiated levels and normalized by Lamin B. 5344 Nucleic Acids Research, 2007, Vol. 35, No. 16 AB −− ++ siCul4A(100 nM) −− ++ siDDB1 (100 nM) 2 2 − + − + − + − + UV (20 J/m , 1 h) UV (20 J/m , 1 h) Cul4A DDB1 DDB2 DDB2 XPC XPC XPC XPC (exposed (exposed longer) longer) Lamin B Lamin B 120 120 −UV −UV 100 100 +UV +UV 80 80 60 60 40 40 20 20 0 0 −siDDB1 +siDDB1 −siCul4A +siCul4A DDB1 −− ++ −− + + −− −− +++ + Cul4A UV (20 J/m , 1 h) − + −++ − + − DDB1-V5 Cul4A-c-Myc DDB2 ** XPC Lamin B −UV +UV Vector DDB1 Cul4A DDB1+Cul4A Transfection Figure 4. DDB1 and Cul4A protect XPC from degradation upon UV irradiation. (A and B) OSU-2 cells were transfected with DDB1 siRNA (A) or Cul4A siRNA (B) for 48 h. Cells were UV irradiated at 20 J/m and allowed to repair for 1 h. The whole cell lysates were subjected to immunoblotting using anti-DDB1, anti-Cul4A, anti-DDB2, anti-XPC and anti-Lamin B antibodies. Relative amount of total XPC in UV-irradiated cells were quantified relative to the respective unirradiated levels, normalized by Lamin B. (C) HeLa-DDB2 cells were transiently transfected with DDB1-V5, Cul4A-c-Myc or a combination of DDB1-V5 plus Cul4A-c-Myc for 48 h. The cultures were treated with 100 mg/ml of CHX, and then UV irradiated at 20 J/m , or mock treated and further incubated in the medium containing CHX for 1 h. The whole cell lysates were subjected to western blot analysis using anti-XPC, anti-DDB2, anti-V5, anti-c-Myc and anti-Lamin B antibodies. Relative XPC level in UV-irradiated cells were quantified relative to the respective unirradiated levels and normalized by Lamin B. Exogenously expressed DDB2-FLAG-HA; Endogenously expressed DDB2. Relative XPC level (%) Relative XPC level (%) Relative XPC level (%) Nucleic Acids Research, 2007, Vol. 35, No. 16 5345 knocking down the expression of DDB1 or Cul4A in A normal human fibroblasts caused an expected inhibition Position Sequence of the UV-induced DDB2 degradation. However, the K81 VAKVT VKSE NLKVI absence of DDB1 or Cul4A clearly enhanced the XPC K89 ENLKV IKDE ALSDG degradation. Interestingly, there was a simultaneous K113 KKAHH LKRG ATMNE reduction in the UV-induced XPC modifications. K183 ERSEK IKLE FETYL These results indicate that DDB1 and Cul4A are required K655 LKRHL LKYE AIYPE for the stabilization of XPC through an influence on its K917 EEKQK LKGG PKKTK protein modifications. To further confirm this finding, we tested if over-expression of DDB1 and Cul4A can protect XPC from degradation in another cell line, i.e. HeLa-DDB2 cells. It is worthy to note that HeLa Transfection cells have both DDB1 and DDB2 (41) and UV–induced UV (20 J/m , 1 h) XPC modification and degradation in HeLa cells are − +++++ −−−− similar to those of OSU-2 cells [(27) and unpublished modified XPC data]. We over-expressed either V5-tagged DDB1, XPC 125 kDa or c-Myc-tagged Cul4A or both DDB1 and Cul4A in HeLa-DDB2 cells. All transfections involving the LaminB over-expression of Cul4A promoted the degradation of 123456789 10 DDB2. Consistent with a previous report (42), this indicates the normal function of ectopically expressed Transfection Wt K655A K917A ∆ 1-117 Cul4A. Interestingly, over-expression of either DDB1 UV (20 J/m , 1 h) − + − + − + − + or Cul4A or of both DDB1 and Cul4A components in cells dramatically inhibit UV-induced XPC degra- XPC dation (Figure 4C). Taken together, these data Relative amount 1 0.52 1 1.10 1 0.71 1 0.65 indicate that both DDB1 and Cul4A can protect XPC Lamin B from being degraded upon UV irradiation and this effect is mainly through allowing the modifications of XPC protein. Figure 5. K655 is the critical site for UV-induced XPC modification and degradation. (A) Six putative SUMO sites in XPC protein predicted by SUMOplot are depicted. (B and C) Wild-type XPC Sumoylation and degradation involve the same site in and three mutant XPC (K655A, K917A and 1-117) were generated XPC protein and transiently co-transfected with DDB2-FLAG into XP-C cells. Twenty-four hours post-transfection, the cells were split into two plates Our previously published studies indicated that and grown for another 24 h, one plate was mock-irradiated and another UV-induced sumoylation of XPC inhibits its degradation was UV-irradiated at 20 J/m and allowed to repair for 1 h. The whole following UV irradiation (27), suggesting that sumoyla- cell lysates were subjected to immunoblotting using anti-XPC antibody. The blots in ‘B’ were exposed longer to show the modified tion site in XPC may be involved in XPC degradation. protein forms. Relative amounts of total XPC following UV irradiation In order to address this question, we needed to determine were quantified relative to the respective unirradiated levels and and manipulate the potential sumoylation sites in XPC normalized by Lamin B controls. protein. The linkage between SUMO and its target proteins occurs through an isopeptide bond between the C-terminal carboxyl group of SUMO and the e-amino sumoylation-specific lysine. Since immortalized XP-C group of a lysine residue in the substrate. The majority (GM15983) cells exhibited reduced DDB2 level, possibly of the sumoylation sites follow a consensus motif due to disrupted p53 via SV40 large T antigen (data not with c-K-X-E (43,44) or c-K-X-E/D (45), where c is a shown), the XPC constructs were transiently co- large hydrophobic amino acid, generally isoleucine, transfected with DDB2 into these XP-C cells and the leucine or valine; K is the lysine residue that is modified; cells were UV irradiated at 20 J/m followed by 1 h for X is any residue and D or E is an acidic residue. repair. The modified forms of XPC protein were detected This motif is bound directly by Ubc9, the sole SUMO– by western blot analysis. As shown in Figure 5B, conjugating enzyme. We used SUMOplot (http:// mutation of K655 to alanine (K655A) produced a www.abgent.com/doc/sumoplot) to predict the putative XPC that was unable to undergo modifications in vivo sumoylation sites in XPC protein. SUMOplot provides (lanes 5, 6), whereas mutation of K917 to alanine the probability of the SUMO consensus sequence (K917A) and deletion of 1–117 amino acids had no (SUMO-CS) potentially engaged in SUMO attachment. effect on the protein’s modification competence The SUMOplot analysis revealed six putative sumoyla- (lanes 7–10). This result indicates that K655 is the site tion sites in XPC protein, e.g. K81, K89, K113, K183, responsible for sumoylation and other modification of K655 and K917 (Figure 5A). In order to assess the valid XPC protein. Furthermore, we also tested the UV- sumoylation site in XPC, we either mutated the putative induced degradation prowess of various XPC forms. lysine to alanine (K655A and K917A), or used an Upon UV irradiation, ectopically expressed wild-type existing 1–117 amino acids deletion XPC construct XPC could be degraded to the same extent as endogen- (pXPC3, 1–117) (46) to experimentally test the possible ous XPC, whereas K655A XPC does not undergo any XPC-K655A Vector XPC-Wt XPC-K917A XPC(D1–117) 5346 Nucleic Acids Research, 2007, Vol. 35, No. 16 degradation (Figure 5C, lanes 1 and 2 versus 3 and 4). UV irradiation is a result of active XPC degradation. In contrast, other mutations such as K917A and 1-117 UV-induced XPC degradation occurs very early and can did not affect UV-induced XPC degradation (lanes 5–8), be seen for more than 2 h. In the meantime, as reported by suggesting once again that K655 is also an essential other groups, XPC expression is also induced so that the residue for the XPC degradation. new synthesis of XPC becomes an overwhelming event after 4 h and masks the decrease of XPC level invoked Blocking UV-induced XPC degradation compromises NER earlier. At this point, the cumulative measurement of the via inhibition of XPG recruitment to damage sites dual opposing effects is reflected as a net increase. Importantly, we show that UV-induced XPC degradation Since K655A mutation abrogates UV-induced XPC is not triggered by the typical protein ubiquitylation degradation, we used this construct to study the function process. The mechanistic studies reveal that 26S protea- of XPC degradation in NER following UV irradiation. some can directly bind XPC to affect its degradation. XPC-Wt or XPC-K655A constructs were transiently Ubiquitylation and sumoylation of XPC following UV co-transfected with DDB2 into XP-C cells, and the irradiation of cells is already established (26,27), albeit the characteristics of XPC, e.g. its binding to hHR23B and nature of the two independent modifications has not been its recruitment to damaged DNA sites were evaluated. fully resolved. The function of XPC ubiquitylation, which The result indicates that K655A mutation does not has also been studied extensively in vitro, is not for the affect the complex forming ability of XPC and hHR23B purpose of its degradation, but to augment DNA binding (data not shown). In addition, both XPC-Wt and of XPC. However, the function of XPC sumoylation has XPC-K655A could be recruited to CPD sites upon UV so far remained unclear. Since we have found that irradiation (Figure 6A). The recruitment of other NER inhibition of XPC sumoylation increases UV-induced factors, which are placed into the repair complex XPC degradation (27), it can be surmised that at least one subsequent to XPC, was also analyzed. TFIIH (XPB) function of XPC sumoylation is to protect XPC from and XPA exhibit the normal recruitment to the being destroyed. Therefore, XPC undergoes degradation UV-damage sites in both XPC-Wt and XPC-K655A and modifications simultaneously following UV irradia- expressing cells (Figure 6B and C). On the other hand, tion and in essence the degradation of XPC is intimately XPG protein, while recruited as normal to the damage regulated by modifications, i.e. more modifications result- sites in XPC-Wt expressing cells, was severely impaired in ing in lesser degradation. its damage site recruitment in XPC-K655A transfected With regards irradiation-related XPC protein induc- cells (Figure 6D). These results indicate that K655A tion, our data argues that XPG is required for this process mutation-induced abrogation of XPC degradation because, in the absence of XPG, the level of XPC does not hampers the recruitment of XPG to the damage sites. increase following UV irradiation. In addition, the We also evaluated the effect of XPC-K655A mutation on transfection of XPG into XP-G cells restores the XPC the efficiency of NER. XP-C cells with transiently increase after 4 h of UV irradiation. Because XP-A and expressed XPC-Wt or XPC-K655A were UV irradiated XP-F cells exhibit normal XPC degradation and induction at 10 J/m and allowed to repair for a 24 h period. The kinetics, we can rule out the possibility that blocking CPD remaining in DNA were quantified and the repair of XPC induction is due to transcription inhibition from rates compared among different cell types. Figure 6E un-repaired lesions located in the transcribed strand of the shows that the expression of XPC-Wt and XPC-K655 is XPC gene. Therefore, XPG may be an important factor in comparable in two transfected cell lines. As expected, DNA damage-induced XPC expression, and it would CPD were not repaired in XP-C cells transfected with be enlightening to unravel the role of XPG in XPC vector alone (Figure 6F). Moreover, the transfection of production. XPC-K655A was unable to restore the DNA repair ability of XPC cells like that achieved with the XPC-Wt construct. These data suggests that inhibition of XPC DDB–Cul4A components differentially regulate XPC degradation by K655A mutation severely affects the degradation function of XPC in NER. The DDB–Cul4A complex is a new class of cullin- containing ubiquitin E3 ligases (47). Previous studies have indicated that the DDB–Cul4A E3 ligase regulates DISCUSSION the autoubiquitylation and proteolysis of DDB2 in Modification, degradation and induction of XPC occur in response to DNA damage (42,48). In addition, tandem within irradiated cells DDB–Cul4A complex is also required for UV-induced The alterations of XPC levels in cells irradiated with UV ubiquitylation of XPC, but this modification does not have been reported either as no change (26), or as an serve as the signal for proteolysis. Nevertheless, our increase (29,30,33). Our previous work, however, detected present study demonstrates that the subunits of this a decrease in XPC level immediately upon UV irradiation E3 complex, DDB2, DDB1 and Cul4A, also regulate (27). Similarly, a study in Saccharomyces cerevisiae also UV-induced XPC degradation. These regulatory events, demonstrated that Rad4 is degraded upon UV irradiation however, serve different functions. DDB2 is required and (34). In the present study, we carried out an in-depth promotes XPC degradation upon UV irradiation, whereas mechanistic investigation of the fate of XPC and DDB1 and Cul4A protect XPC from being degraded. confirm that the observed decrease of XPC following DDB2 has been shown to be a critical factor in the Nucleic Acids Research, 2007, Vol. 35, No. 16 5347 AB Vector XPC-Wt XPC-K655A Vector XPC-Wt XPC-K655A XPC V5(XPC) CPD XPB Merge Merge CD Vector XPC-Wt XPC-K655A Vector XPC-Wt XPC-K655A XPC XPC XPA XPG Merge Merge F 120 Transfection XPC Lamin B 12 3 Empty vector XPC-Wt XPC-K655A 0 5 10 15 20 25 Repair time (h) Figure 6. K655A mutation compromises NER of UV-induced CPD through inhibiting XPG recruitment. (A–D) XP-C cells grown on coverslips were co-transfected with DDB2-FLAG and either wild-type or K655A mutant XPC for 48 h, then UV irradiated through a 5mm micropore filter at 100 J/m . After incubation for another 30 min, the cells were fixed, permeabilized and then subjected to dual immunofluorescent staining with rabbit anti-XPC and mouse anti-CPD antibodies (A), or mouse anti-V5 (for XPC) and rabbit anti-XPB antibodies (B), or rabbit anti-XPC and mouse anti- XPA antibodies (C), or rabbit anti-XPC and mouse anti-XPG antibodies (D). (E and F) XP-C cells were co-transfected with DDB2-FLAG and either wild type or K655A mutant XPC for 24 h, then the cells were split into five 60 mm plates and incubated for another 24 h. Whole cell lysates prepared from one plate of each transfection were subjected to immunoblotting using anti-XPC antibody to confirm the expression of XPC (E). Cells in other plates were UV irradiated at 10 J/m and allowed to repair for the indicated times. Genomic DNA was isolated and the identical amount of DNA was subjected to immuno-slot blotting using anti-CPD antibody to detect the CPD remaining in each samples (F). removal of CPD, most likely by allowing the recruitment its mutant forms, has the ability to trigger UV-induced of XPC to the damage sites (17,18). For instance, in XPC degradation. Since mutated DDB2 cannot bind to DDB2-deficient XP-E cells, XPC cannot be recruited to UV-damaged DNA, we propose that XPC degradation the damage sites and consequently XPC cannot be occurs at the damage sites, and the role of DDB2 in this degraded. In addition, only the wild-type DDB2, but not event is to help promptly recruit XPC to UV lesions. Wt K655A Vector Remaing of CPD (%) 5348 Nucleic Acids Research, 2007, Vol. 35, No. 16 DDB1 and Cul4A have been reported to be involved to 6-4PP in the absence of XPC degradation. Structural in the proteolysis of several proteins, such as DDB2 (42), analysis of DNA lesions has revealed that 6-4PP induces Kip1 significant helix distortion (54), including disruption of p27 (49) and CDT1 (50). However, in this study, we base pairing and this structural distortion accommodates demonstrate that DDB1 and Cul4A did not promote all needed proteins to allow the required assembly of the XPC degradation, but instead protect XPC from destruc- repair machinery. Thus, it seems that XPC degradation is tion by the proteasome. In addition, knocking down the not necessary here to make space for incoming XPG. expression of either DDB1 or Cul4A impairs UV-induced In contrast, the distortion induced by CPD is much less XPC modifications. In light of the earlier observation that pronounced (54,55). The DNA helix distortion induced by Ubc9 knockdown impaired UV-induced XPC modification CPD could be too subtle to render sufficient space for all while promoting its degradation (27), we conclude that the NER factors to simultaneously congregate at the both XPC ubiquitylation and sumoylation can prevent damage site. In this case, XPC–hHR23B complex will XPC degradation upon UV irradiation. The fact that XPC have to leave the damage site and, therefore, after serving modifications as well as degradation involve the same lysine the damage recognition function XPC is degraded to make the space needed for XPG recruitment. residue of the XPC protein reinforces this conclusion. In summary, this study demonstrates that XPC can be Based on this and other studies, we proposes that degraded independent of ubiquitylation upon UV irradia- DDB2 has two distinct functions in UV-induced XPC tion. The level of XPC is very important for cells to degradation. On the one hand, DDB2 helps the recruit- execute GGR, even though XPC degradation is necessary ment of XPC to the UV lesions and XPC has to undergo for efficient removal of CPD. Moreover, the UV-induced degradation to execute repair. On the other hand, DDB2 XPC degradation is controlled by XPC modifications to brings DDB–Cul4A E3 ligase to the damage sites to allow avoid excessive depletion of XPC from the cells. protective ubiquitylation of XPC so as to prevent its Meanwhile, XPC expression is also induced following degradation before repair is complete. Thus, the preven- UV irradiation so that the new synthesis replenishes the tion of inappropriate degradation of XPC by the DDB– depleted XPC to ensure the presence of sufficient XPC for Cul4A E3 activity enables XPC to execute its function in the upcoming rounds of damage removal. genomic repair. XPC degradation has an important role in NER SUPPLEMENTARY DATA In this study, we mutated the XPC K655 to alanine to Supplementary Data are available at NAR Online. understand the function of UV-induced XPC degradation in NER. Mutation at this site blocked both UV-induced XPC modifications as well as its degradation. As described ACKNOWLEDGEMENTS above, XPC sumoylation is believed to inhibit XPC degradation while XPC ubiquitylation is shown to We thank Drs Michael Tainsky, Yoshihiro Nakatani, Gan enhance the binding of XPC to damaged DNA as well Wang, Karlene Cimprich and Harvey Ozer for cell lines; as inhibit XPC degradation. It may be noted that Dr Yue Xiong for plasmids and antibodies; Drs Gilbert ubiquitylation of XPC was not found to promote the Chu and Andrew Paterson for plasmids and Drs Yue Zou, dual incision in a reconstituted NER reaction with Kelly Trego and John Turchi for purified XPC and purified proteins (26). Similarly, inhibition of XPC XPC–hHR23B proteins. Critical reading of the manu- sumoylation, by knockdown of Ubc9 expression, did not script by Dr Mark Parthun is gratefully acknowledged. affect the efficiency of NER (27). Therefore, it can be This work is supported by NIH grants ES2388, reasoned that the observed effect of K655A mutation on ES12991 and CA93413 to A.A.W. Funding to pay Open DNA repair is a consequence of eliminating its ability to Access publication charges for this article was provided by degrade XPC. National Institute of Health. It has already been reported that during assembly of Conflict of interest statement. None declared. NER factors, XPC–hHR23B and XPG cannot simulta- neously exist in the repair complex and that the entry of XPG into the complex coincides with XPC–hHR23B REFERENCES leaving the complex (51). In contrast, XPC–hHR23B and XPA–RPA complexes can simultaneously bind to distort- 1. De Laat,W.L., Jaspers,N.G. and Hoeijmakers,J.H. (1999) ing DNA lesions (52). 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Nucleic Acids Research – Oxford University Press
Published: Aug 9, 2007
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