p53 responsive nucleotide excision repair gene products p48 and XPC, but not p53, localize to sites of UV-irradiation-induced DNA damage, in vivo

Maureen E. Fitch, Irina V. Cross and James M. Ford1

Departments of Medicine and Genetics, Division of Oncology, 1115 CCSR Bldg, 269 Campus Drive, Stanford University Medical School, Stanford, CA 94305-5151, USA

1 To whom correspondence should be addressed Email: jmf{at}stanford.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The p53 tumor suppressor gene is an important mediator of the cellular response to ultraviolet (UV)-irradiation-induced DNA damage and affects the efficiency of the nucleotide excision repair (NER) pathway. The mechanism by which p53 regulates NER may be through its ability to act as a transcription factor, and/or through direct interactions with damaged DNA or the repair machinery. p53 has been shown to regulate the expression of the DDB2 gene (encoding the p48 protein) and the XPC gene, two important components of the NER pathway involved in DNA damage recognition. In this study, a localized UV-irradiation technique was used to examine the localization of p53, p48 and XPC proteins in relation to sites of UV photoproducts, in vivo. We did not observe any specific co-localization of p53 with sites of UV-induced DNA damage, but did observe rapid co-localization of both p48 and XPC to these sites. p48 bound to UV photoproducts in cells mutant or deficient for either p53, XPC or XPA, and p48 enhanced XPC binding to lesions, suggesting that p48 is a very early recognition factor of DNA damage. We propose that p53 functions to transcriptionally regulate the DDB2 and XPC NER genes, but does not activate the NER pathway through direct interactions with UV-induced damaged DNA or other repair factors.

Abbreviations: CPD, cyclobutane pyrimidine dimer; GGR, global genomic repair; NER, nucleotide excision repair; TCR, transcription coupled repair; Tet, tetracycline; UV, ultraviolet; 6–4PP, pyrimidine (6–4) pyrimidone photoproduct; UV-DDB, UV-damaged DNA binding factor; wt, wild-type; XP, xeroderma pigmentosum


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Preservation of genetic integrity is critical to an organism's survival, and complex repair pathways exist to remove any exogenously or endogenously introduced lesions from the DNA. Nucleotide excision repair (NER) is one highly conserved pathway that serves to remove several types of bulky lesions in DNA, including the major photoproducts induced by ultraviolet (UV) radiation, the cyclobutane pyrimidine dimer (CPD) and the 6–4 photoproduct (6–4PP) (reviewed in ref. 1). NER is the only pathway in humans that removes UV-induced photoproducts. Mutations in NER genes cause the inherited cancer-prone syndrome xeroderma pigmentosum (XP), and result in the developmental and neurological abnormalities seen in Cockaynes syndrome (CS) and trichothiodystrophy (2).

Characterization of the defects in XP cells, which can be grouped into eight complementation groups (XP-A through XP-G and XP-V), has led to a greater understanding of the biochemical events involved in NER. NER proceeds through two distinct, yet overlapping pathways; transcription coupled repair (TCR) that preferentially removes lesions from the transcribed strand of active genes (3), and global genomic repair (GGR) that removes lesions from the overall genome and non-transcribed strands (1). TCR is believed to be activated by the recognition of a stalled RNA polymerase II that acts to recruit the NER machinery (4). The damage recognition steps of GGR are not as well understood, but there are several candidate factors that may be responsible for sensing the damage that is repaired through GGR. The XPC gene product, together with hHR23B, forms a heterodimeric complex that has strong affinity for damaged DNA in vitro (5). Mutations or loss of XPC causes complete inhibition of GGR of both CPDs and 6–4PPs in vitro and in vivo, whereas TCR is unaffected (6,7). The XP-E phenotype is caused by mutations in the DDB2 gene (8). The protein product of this gene, p48, interacts with the p127 protein (the product of the DDB1 gene), and together form a UV-damaged DNA binding complex (termed UV-DDB) (9,10). UV-DDB is the most readily detectable UV-damaged DNA binding activity in extracts from human cells, and has a greater affinity for UV-damaged DNA substrates in vitro than does the XPC–hHR23B complex or XPA (5). However, the role that p48 and UV-DDB play in NER in vivo is not clear because UV-DDB is not required for NER in vitro (11), and cells with DDB2 mutations exhibit only partially diminished GGR rates in vivo, with CPD repair affected to a much greater extent than 6–4PP repair (12). UV-DDB does stimulate repair rates in vivo when it is microinjected into XP-E cells, and so its function has been proposed to be important for repairing damaged DNA in the context of chromatin (13). XPC–hHR23B also binds UV-damaged DNA with high affinity, and because loss of XPC completely inhibits GGR, it has also been proposed to be a DNA-damage recognition factor (14,15). Key questions remain about how these factors potentially interact with each other during the damage recognition step or activate the remaining enzymes involved in the repair machinery.

We and others have demonstrated that loss of the tumor suppressor protein p53 leads to decreased rates of GGR, but not TCR, following UV-C irradiation and affects CPD repair much more than 6–4PP repair (1618). This phenotype is reminiscent of p48 deficiency in XP-E cells, and we have in fact demonstrated that p53 regulates both basal and UV-inducible levels of DDB2 expression (12,19). We have also recently demonstrated that p53 regulates the expression of the XPC gene, again affecting both the basal and inducible levels (20). These data suggest that p53 regulates NER through its activities as a transcriptional regulator of genes involved in DNA damage recognition. However, p53 has also been shown to bind to certain repair factors, including XPB and XPD, as well as damaged DNA itself, thus suggesting that it may act as a direct repair factor (2123).

We have used the newly developed micropore filter local UV-irradiation technique to examine the in vivo localization of p53, UV-DDB and XPC after UV-C irradiation to address the question of whether or not these proteins are involved in binding lesions in vivo (24,25). This assay is a powerful method to explore protein localization after UV damage because only portions of the nucleus are irradiated through the micropore filter, while the remaining portion of the nucleus that is covered by the filter is blocked from the radiation. We have determined that p53 does not localize to sites of DNA damage at any time following UV irradiation, but that p48 and p127 do so very rapidly. XPC also localizes to lesions, but with apparently slower kinetics than p48. We further demonstrate that the presence of p48 can enhance the binding of XPC to sites of DNA damage and that p53 is not required for the in vivo localization of these proteins. We propose that both XPC and p48 are UV-damaged DNA recognition factors in vivo, and that p48 particularly functions to seek out CPDs that are located within chromatin structures by its very high affinity for damaged DNA.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell lines
All cells were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine and antibiotics, and were incubated at 37°C and 5% CO2. Early passage WI38 cells (Repository # GM01604A), which express wild-type (wt) p53, were obtained from the NIGMS Human Genetic Cell Repository (Coriell Institute for Medical Research, Camden, NJ). WI38 cells were maintained in media as described above, but with 20% FBS, vitamins and non-essential amino acids. 041 TR cells, a subclone of TR9-7 cells obtained from Dr George Stark (Cleveland Clinic Foundation, Cleveland, OH), were constructed from Li–Fraumeni Syndrome 041 human fibroblasts, mutant for p53, into which a tetracycline (Tet)-regulated system for expression of wt p53 was stably transfected (26). 041 TR cells were grown in the presence of 600 µg/ml G418 and 50 µg/ml hygromycin, and maintained in 2 µg/ml of Tet when suppression of wt p53 expression was desired. XP-C cells (XP1MI) have been described (27,28), as have XP-A cells (SV40 XP12RO) (29,30).

Antibodies
Primary antibodies used for immunoblot analysis were mouse monoclonal anti-p53 at a 1:2000 dilution (DO-1, Santa Cruz), mouse anti-p21 at 1:500 (#556430, Pharmingen, CA), mouse anti-tubulin at 1:15,000 (B-5-1-2, Sigma-Aldrich), and mouse anti-XPC at 1:5 (A gift of Eva Lee, University of California, Irvine, CA). Secondary antibody was goat anti-mouse IgG conjugated to HRP diluted 1:5000 (Pierce Biotechnologies, IL). Primary antibodies for immunofluorescence were mouse monoclonal anti-CPD at 1:1500 [TDM2, a gift from Toshio Mori, Nara Medical University, Nara, Japan (31)], rabbit polyclonal anti-p53 at 1:100 (FL393, Santa Cruz, CA), and mouse anti-V5-FITC conjugated at 1:500 (Invitrogen, CA). Secondary antibodies were Alexa Fluor 594 goat anti-mouse and Alexa Fluor 488 goat anti-rabbit, both used at 1:500 (Molecular Probes, OR).

Immunoblot
Briefly, cells were grown in a 150 mm dish, washed with PBS, and then some dishes were covered by a 150 mm 3 µm polycarbonate filter (Millipore, MA). The cells were irradiated with the indicated UV dose, the filter was removed, media was replaced, and the cells were returned to the incubator for 16 or 48 h. Cells were harvested in 150 mM NaCl, 10 mM Tris pH 7.4, 5 mM EDTA pH 8.0, 1% Triton X-100, 0.5 mM Pefabloc, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µM DTT. Lysates were incubated on ice for 1 h, then sonicated for 20 min at 4°C in a Branson 1510 water bath sonicator. Debris was spun out by 14 000 g spin for 10 min. Protein concentration was quantified by the BCA kit (Pierce). Fifty micrograms of freshly boiled lysate was loaded onto a 12% reducing polyacrylamide gel, transferred to Hybond ECL paper (Amersham Biosciences, NJ), probed with the indicated antibodies, visualized by chemiluminescence (Super Signal, Pierce) and exposure to autoradiography film (Eastman Kodak Company).

Immunoslotblot analysis of CPD lesions
Cells were irradiated at the indicated doses in a similar manner as for the immunoblot analysis. Cells were immediately lysed after UV irradiation in 10 mM Tris–HCl, pH 8.0, 1 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K and 0.1 mg/ml RNase. Genomic DNA was isolated by phenol–chloroform extraction, followed by ethanol precipitation, and the concentration was determined. Twenty-five nanograms of sheared genomic DNA was denatured and slotted onto Hybond N+ nylon membrane (Amersham Biosciences) and CPDs were detected by monoclonal antibody as described previously (16,32). Images were analyzed by Quantity One software (Bio-Rad, CA). The relative amounts of CPDs were normalized to the amount of CPDs in the 20 J/m2 sample.

UV irradiation
For local UV irradiation, cells were grown overnight on glass coverslips. Prior to irradiation, the media was aspirated, and the cells were washed in PBS. For every experiment using localizing irradiation, an isopore polycarbonate filter of 3 µm size (Millipore) pre-soaked in PBS was placed over the cells, and the cells were irradiated through the filter with varying doses of UV-C (predominantly 254 nm wavelength) from a germicidal lamp calibrated to deliver 10 J/m2/s. The membrane was removed and the cells were either fixed directly after UV, or medium was replaced and the cells put back in the 37°C incubator for the indicated times.

Immunofluorescence
Cells were grown as indicated on coverslips in a 35 mm dish, washed in PBS, then fixed by 2% formaldehyde in 0.2% Triton X-100/PBS for 10 min on ice. Cells were washed 3x in PBS, then the DNA was denatured by incubation in 2 N HCl for 5 min at 37°C. Cells were incubated in 20% FBS in washing buffer (WB-0.1% Triton X-100 in PBS) for 30 min at room temperature to block non-specific binding. Primary and secondary antibodies were made up in 1% BSA in WB and incubated for 45 min at room temperature. After each antibody step, cells were washed three times for 5 min in WB. When staining for both CPD and the V5 epitope-tagged proteins, a second blocking step of 5 µg/ml mouse IgG (Sigma) was added for 30 min after the CPD and the goat-anti mouse antibodies had been incubated, to block non-specific interactions between them and the V5 antibody. Anti-V5 FITC conjugated antibody was added after the IgG step and incubated for 45 min at room temperature. Coverslips were mounted in VectaShield with DAPI (Vector Laboratories, CA). Images were captured by a Nikon Eclipse E800 microscope using an RT Slider CCD camera (Diagnostic Instruments, MI), analyzed by Spot RT 3.0 software (Diagnostic Instruments) and further adjusted in Adobe Photoshop 6.0.

Transfections
Cells were plated on coverslips in a 35 mm dish 24 h prior to transfection. The DDB2 full-length cDNA (-20 to 1281 bp not including the final stop TGA) was cloned in frame into the pcDNA3.1 vector (Invitrogen) to pick up the coding sequence of the V5 and His epitope tags at the C-terminus by the Invitrogen TOPOTM method. The cDNA was generated by RT–PCR using RNA isolated from GM38 normal human fibroblasts. XP2RO and XP82TO mutations were introduced into the DDB2 gene by the use of the QuikChangeTM Site Directed mutagenesis kit (Stratagene, CA). To generate the 2RO mutation, codon 273 was changed from CGC to CAC (R>H); for the 82TO mutation, codon 244 was changed from AAA to GAA (K>E). The full-length p127 (-12 to 3432 bp) and full-length XPC (-15 to 2820 bp) cDNAs were cloned into the same vector in a similar manner. p127 cDNA was a gift from Gilbert Chu (Stanford University). XPC cDNA was generated by RT–PCR using RNA from WI38 normal human fibroblasts. All transfections were done using Lipofectamine 2000 (Invitrogen) according to their instructions. When performing double transfections, the p127-V5 or XPC-V5 expressing vectors were transfected with a 5-fold excess of a p48 cDNA (no tag) in a pcDNA3.1 vector. Cells were irradiated 24 h post-transfection.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
p53 activation after local UV irradiation
We first established whether p53 is activated by the amount and distribution of DNA damage induced through the micropore filter following UV-C irradiation. The amount of DNA damage induced by varying amounts of UV irradiation delivered through a 3 µm isopore filter was quantified by immunoslotblot analysis of total genomic DNA using a monoclonal antibody against the CPD, and expressed as a percentage of that resulting from 20 J/m2 UV delivered without a filter (Figure 1A). A UV dose of 100 J/m2 delivered through the filter induced fewer lesions per cell equivalent than a dose of 20 J/m2 without a filter. The average number of CPDs increased proportionally with the dose, such that 400 J/m2 UV through the filter induced just over 100% of the lesions observed from 20 J/m2. We examined p53 protein stabilization after local irradiation in p53 wt WI38 normal human fibroblasts using an immunoslotblot analysis (Figure 1). p53 protein levels increased in proportion to the total number of UV-induced lesions per cell, with similar p53 levels observed following 400 J/m2 under the filter and 20 J/m2 without a filter at both 16 and 48 h post-irradiation. Levels of the p53-responsive gene product p21 (Cip1/Waf-1) also rose in a dose-dependent manner at both time points examined. Levels of the p53-responsive gene product XPC did not change greatly at the 16 h time point (data not shown). However, by 48 h, we observed a significant increase in XPC protein levels in both the 20 J/m2 dose and the 400 J/m2 dose under the filter (Figure 1B). This late induction is similar to our previous results in WI38 primary human fibroblasts (20). Stabilization of p53 and up-regulation of these known p53-response genes are strong indicators that the p53 activation mechanism that responds to DNA damage is activated by local irradiation, and that the mechanism responds proportionally to the total number of lesions in the cell, not their overall distribution.



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Fig. 1. Response of p53 to local DNA damage in normal human fibroblasts. WI38 cells were irradiated without (lane 2) or with a 3 µm filter at the indicated dose [lanes 3–6 in (A) and * in (B)]. Cells were harvested (A) 16 h post-UV or (B) 48 h post-UV, and lysates were analyzed by immunoblot to detect p53, p21, XPC and tubulin levels after UV irradiation. Images were compiled using Adobe Photoshop. The relative number of CPDs was quantified by immunoslotblot as described in the Materials and methods.

 
p53 localization after local UV irradiation
We next examined p53 localization in vivo after UV damage in WI38 cells. A dose of 300 J/m2 was chosen to irradiate the cells through a 3 µm filter because that combination activated p53 sufficiently to detect by immunofluorescence, and the immunoblot analysis confirmed that this dose increased p53 levels and increased levels of known p53 response genes (Figure 1). UV-damaged sites in the nucleus were visualized by a monoclonal antibody to the CPD, and cells typically contained three to 10 discrete irradiated sites that were consistent with the size of the pores in the 3 µm filter. p53 was undetectable by immunofluorescence prior to UV irradiation, presumably because the levels were low in unstimulated cells, and its location was primarily cytoplasmic (data not shown). Once nuclear p53 levels rose to high enough levels to detect by immunofluorescence, between 6 and 20 h post-UV, we were able to assess if p53 localized to areas of damaged DNA to a greater degree than non-damaged areas (Figure 2A). Six hours following UV irradiation, p53 distributed in a punctate pattern in the nucleus, but did not accumulate to a greater degree in areas that had been irradiated, as indicated by the staining pattern of the CPD antibody (Figure 2A). This suggests that p53 does not specifically interact with UV photoproducts or associated NER repair complex proteins or intermediates at this time point. Because p53 was not detected at early times after UV in WI38 cells, and a significant amount of repair occurs soon after UV irradiation, we examined the localization of wt p53 in 041 TR cells, a human cell line derived from a Li–Fraumeni patient that is mutant for p53, but that contains a stably integrated Tet-regulated wt p53 gene (26). We have shown previously that 041 TR cells induce NER and p53-dependent transcriptional regulation of the DDB2 and XPC genes following removal of Tet (12,16,20). Tet was removed from the cells 20 h prior to UV irradiation, and p53 localization examined immediately after UV, and at several time points thereafter. No localization of p53 to areas of damage occurred either 15 min (Figure 2B) or 2 h (Figure 2C) after irradiation, but a punctate pattern following UV irradiation was observed. The levels of p53 were higher in this cell line due to induced overexpression in comparison with the 6 h time point in WI38 cells, and thus the punctate pattern appeared more pronounced over that observed in WI38 cells. These foci were not distributed evenly throughout the nucleus, but rather appeared diminished and even absent from areas of the nucleus that had been irradiated (Figure 2B and C). To verify that the pattern of distribution observed was characteristic of functional p53, we also examined the localization of mutant p53 after UV irradiation. Li–Fraumeni 087 fibroblasts, that constitutively overexpress a mutant p53 protein containing a point mutation at amino acid position 248 that renders it incapable of specific DNA binding (33), were irradiated with 300 J/m2 through a filter and again examined for localization of p53 at various times after UV. At several time points examined, the mutant p53 was distributed throughout the nucleus, and there was no specific accumulation seen at UV-irradiated sites, nor did we observe the bright foci appearance (Figure 2D). Taken together, our results with cells expressing wt and transcriptionally deficient p53 suggest that following localized UV irradiation, wt p53 is activated, and may function as a transcription factor at foci, but does not localize to sites of DNA damage where transcription is transiently inhibited.



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Fig. 2. p53 does not localize to sites of UV damage. (A) WI38 normal human fibroblasts were fixed 6 h after 300 J/m2 UV delivered through a 3 µm filter. (B and C) 041 TR cells were induced to express p53 20 h prior to UV-irradiation by the withdrawal of Tet. Cells were fixed (B) 15 min or (C) 2 h post 300 J/m2 UV delivered through a 3 µm filter. (D) 087 fibroblasts mutant for p53 were fixed 2 h after 300 J/m2 UV delivered through a 3 µm filter. UV irradiated sites were visualized by an antibody to the CPD. All fluorescent images were compiled in Adobe Photoshop.

 
Co-localization of the p53 target gene product p48 to sites of UV-induced DNA damage
We next examined if several known NER proteins localized to sites of UV damage, and in particular the p53 responsive gene products p48 and XPC. To examine p48 protein localization, we established a human 041 cell line null for p53 that stably expressed a V5 epitope-tagged DDB2 cDNA (clone 041-p48.6) (33a). We have shown previously that this cell line contains lower levels of endogenous DDB2 mRNA in comparison with WI38 fibroblasts, and that the mRNA levels do not change following UV-C irradiation (12). Following UV irradiation through a 3 µm filter with 200 J/m2, p48 was observed to immediately co-localize with UV-induced damage sites, and remained associated with lesions for over 90 min (Figure 3A, and data not shown). By 2 h, the association of p48 with UV lesions was no longer detectable by immunofluorescence. We have observed by western blotting that p48 protein levels decrease significantly in 041-p48.6 cells within 2 h of UV irradiation, and that this loss can be attenuated by the addition of proteasome inhibitors (33a, 34). Therefore, we cannot be certain at times >90 min if p48 is no longer bound to lesions, or is simply undetectable because the majority of the protein has been degraded. We also examined the localization of p48 to sites of DNA damage in NER deficient cell lines using transient transfections with a V5-tagged DDB2 cDNA in a pcDNA 3.1 expression vector. Transfected p48 co-localized to sites of damage in both XP-C cells and XP-A cells immediately after UV irradiation and remained associated with the lesions for up to 2 h (Figure 3B and C and data not shown). Notably, both of these cell lines are also SV40 T antigen transformed, and so functionally p53 deficient as well, confirming the results from the 041 cells that p48 does not require wt p53 protein to associate with UV lesions. The results in these cell lines also demonstrate that p48 does not require XPC or XPA to bind to lesions.



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Fig. 3. p48 associates with UV lesions immediately after UV irradiation. (A) 041-p48.6 cells stably expressing p48 were fixed immediately after 200 J/m2 UV delivered through a 3 µm filter. (B) XP-C and (C) XP-A cells were transfected with a DDB2-V5 cDNA 24 h prior to UV treatment and then fixed immediately after UV-irradation. p48 was visualized by an antibody to the V5 epitope.

 
Clinically occurring mutants of DDB2 do not associate with UV lesions
XP2RO and XP82TO are XP-E cell lines that each has a point mutation in the DDB2 gene (R273H and K244E, respectively) that affects several known p48 functions. For example, both 2RO and 82TO mutant p48 proteins are greatly diminished in their ability to induce the nuclear import of p127 (35,36), and both display no UV-DDB activity in vitro (37). We further show that they fail to bind UV-lesions in vivo (Figure 4). The localization of each mutant protein was examined at several time points post-UV, and no specific co-localization with lesions was observed, although as described previously (35,37), these proteins do readily localize to the nucleus (Figure 4A and B).



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Fig. 4. p48 mutant proteins do not bind to UV lesions. XP-C cells were transfected 24 h prior to 200 J/m2 UV-irradiation delivered through a 3 µm filter with either a p48 mutant (A) 2RO or (B) 82TO cDNA expressing plasmid. Cells were fixed 30 min after 200 J/m2 UV delivered through a 3 µm filter. Both mutant p48 proteins had a C-terminal V5 epitope tag.

 
p127 requires p48 for efficient binding to UV lesions
We next examined the localization of p48's heterodimeric binding partner, p127, following UV-induced DNA damage. XP-A cells were transiently transfected with a V5-tagged p127 cDNA, irradiated through a 3 µm filter with 200 J/m2, and examined for localization of p127 at several time points post-UV (Figure 5A). We did not observe any specific co-localization of p127 with CPDs at any time point after UV irradiation. The XP-A cells used in this study were SV40 T-antigen transformed, and thus functionally p53 deficient. Because p53 is known to affect the basal as well as inducible expression levels of DDB2 (12), we examined the basal DDB2 mRNA levels in these cells. DDB2 mRNA levels were lower in these cells in comparison with wt fibroblasts (data not shown), and thus we inferred that endogenous p48 protein levels may also be diminished. p48 has been shown to enhance import of p127 into the nucleus (35), and so p127 may not be present in the nucleus in sufficient enough levels to detect co-localization with UV lesions in these cells. To potentially enhance any binding activity of p127, we performed co-transfection experiments of both DDB1 and DDB2 into XP-A cells. When p48 was overexpressed, p127 readily co-localized with lesions within minutes (Figure 5B). As expected, the 2RO and 82TO mutants of p48 were not able to complement p127 binding to lesions when both were transfected into cells (data not shown).



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Fig. 5. p127 associates with DNA damage when p48 is present in sufficient levels. XP-A cells were transfected with p127-V5 only (A) or with p127-V5 and DDB2 (B). Cells were UV-irradiated with 200 J/m2 24 h post-transfection and fixed 10 min post-UV. p127 was visualized by an antibody to the V5 epitope present on the transfected p127.

 
Characterization of XPC binding to UV lesions
Volker et al. have reported previously that XPC binding occurs within 15 min in XP-A cells after 30 J/m2 delivered through a 3 µm filter (14). We used a much higher dose of 200 J/m2 through a 3 µm filter and observed co-localization of XPC with UV lesions in XP-A cells within 5 min of irradiation (data not shown). However, we did not observe detectable co-localization immediately after UV like that seen for p48 (Figure 6A). To determine if XPC localization, like p127, could be accelerated by increasing the amount of p48 in these cells, we performed co-transfection studies with XPC and DDB2. When p48 was overexpressed along with XPC in XP-A cells, we observed an enhancement of XPC binding to lesions, so that there was significant XPC binding detectable immediately post-UV in XP-A cells (Figure 6B). We observed the same effects of XPC binding when it was transfected into an XP-C cell line. XPC transfected alone did not co-localize to UV lesions immediately after UV irradiation (Figure 6C). When p48 was co-transfected with XPC into XP-C cells, we observed XPC binding to lesions immediately post-UV like what was observed in XP-A cells (Figure 6D), confirming that p48 can enhance XPC binding to UV-induced lesions.



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Fig. 6. p48 enhances XPC binding to UV lesions. XP-A cells transfected with either (A) XPC-V5 only or (B) XPC-V5 and DDB2; XP-C cells transfected with either (C) XPC-V5 only or (D) XPC-V5 and DDB2. All cells were irradiated with 200 J/m2 UV 24 h post-transfection and then fixed immediately after UV-irradiation. XPC was visualized by a V5 epitope on the transfected XPC.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have used the newly developed technique of micropore filter mediated local UV irradiation to directly examine the localization of several important proteins involved in NER to areas of UV-induced DNA damage in vivo. p53 is an important contributor to the cell's response to UV damage, and yet its role in DNA repair is not fully understood. It has been proposed that p53 can directly bind to damaged DNA through its non-specific DNA binding domain, and/or that it can directly interact with other DNA repair factors. There is evidence that p53 activates base excision repair (BER) through such direct interactions (38). Addition of p53 to repair lysates can stimulate BER in vitro, and p53 interacts through its N-terminus with the BER enzymes DNA polymerase ß, and the 5' AP endonuclease APE1 (39,40). In contrast, p53 does not stimulate NER in vitro when added to lysates (41,42). p53 interacts in vitro with XPB, XPD (components of the transcription factor TFIIH), and CSB (21); however, deficiencies in these proteins lead to defects in TCR. We have shown previously that cells deficient in p53 do not show significant defects in their TCR pathway following UV-C irradiation, and so interactions with these proteins do not readily explain p53's primary role in global genomic NER (17). Several studies have suggested that p53 has an affinity for damaged or altered DNA structures, but these studies used cell extracts on artificial substrates in vitro (22,23). A study by Jackson et al. (43) examined p53 localization after whole cell UV irradiation using a biotin-dUTP labeling system to indicate sites of DNA repair. Their data were suggestive that p53 does not localize to sites of NER. However, because the whole cell was irradiated, the experiments were difficult to interpret. We show very clearly by the use of local UV irradiation of portions of the cell nucleus that p53 does not localize specifically to sites of UV-induced DNA damage. On the contrary, we see a relative lack of p53 protein in areas of chromatin that have recently been irradiated. A study by Moné et al. (25) using this technique showed that transcription is almost completely inhibited in areas that have been irradiated, while chromatin outside of the damaged areas continue to support normal levels of transcription. In fact, our data (Figure 1) suggest that the transcriptional response to UV irradiation is different following local DNA damage than when a similar number of lesions are distributed uniformly throughout the nucleus. For example, Figure 1 demonstrates higher p21 levels in cells 16 h following irradiation with 400 J/m2 under the filter in comparison to cells irradiated uniformly with 20 J/m2, even though the p53 levels are lower in the locally irradiated cells. This pattern is also observed for XPC levels 48 h post-irradiation. This may reflect that although the absolute number of lesions is comparable between the uniformly irradiated cells and the locally irradiated cells, the damage incurred through the filter is concentrated in discrete regions of the nucleus, leaving the majority of active chromatin without DNA damage and unhindered to perform transcription. However, XPC levels were not different between local and uniform irradiation at 16 h, and p21 levels were higher in the uniform irradiated cells at 48 h than the locally irradiated. Therefore, whether these observations are due to differential activation of transcription, or effects from differing damage in genomic regions will require further investigation. Taken together, the data strongly suggest that p53 functions primarily as a transcription factor after UV-induced DNA damage, and not as a UV damage recognition factor, and further that, like TFIIH, it is responsive to the mechanisms in the cell that prevent transcription in areas that have been damaged.

We have further characterized the initial cellular activities of several p53-regulated NER proteins, namely the UV-DDB complex made up of p48 and p127, and XPC. We find that p48 binds very rapidly to DNA lesions induced by 200 J/m2 through a 3 µm filter and can be detected at these sites immediately after irradiation, and up to 90 min later. The immediate binding of p48 to UV lesions occurs regardless of the presence of p53 or of the other repair factors XPC or XPA, consistent with a previous report by Wakasugi et al. (44). p127 was also found to localize immediately to sites of DNA damage, but only in the presence of high levels of p48. As there are no known p127 deficient cell lines, we did not determine if p48 equally requires p127 for DNA binding. However, we speculate that either p48 alone binds to UV lesions and then recruits p127, or that together, p48 and p127 bind to lesions. In vitro evidence would support the latter hypothesis, as p48 by itself does not have as much UV-DDB activity as p48 with p127 (37). The fact that the p48 2RO and 82TO mutant proteins, which do not efficiently bind to p127, fail to bind to UV lesions also suggests that UV-DDB complex formation is needed for binding to lesions. p127 is not induced after UV irradiation like DDB2 (12), and so regulation of p48 levels by p53, and possibly the proteasome [(34) and unpublished data], may suffice for control of this step of DNA repair.

We have further characterized XPC binding to UV lesions. We observed XPC localization to sites of DNA damage in XP-A cells within 5 min of irradiation, yet this clearly occurred with slower kinetics than that observed for p48. Overexpression of p48 accelerated the binding of XPC to lesions by several minutes. This suggests that XPC by itself is fully capable of binding lesions, yet p48/UV-DDB is also a recognition factor that has some activity that can stimulate XPC binding. There is conflicting evidence in the literature about UV-DDB's ability to stimulate NER in vitro. One of the first NER reconstitution experiments using purified components by Aboussekhra et al. (11) saw little effect with the addition of UV-DDB to the other core NER components on the repair of a UV-damaged substrate. A more recent study has demonstrated that addition of recombinant UV-DDB to the other core components stimulated excision of a CPD by up to 17-fold, but had little or no effect on a 6–4PP containing substrate (44). We now demonstrate that p48 can stimulate XPC binding to lesions in vivo. Taken together with the in vitro data, our results suggest that UV-DDB may stimulate the rate of GGR by increasing the recognition of UV lesions, and in particular, CPDs. UV-DDB may increase recognition through its actions as a chromatin remodeling factor, for which there is accumulating evidence based on the phenotype of p48 deficient cells, and UV-DDBs ability to stimulate repair of a nucleosome bound damaged substrate (13). 6–4PPs occur with much less frequency in nucleosome bound DNA than CPDs, thus requiring less remodeling for their repair (45). 6–4PPs are more distorting to the overall structure of DNA than CPDs, and so 6–4PPs may be recognized easier and not require a specialized recognition factor that can work in the context of chromatin structure. The enhanced XPC binding we observed could be due to p48 facilitating a more open conformation of the chromatin around UV lesions, CPD's in particular, thus allowing greater access to the lesions.

In this study, an antibody to CPDs was used to identify the areas that have been UV-irradiated. However, 6–4PPs will also be found at these sites depending on the time post-UV and the repair capabilities of the cell type. Therefore, we cannot conclude which lesion is the primary recruiting factor for p48 or XPC. Repair of 6–4PPs is almost complete after several hours in normal cells, while complete CPD removal can take over 24 h. As we indicated, detectable p48 remains associated with the lesions for only a few hours in repair proficient cells, perhaps suggesting that 6–4PPs are the major recruiting lesion. p48 and XPC both bind 6–4PPs with more avidity than CPDs in vitro (46). It will be of interest to learn if there is a difference in binding preferences of the proteins to the different photoproducts in vivo.

One interesting question that remains to be answered regarding the role of p48 in NER is why DDB2 basal levels are typically very low and are inducible following UV irradiation, and why p48 is degraded in response to UV irradiation. This degradation potentially occurs due to the ability of p48 to interact with the specific E3 ubiquitin ligase Cul-4A (47,48). Taken together, one can speculate that the activities of p48 are tightly regulated and potentially harmful to the cell if left unchecked. For example, p48 may have non-specific DNA binding activity leading to gratuitous repair replication and the potential for mutagenesis. If UV-DDB is a chromatin remodeling factor, perhaps it can cause non-specific ‘loosening’ of chromatin structures that potentially lead to a general deregulation of gene expression because many genes are controlled by their level of chromatin structure. UV-DDB has also been proposed to have transcription activating activities through its ability to interact with E2F1 (35), and these activities may need to be tightly regulated. Another possible explanation for the degradation of p48 is that this process is somehow integral to facilitating NER. Intriguingly, XPC and hHR23B levels have also recently been suggested to be regulated by the proteasome (49), lending further credence to the idea that the proteasome may function in regulating NER, and that replenishment of the levels of these proteins is critical to completion of timely repair.

Another interesting question is how p53 regulated transcription of NER genes is important to the cells response to damage. p53 appears to regulate basal levels of p48 and XPC independent of UV-inducible responses, maintaining higher levels of these gene products in wt cells for immediate use in the early steps of NER (12,19,20). p53's induction of p48 and XPC following DNA damage is somewhat late in the UV response, typically reaching maximal levels for both after 24 h. However, transcriptional induction of p48 and XPC in response to UV may help maintain a critical cellular level of these proteins that have been depleted by proteasomal degradation at sites of DNA damage. In addition, it may also function to ensure recognition of any lingering CPD lesions that have as yet gone unrepaired, and in particular those lesions remaining in less accessible regions of chromatin. Understanding the biochemical functions of UV-DDB and XPC in NER and chromatin remodeling will lead to a greater understanding of the need for transcriptional control of DNA repair genes by factors like p53.


    Acknowledgments
 
We thank Drs Phil Hanawalt and Ann Ganesan for their helpful discussions and critical reading of this manuscript. We thank Anne Tecklenberg and Janet Jin for technical assistance. This work was supported by an American Cancer Society Postdoctoral fellowship (to M.E.F.) and by a National Institutes of Health Award RO1 CA83889, a Sidney Kimmel Foundation for Cancer Research Scholar Award, and a Burroughs Wellcome Fund New Investigator Award in Toxicological Sciences (to J.M.F.).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received November 12, 2002; revised February 11, 2003; accepted February 21, 2003.