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• W2062150992 abstract "Article7 September 2006free access New functions of XPC in the protection of human skin cells from oxidative damage Mariarosaria D'Errico Mariarosaria D'Errico Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Eleonora Parlanti Eleonora Parlanti Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Massimo Teson Massimo Teson Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy Search for more papers by this author Bruno M Bernardes de Jesus Bruno M Bernardes de Jesus Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM, Illkirch, CU, Strasbourg, France Search for more papers by this author Paolo Degan Paolo Degan Istituto Nazionale per la Ricerca sul Cancro, Department of Translational Oncology, Genova, Italy Search for more papers by this author Angelo Calcagnile Angelo Calcagnile Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Pawel Jaruga Pawel Jaruga Chemical and Biochemical Engineering Department, University of Maryland Baltimore County, Baltimore, MD, USA Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA Search for more papers by this author Magnar Bjørås Magnar Bjørås Department of Molecular Biology, Institute of Medical Microbiology and Centre of Molecular Biology and Neuroscience, University of Oslo, Rikshospitalet Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Marco Crescenzi Marco Crescenzi Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Antonia M Pedrini Antonia M Pedrini Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy Search for more papers by this author Jean-Marc Egly Jean-Marc Egly Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM, Illkirch, CU, Strasbourg, France Search for more papers by this author Giovanna Zambruno Giovanna Zambruno Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy Search for more papers by this author Miria Stefanini Miria Stefanini Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy Search for more papers by this author Miral Dizdaroglu Miral Dizdaroglu Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA Search for more papers by this author Eugenia Dogliotti Corresponding Author Eugenia Dogliotti Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Mariarosaria D'Errico Mariarosaria D'Errico Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Eleonora Parlanti Eleonora Parlanti Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Massimo Teson Massimo Teson Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy Search for more papers by this author Bruno M Bernardes de Jesus Bruno M Bernardes de Jesus Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM, Illkirch, CU, Strasbourg, France Search for more papers by this author Paolo Degan Paolo Degan Istituto Nazionale per la Ricerca sul Cancro, Department of Translational Oncology, Genova, Italy Search for more papers by this author Angelo Calcagnile Angelo Calcagnile Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Pawel Jaruga Pawel Jaruga Chemical and Biochemical Engineering Department, University of Maryland Baltimore County, Baltimore, MD, USA Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA Search for more papers by this author Magnar Bjørås Magnar Bjørås Department of Molecular Biology, Institute of Medical Microbiology and Centre of Molecular Biology and Neuroscience, University of Oslo, Rikshospitalet Radiumhospitalet HF, Oslo, Norway Search for more papers by this author Marco Crescenzi Marco Crescenzi Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Antonia M Pedrini Antonia M Pedrini Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy Search for more papers by this author Jean-Marc Egly Jean-Marc Egly Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM, Illkirch, CU, Strasbourg, France Search for more papers by this author Giovanna Zambruno Giovanna Zambruno Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy Search for more papers by this author Miria Stefanini Miria Stefanini Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy Search for more papers by this author Miral Dizdaroglu Miral Dizdaroglu Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA Search for more papers by this author Eugenia Dogliotti Corresponding Author Eugenia Dogliotti Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy Search for more papers by this author Author Information Mariarosaria D'Errico1, Eleonora Parlanti1,‡, Massimo Teson2,‡, Bruno M Bernardes de Jesus3,‡, Paolo Degan4, Angelo Calcagnile1, Pawel Jaruga5,6, Magnar Bjørås7, Marco Crescenzi1, Antonia M Pedrini8, Jean-Marc Egly3, Giovanna Zambruno2, Miria Stefanini8, Miral Dizdaroglu6 and Eugenia Dogliotti 1 1Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Rome, Italy 2Laboratory of Molecular and Cell Biology, Istituto Dermopatico dell'Immacolata, IRCCS, Rome, Italy 3Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS/INSERM, Illkirch, CU, Strasbourg, France 4Istituto Nazionale per la Ricerca sul Cancro, Department of Translational Oncology, Genova, Italy 5Chemical and Biochemical Engineering Department, University of Maryland Baltimore County, Baltimore, MD, USA 6Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD, USA 7Department of Molecular Biology, Institute of Medical Microbiology and Centre of Molecular Biology and Neuroscience, University of Oslo, Rikshospitalet Radiumhospitalet HF, Oslo, Norway 8Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, Italy ‡These authors contributed equally to this work *Corresponding author. Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel.:+39 06 4990 2580; Fax: +39 06 4990 3650; E-mail: [email protected] The EMBO Journal (2006)25:4305-4315https://doi.org/10.1038/sj.emboj.7601277 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA-distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP-C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA-oxidizing agents and this effect is reverted by expression of wild-type XPC. Upon oxidant exposure, XP-C primary keratinocytes and fibroblasts accumulate 8,5′-cyclopurine 2′-deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8-hydroxyguanine (8-OH-Gua) is also observed. We demonstrate that XPC–HR23B complex acts as cofactor in base excision repair of 8-OH-Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC-HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP-C patients. Introduction Different types of damage require different repair mechanisms but several lines of evidence indicate that functional overlap between pathways occurs frequently to guarantee genomic stability. An example is the repair of oxidative DNA damage, which is a ubiquitous type of damage. Oxidative stress from ionizing radiations (IR) and oxidants, and normal cellular metabolism cause oxidative damage to bases and sugar-phosphates, as well as single- and double-strand breaks in DNA and formation of DNA–protein crosslinks (reviewed in Evans et al, 2004). Base oxidation products are cytotoxic and mutagenic, and several studies suggest that they may contribute to ageing and human pathologies, including neurodegeneration and cancer (reviewed in Evans et al, 2004). Base excision repair (BER) is the major mechanism for repair of DNA base damage by reactive oxygen species (ROS), but other repair pathways like NER, mismatch repair and recombination may be involved too. Specific DNA glycosylases are responsible for modified base removal by BER followed by formation of a single-stranded gap, filling of the gap and ligation by either short- or long-patch BER (reviewed in Fortini et al, 2003). The key role of BER in oxidative DNA damage repair is testified by the high risk of colorectal tumours in individuals carrying inherited mutations of the MYH DNA glycosylase that specifically excises adenine mispaired with 8-OH-Gua (reviewed in Sampson et al, 2005). The major function of NER is to repair a wide range of lesions, including UV photoproducts, all resulting in large local distortions of the DNA structure. Impaired NER activity is associated with the rare autosomal recessive disorders, like xeroderma pigmentosum (XP), Cockayne syndrome (CS) and tricothiodystrophy (TTD) (reviewed in Lehmann, 2003). XP is a cancer-prone disorder, whereas CS and TTD are multisystemic diseases characterized by developmental and neurological abnormalities and premature ageing. NER operates through two subpathways that differ primarily in the recognition of base damage. In the transcription-coupled repair (TCR), it is the stalling of RNA polymerase II that triggers rapid repair of lesions from the transcribed strand of active genes. In the global genome repair (GGR), specific protein factors bind to damage sites and initiate repair on the genome overall (reviewed in Hoeijmakers, 2001; Hanawalt, 2002). XPC plays a key role as GGR-specific damage recognition protein. The human XPC protein in vivo is a heterotrimeric complex including HR23B and centrin 2 proteins (Araki et al, 2001). This complex binds to various types of NER lesions by recognizing alterations in the DNA structure more than the lesions themselves (Sugasawa et al, 2002). In agreement with its role as structure-specific DNA-binding factor, XPC has been involved in the recognition of structurally unrelated lesions. In vitro experiments have shown that XPC-containing complexes are able to excise oxidative DNA lesions such as free radical-induced 8,5′-cyclopurine 2′-deoxynucleosides (Brooks et al, 2000; Kuraoka et al, 2000) that are then repaired via NER. Recent evidence points to an additional, unexpected role of XPC in BER of endogenous lesions. The XPC–HR23B complex functionally interacts with 3-methyadenine DNA glycosylase (Miao et al, 2000) and thymine DNA glycosylase (Shimizu et al, 2003) that initiate BER of alkylation and deamination products, respectively. It has been suggested that the involvement of XPC in repair of oxidative DNA damage is responsible for the high frequency of spontaneous lung tumours recently described in XPC mutant mice (Hollander et al, 2005). Whether a defect in XPC leads to an altered response to ROS-generating agents in human cells, thus contributing to cancer development, is unknown. Here, we analysed the role of XPC in the response to oxidizing agents by using human primary keratinocytes and fibroblasts from the same skin biopsy as a model cell system. We show that XPC protects human skin cells from the killing effects of X-rays and potassium bromate (KBrO3). We provide the first in vivo evidence that XPC is involved in the repair of 8,5′-cyclopurine 2′-deoxynucleosides and major oxidized DNA bases, 8-OH-Gua and 8-hydroxyadenine (8-OH-Ade). By in vitro reconstitution experiments, we uncover a new role of XPC as a cofactor for the efficient cleavage of 8-OH-Gua by OGG1. XPC complex might contribute to cancer prevention by participating in BER of 8-OH-Gua and other oxidative DNA lesions. Results XP-C keratinocytes and fibroblasts are hypersensitive to the killing effects of oxidizing agents XP-C primary fibroblasts and keratinocytes were exposed to X-rays and cell sensitivity measured by a clonal assay. As shown in Figure 1A and B, both XP-C cell types were more sensitive to X-rays (approximately 2 fold on the basis of D37) than normal cells. Similarly to what previously reported for UVB (D'Errico et al, 2005), fibroblasts appear to be more sensitive to oxidative damage than keratinocytes. To determine whether X-ray sensitivity originated from oxidatively induced DNA lesions, cells were treated with KBrO3 that is known to produce ROS, lipid peroxidation and oxidative DNA damage (Ballmaier and Epe, 1995). A clear hypersensitivity to the killing effects of KBrO3 was observed in XP-C keratinocytes and fibroblasts when compared with normal cells (Figure 1C and D). This finding suggests that the hypersensitivity to IR of XP-C skin cells involves oxidative DNA damage. Figure 1.XP-C keratinocytes and fibroblasts are hypersensitive to the killing effects of X-rays and KBrO3. Survival of primary keratinocytes (A, C) and fibroblasts (B, D) from two normal (N1RO and N2RO, closed symbols) and two XP-C (XP26PV and XP28PV, open symbols) donors after X-rays and KBrO3 treatment. (A–B) Survival of keratinocytes and fibroblasts after exposure to X-rays. (C–D) Survival of keratinocytes and fibroblasts after exposure to KBrO3. Survival was determined by colony formation assay. The reported values are the mean of at least two independent experiments, each performed in triplicate with standard errors always <10%. Download figure Download PowerPoint Lack of XPC is responsible for the hypersensitivity to DNA-oxidizing agents of human fibroblasts To further investigate the correlation between absence of a functional XPC protein and hypersensitivity to oxidizing agents, normal and XP-C fibroblasts were analysed for their sensitivity to KBrO3 following transfection with an expression vector carrying the human EGFP-XPC fusion cassette (pEGFP-XPC) or an empty vector (pEGFP-C1). The efficiency of transfection was usually lower in XP-C (3–4%) as compared with normal cells (10–12%), independently of the type of vector used. The median EGFP intensity of both normal and XP-C cells expressing the XPC chimera was lower as compared with cells expressing the control EGFP, likely reflecting cytotoxic effects and/or decreased intrinsic fluorescence of the ectopically expressed protein. In each experiment, cell killing by KBrO3 was evaluated as percentage of dead cells 24 h post-treatment by enumerating propidium iodide (PI)-positive cells (i.e. red cells) in at least 100 transfected cells, identified as green cells expressing the EGFP protein (Figure 2A–D). Examples of cells showing nuclear accumulation of the green fluorescent signal of the XPC chimera (Figure 2A and C), and either sensitive (Figure 2B) or resistant (Figure 2D) to KBrO3 are shown. The overexpression of XPC in normal cells was slightly toxic (Figure 2E). At 24 h after treatment with two doses of KBrO3, the XP-C fibroblasts expressing normal XPC showed significant protection from cell killing (approximately 50% recovery; p<0.05) as compared with fibroblasts transfected with the empty vector (Figure 2E). Figure 2.The lack of XPC is responsible for the hypersensitivity to KBrO3 of human fibroblasts. Normal and XP-C fibroblasts were transiently transfected with an expression vector encoding for the EGFP-XPC fusion protein (pEGFP-XPC) or with the empty vector encoding for EGFP only (pEGFP-C1). At 24 h after transfection, cells were either untreated (control) or exposed to different KBrO3 doses (5 and 10 mM). At 48 h after transfection, cells were stained with PI to label in red dead cells, cytocentrifuged on slides and then analysed by fluorescence microscopy. (A–D) The photographs are representative fields of normal fibroblasts expressing the EGFP-XPC chimera (green fluorescence) and stained with PI to evaluate their viability following KBrO3 treatment (red fluorescence). Examples of cells expressing the ectopic protein and either dead (A–B) or alive (C–D). (E) The histograms report the percentages of dead cells following KBrO3 treatment of normal (F N1RO) and XP-C (F XP26PV) cells transfected with either the pEGFP-XPC or the pEGFP-C1 plasmid DNA. The reported values are the mean of at least three independent experiments and standard deviations are indicated. Download figure Download PowerPoint The restoration of normal sensitivity to KBrO3 in XP-C fibroblasts upon wild-type XPC expression provides evidence that XPC is directly involved in cell protection from the lethal effects of oxidizing agents. XP-C keratinocytes are defective in the repair of oxidatively induced DNA lesions The protection by XPC from oxidative stress-induced cell killing strongly suggested an involvement of this protein in the repair of oxidative DNA damage. Oxidative stress generates different kinds of lesions in DNA (reviewed in Evans et al, 2004). A unique reaction of hydroxyl radical-induced sugar radicals leads to the formation of 8,5′-cyclopurine 2′-deoxynucleosides that involves a concomitant damage to the base and sugar moieties of a purine nucleoside in DNA. One of these lesions, that is, (5′S)-8,5′-cyclo-2′-deoxyadenosine [(5′S)-cdA] has been shown to be repaired in vitro by NER (Kuraoka et al, 2000). The induction of (5′S)-cdA, (5′R)-8,5′-cyclo-2′-deoxyguanosine [(5′R)-cdG] and (5′S)-8,5′-cyclo-2′-deoxyguanosine [(5′S)-cdG] by a low dose of X-rays (5 Gy) and their repair was assayed by liquid chromatography/mass spectrometry (LC/MS) or gas chromatography/MS (GC/MS) in primary keratinocytes of two normal subjects (K N1RO, K N2RO) and two XP-C patients (K XP26PV, K XP28PV). Keratinocytes were selected for this analysis because the steady-state level of oxidative DNA base damage in this cell type is significantly lower than in fibroblasts (see below), thus allowing a better estimate of oxidatively induced DNA lesions. As shown in Figure 3A, the steady-state levels of selected 8,5′-cyclopurine 2′-deoxynucleosides were similar in normal and XP-C keratinocytes. Upon irradiation with 5 Gy, (5′S)-cdA, (5′R)-cdG and (5′S)-cdG were induced at statistically significant levels (P<0.05) and then repaired to completion in 2 h in normal keratinocytes, whereas they were not repaired in XP-C cells. This is the first evidence that XPC–NER complexes are required for the repair of 8,5′-cyclopurine 2′-deoxynucleosides in vivo, as previously shown in vitro (Brooks et al, 2000; Kuraoka et al, 2000). Figure 3.XP-C keratinocytes accumulate cyclopurines and oxidized DNA bases induced by X-rays. Levels of oxidatively modified nucleosides in DNA of keratinocytes from normal (K N1RO) and XP-C (K XP26PV and K XP28PV) donors were measured by LC/MS or GC/MS. For each data point, DNA samples isolated from three independent experiments for cell strain were used. The chemical structures of the modified nucleosides are illustrated. (A) Induction by X-rays and repair of (5′S)-cdA, (5′R)-cdG and (5′S)-cdG. (B) Induction by X-rays and repair of 8-OH-dG and 8-OH-dA. DNA samples were isolated from untreated cells (control), cells exposed to X-rays (5 Gy), and exposed to X-rays and allowed to repair for 2 h (5 Gy+2 h rep). The data represent the mean of three independent experiments and standard deviations are reported. Statistical analysis was performed using one-way analysis of variance. The stars indicate statistically significant differences between control and 5 Gy, or control and 5 Gy+2 h rep with a P<0.05. Download figure Download PowerPoint The levels of other major oxidatively induced DNA bases induced by X-rays, 8-OH-Gua and 8-OH-Ade, were also measured by LC/MS as their nucleosides 8-hydroxy-2′-deoxyguanosine (8-OH-dG) and 8-hydroxy-2′-deoxyadenosine (8-OH-dA), respectively. As shown in Figure 3B, the steady-state levels were similar in normal and XP-C keratinocytes. A significant increase in the level of these products was detected immediately after X-ray exposure in both normal and XP-C keratinocytes. Normal keratinocytes repaired to completion both modified DNA bases within 2 h after treatment, as expected on the basis of their repair rate in vivo (Tuo et al, 2003). In contrast, the repair of both lesions was defective in XP-C keratinocytes. The repair kinetics of 8-OH-dG was investigated in keratinocytes and fibroblasts following treatment with KBrO3 by using high-performance liquid chromatography/electrochemical detection (HPLC-ED) methodology. This technique was suited to estimate oxidatively induced 8-OH-dG in both cell types. As shown in Figure 4A and B, the steady-state level of 8-OH-dG was significantly higher (P<0.05) in fibroblasts than in keratinocytes. This effect is associated with a higher antioxidant capacity of keratinocytes as compared with fibroblasts (D'Errico et al, 2006). It should be noted that the background levels of 8-OH-dG measured in keratinocytes by HPLC-ED differ from those measured by LC/MS. This is not unexpected as two different methods have been used; however, the observed values are both within the range of the internationally accepted background levels of 8-OH-dG in cells (ESCODD, 2003; Collins et al, 2004). Therefore, the comparison of the results obtained by these two techniques can only be qualitative but not quantitative. Upon treatment with KBrO3 (40 mM), a statistically significant increase in DNA levels of 8-OH-dG was induced in both cell types (P<0.001) (Figure 4A and B), in agreement with a significant production of this damaged base by this agent (Ballmaier and Epe, 1995). At this high oxidant dose, the levels of induced 8-OH-dG in the two cell types were similar although the steady-state levels were significantly different. At lower KBrO3 doses (up to 20 mM), higher induced levels of 8-OH-dG were detected in fibroblasts than in keratinocytes (data not shown) in agreement with an additive effect of the background and induced levels. It is likely that under massive ROS production (high oxidant doses), the cellular antioxidant defence mechanisms are saturated, and the genetic background of the cell system does not influence the DNA oxidation levels anymore. When cells were allowed to repair after DNA damage, 8-OH-dG levels decreased significantly during the time course both in normal and XP-C keratinocytes and fibroblasts (P<0.001) (Figure 4A and B). However, in the absence of XPC, the levels of 8-OH-dG at each repair time were significantly higher than in normal cells in both cell types (P<0.001). At 2 h post-treatment, when most lesions were repaired in normal cells, residual damage was still present in XP-C cells. When the levels of 8-OH-dG at different repair times were expressed as percentage of the levels immediately after treatment (Figure 4C and D), the repair rate of XP-C keratinocytes and fibroblasts was slower than that of their normal counterparts at early repair times. This is remarkable in the case of keratinocytes that repaired 30% of 8-OH-dG lesions within the first 10 min after treatment but, when XPC is defective, they did not show any repair of this oxidized base within the same time frame (Figure 4C). Figure 4.Repair of 8-OH-Gua is slower in XP-C keratinocytes and fibroblasts as compared with normal cells. 8-OH-dG levels were analysed in normal (N1RO) and XP-C (XP26PV) human fibroblasts and keratinocytes after exposure to 40 mM KBrO3 (1 h). Aliquots of cells were taken at the indicated times and the levels of 8-OH-Gua were measured by HPLC-ED. (A–B) 8-OH-dG levels in keratinocytes (A) and fibroblasts (B). (C–D) 8-OH-dG levels present at different repair times in keratinocytes (C) and fibroblasts (D) expressed as percentage of amounts at time 0.The data represent the mean of three independent experiments, and standard deviations (A–B) or standard errors (C–D) of the means are reported. Statistical analysis was performed using one- and two-way analyses of variance. Download figure Download PowerPoint The contribution of XPC to 8-OH-Gua repair was, therefore, confirmed in both cell types. In addition, the analysis of the repair kinetics allowed estimating the relative contribution of the XPC-dependent mechanism to the overall repair of 8-OH-Gua. This discloses a role for XPC as cofactor in the repair of this oxidized base. A deficiency in XPC leads to a reduction in cleavage of 8-OH-Gua-containing oligonucleotides by keratinocyte extracts It is well known that 8-OH-Gua is repaired via BER pathway initiated by a specific DNA glycosylase, OGG1 (reviewed in Fortini et al, 2003; Evans et al, 2004). To gain insights into the mechanism of oxidative DNA base damage accumulation in XP-C keratinocytes, we examined the incision of 8-OH-Gua-containing duplex oligonucleotides by nuclear extracts of normal and XP-C keratinocytes. The incision activity was estimated by measuring the relative yield of uncleaved and cleaved products by PAGE analysis. A significant reduction (approximately 50%, P<0.05) in 8-OH-Gua cleavage was recorded when XP-C extracts were compared with normal extracts (Figure 5A). To test whether the XP-C defect was indeed responsible for the reduced cleavage efficiency of XP-C extracts, the assay was repeated in the presence of increasing concentrations of purified recombinant XPC–HR23B complex. As shown in Figure 5B, the correction of the defect was achieved in both cell extracts by adding 80 fmol of XPC–HR23B. The addition of the same amount of XPC–HR23B to wild-type extracts did not change the cleavage efficiency (data not shown). Figure 5.Cell extracts from XP-C keratinocytes are defective in 8-OH-Gua cleavage, but addition of purified XPC–HR23B restores normal cleavage activity. 30-mer duplex oligonucleotides (50 fmol) containing 8-OH-Gua were incubated with nuclear extracts (5 μg) of XP-C keratinocytes at 37°C for different periods of time as indicated. The 5′ end-labelled oligonucleotide was the 8-OH-Gua-containing strand. The products were separated by denaturing 20% PAGE. (A) Keratinocyte extracts from normal (K N1RO) and XP-C (K XP26PV and K XP28PV) donors. The relative percentages of 8-OH-Gua cleavage were obtained by electronic autoradiography of the gel (Instant Imager, Packard). Two or three independent extracts per cell strain were tested. (B) The 8-OH-Gua cleavage reaction by XP-C keratinocyte extracts was performed in the presence of varying concentrations of XPC–HR23B as indicated. Download figure Download PowerPoint Thus, the defect in 8-OH-Gua cleavage is likely to account for its accumulation in the genome of XP-C keratinocytes. XPC–HR23B stimulates the activity of OGG1 To determine the effect of XPC–HR23B on OGG1-catalysed 8-OH-Gua excision, a limiting amount of OGG1 (60 fmol) was incubated with an 8-OH-Gua-containing duplex oligonucleotide in the presence of varying amounts of XPC–HR23B. In this assay, the OGG1-processed DNA was treated with alkali to convert AP sites to nicks before denaturing PAGE. As shown in Figure 6A, the activity of OGG1 increased as a function of the concentration of XPC–HR23B (lanes 2 and 3). A three-fold stimulation of OGG1 activity was observed after 30 min incubation in the presence of 200 fmol of XPC–HR23B. Purified XPC–HR23B by itself showed no detectable DNA glycosylase activity on the 8-OH-Gua-containing oligomer (lane 4). No stimulation of OGG1 activity was detected upon addition to the cleavage reaction of another NER protein, XPA (lanes 8 and 9), or following addition of bovine serum albumin (BSA) (data not shown). To examine which subunit of the complex was responsible for the stimulation, HR23B was added alone. This subunit had a minor effect on OGG1 activity (lanes 5 and 6) and no relation with the dose was detected. The involvement of XPC in OGG1 stimulation was further demonstrated by the similar level of stimulation by XPC alone compared to that induced by the XPC–HR23B complex (Figure 6C, lanes 2 and 3). These findings clearly indicate that XPC is the subunit of the complex responsible for the stimulation of the OGG1 activity. The use of the alkali treatment does not allow to test which is the activity of OGG1 that is stimulated by XPC–HR23B. However, as the AP lyase activity of OGG1 is very weak, it is likely that the stimulation involves the DNA glycosylase activity of OGG1. Figure 6.XPC–HR23B stimulates the activity of OGG1. DNA fragments (210 bp) containing a single 8-OH-Gua lesion were incubated with purified proteins, as indicated, at 37°C for 30 min. The 5′ end-labelled strand was the 8-OH-Gua-containing stran" @default.
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• W2062150992 title "New functions of XPC in the protection of human skin cells from oxidative damage" @default.
• W2062150992 cites W1501611632 @default.
• W2062150992 cites W1505189576 @default.
• W2062150992 cites W1552620600 @default.
• W2062150992 cites W1824179904 @default.
• W2062150992 cites W1975248407 @default.
• W2062150992 cites W1975418206 @default.
• W2062150992 cites W1978122209 @default.
• W2062150992 cites W1979123830 @default.
• W2062150992 cites W1988699070 @default.
• W2062150992 cites W1990849368 @default.
• W2062150992 cites W2007709159 @default.
• W2062150992 cites W2013615285 @default.
• W2062150992 cites W2019369653 @default.
• W2062150992 cites W2020543172 @default.
• W2062150992 cites W2027604638 @default.
• W2062150992 cites W2027755817 @default.
• W2062150992 cites W2030666445 @default.
• W2062150992 cites W2032128602 @default.
• W2062150992 cites W2036794974 @default.
• W2062150992 cites W2038389845 @default.
• W2062150992 cites W2048527989 @default.
• W2062150992 cites W2050662558 @default.
• W2062150992 cites W2070357156 @default.
• W2062150992 cites W2070572828 @default.
• W2062150992 cites W2070847429 @default.
• W2062150992 cites W2071628857 @default.
• W2062150992 cites W2074552403 @default.
• W2062150992 cites W2080605611 @default.
• W2062150992 cites W2092525424 @default.
• W2062150992 cites W2092770418 @default.
• W2062150992 cites W2093540734 @default.
• W2062150992 cites W2094884032 @default.
• W2062150992 cites W2096136751 @default.
• W2062150992 cites W2106443150 @default.
• W2062150992 cites W2113014296 @default.
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• W2062150992 cites W2128004417 @default.
• W2062150992 cites W2131165436 @default.
• W2062150992 cites W2134371533 @default.
• W2062150992 cites W2144466703 @default.
• W2062150992 cites W2155666009 @default.
• W2062150992 cites W2159036652 @default.
• W2062150992 cites W2160696924 @default.
• W2062150992 cites W2168882862 @default.
• W2062150992 cites W2183682051 @default.
• W2062150992 cites W2319228790 @default.
• W2062150992 cites W3024576753 @default.
• W2062150992 cites W4293719097 @default.
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