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Chuck C.-k. Chao - One of the best experts on this subject based on the ideXlab platform.

  • The Role of DDB2 in Regulating Cell Survival and Apoptosis Following DNA Damage - A Mini-Review
    DNA Repair, 2011
    Co-Authors: Chuck C.-k. Chao
    Abstract:

    Nucleotide excision repair (NER) represents a central cellular process for the removal of structurally and chemically diverse DNA lesions [Friedberg et al., 2006]. Mutations in genes involved in NER are associated with rare autosomal recessive syndromes such as xeroderma pigmentosum (XP), a condition characterized by sensitivity to UV light, neurological abnormalities, and a propensity to develop skin cancer (Cleaver, 2005). The observation that cells from XP subgroup E (XP-E cells XP2RO and XP3RO) are defective in recognizing damaged DNA and performing NER highlighted the physiological importance of the protein termed DNA damage-binding protein, or DDB [Chu & Chang, 1988]. The DDB protein, sometimes also referred to as UV-DDB due to its high affinity and specificity for UV-damaged DNA, contains two principal subunits, DDB1 and DDB2 [Grossman, 1976; Keeney et al., 1993; Takao et al., 1993]. The DDB protein complex also binds to non-UVdamaged DNA, like cisplatin-modified DNA, although with much lower affinity. Although the history of DDB spans more than two decades, the complete understanding of its physiological functions remains to be clarified. The activity of DDB has been repeatedly described in crude mammalian cell extracts by electrophoretic mobility shift assays or filterbinding assays performed by different laboratories since the first report of its discovery [Feldberg & Grossman, 1976]. Notably, micro-injections of DDB complexes into the nucleus of XP-E cells restored NER activity [Keeney et al., 1994], supporting the notion that DDB participates in chromatin NER. The DDB1 gene from simian cells was the first DDB gene to be identified [Takao et al., 1993]. The human DDB1 and DDB2 genes were subsequently sequenced [Dualan et al., 1995; Lee et al., 1995]. Soon after, DNA sequencing from Linn’s laboratory revealed that DDB2 is mutated in XP-E cells which lack DDB activity [Nichols et al., 1996; Tang & Chu, 2002]. The predicted DDB2 protein sequence was shown to contain several functional domains, including WD40 repeats, post-translation modification sites (e.g. acetylation, phosphorylation, and ubiquitination), DDB1and DNA-binding sites, as well as a DWD box. Notably, in a majority of XP-E cell lines, DDB2 was found to be altered at domains other than the one required for binding DNA. Thus, DDB appears to be regulated at several levels in UV-irradiated cells, including by transcriptional activation of DDB2 mRNA, post-translational modification, translocation to the nucleus, complex formation,

  • Damaged DNA-binding protein 2 (DDB2) protects against UV irradiation in human cells and Drosophila
    Journal of biomedical science, 2010
    Co-Authors: Nian-kang Sun, Chun-ling Sun, Chia-hua Lin, Li-mai Pai, Chuck C.-k. Chao
    Abstract:

    We observed previously that cisplatin-resistant HeLa cells were cross-resistant to UV light due to accumulation of DDB2, a protein implicated in DNA repair. More recently, we found that cFLIP, which represents an anti-apoptotic protein whose level is induced by DDB2, was implicated in preventing apoptosis induced by death-receptor signaling. In the present study, we investigated whether DDB2 has a protective role against UV irradiation and whether cFLIP is also involved in this process. We explored the role of DDB2 in mediating UV resistance in both human cells and Drosophila. To do so, DDB2 was overexpressed by using a full-length open reading frame cDNA. Conversely, DDB2 and cFLIP were suppressed by using antisense oligonucleotides. Cell survival was measured using a colony forming assay. Apoptosis was monitored by examination of nuclear morphology, as well as by flow cytometry and Western blot analyses. A transcription reporter assay was also used to assess transcription of cFLIP. We first observed that the cFLIP protein was upregulated in UV-resistant HeLa cells. In addition, the cFLIP protein could be induced by stable expression of DDB2 in these cells. Notably, the anti-apoptotic effect of DDB2 against UV irradiation was largely attenuated by knockdown of cFLIP with antisense oligonucleotides in HeLa cells. Moreover, overexpression of DDB2 did not protect against UV in VA13 and XP-A cell lines which both lack cFLIP. Interestingly, ectopic expression of human DDB2 in Drosophila dramatically inhibited UV-induced fly death compared to control GFP expression. On the other hand, expression of DDB2 failed to rescue a different type of apoptosis induced by the genes Reaper or eiger. Our results show that DDB2 protects against UV stress in a cFLIP-dependent manner. In addition, the protective role of DDB2 against UV irradiation was found to be conserved in divergent living organisms such as human and Drosophila. In addition, UV irradiation may activate a cFLIP-regulated apoptotic pathway in certain cells.

  • Potential attenuation of p38 signaling by DDB2 as a factor in acquired TNF resistance.
    International journal of cancer, 2005
    Co-Authors: Chun-ling Sun, Chuck C.-k. Chao
    Abstract:

    Our previous study demonstrated that DDB2, a DNA repair protein, attenuates cell surface membrane-associated death signal induced by UV or FasAb; DDB2 is overexpressed in cisplatin-selected cells. However, the molecular mechanism underlying the protective role of DDB2 along the apoptotic pathway remains unknown. Our study identified the cross-resistance of the cisplatin-selected cells to tumor necrosis factor-α (TNF-α). Since knock-down of the DDB2 level rendered cells (HR18) sensitive to the treatment, the cell sensitivity to TNF-α appears inversely proportional to the cellular level of DDB2. Treatment of HeLa cells with TNF-α transiently induced activation of p38MAPK signal, but this induction was significantly reduced in the resistant cells. Overexpression of DDB2 attenuated the activation of p38 in cells. TNF-α-induced apoptotic signals, represented by caspase-8 and downstream substrate cleavage, were reduced in resistant cells compared to their sensitive counterparts. Inhibition of p38 signal by SB202190 clearly attenuated TNF-α-induced apoptotic signals. Moreover, overexpression of DDB2 in HR18 cells also attenuated TNF-α induced caspase activation. These results suggest that p38MAPK activation may be a key upstream signal of TNF-α-induced apoptosis and that attenuation of p38 signal by DDB2 overexpression may be responsible for acquired TNF-α resistance. © 2005 Wiley-Liss, Inc.

  • Cross-Resistance to Death Ligand-Induced Apoptosis in Cisplatin-Selected HeLa Cells Associated with Overexpression of DDB2 and Subsequent Induction of cFLIP
    Molecular Pharmacology, 2005
    Co-Authors: Chuck C.-k. Chao
    Abstract:

    This work reports the involvement of damaged DNA-binding protein 2 (DDB2), a component involved in the genomic repair of UV damage, in the cross-resistance of cisplatin-selected cell lines to death ligand-mediated apoptosis. The cisplatin-resistant cell line (HR3) exhibits enhanced expression of DDB2 and cross-resistance to UV-induced activation of apoptosis and caspases. This investigation further demonstrates that HR3 cells also exhibited cross-resistance to death ligands [Fas-inducing antibody and tumor necrosis factor (TNF)-α]. Depletion of the elevated DDB2 in HR3 cells sensitizes Fas-inducing antibody-induced and TNF-α-induced apoptosis. In contrast, the overexpression of DDB2 induces cellular FLICE-like inhibitory protein (cFLIP) expression and further attenuates death ligand-induced apoptosis. Moreover, reverse transcription-polymerase chain reaction and reporter assay indicated that DDB2 could increase both endogenous and exogenous cFLIP mRNA levels. Accordingly, the elimination of cFLIP by antisense oligonucleotides suppresses DDB2 protection. These findings reveal that DDB2 regulates TNF signaling-mediated apoptosis via cFLIP and contributes to acquired cross-resistance. DDB2, while participating in DNA repair, functions as a negative regulator of apoptosis and may therefore have a pivotal role in regulating immune response and cancer-therapeutic efficacy.

  • Restoration of UV sensitivity in UV-resistant HeLa cells by antisense-mediated depletion of damaged DNA-binding protein 2 (DDB2).
    FEBS Letters, 2002
    Co-Authors: Nian-kang Sun, Pachiyappan Kamarajan, Haimei Huang, Chuck C.-k. Chao
    Abstract:

    Damaged DNA-binding activity comprises two major protein components, DDB1 and DDB2, which are implicated in the repair of ultraviolet (UV) radiation-induced DNA damage. The possible role of DDB2 as a determinant of cellular sensitivity to UV was investigated. The abundance of DDB2 in UV-resistant HeLa cell lines was increased compared with that in the parental UV-sensitive cells. Stable transfection of the resistant cells with DDB2 antisense cDNA resulted in marked depletion of DDB2 protein and restored cellular sensitivity to UV-induced apoptosis. Whereas the extent of UV-induced activation of apoptosis executioners, including DNA fragmentation factor, and caspase-3 were reduced in the UV-resistant cells compared with those apparent in the sensitive cells, depletion of DDB2 from the resistant cells restored the normal activation patterns for these proteins. In contrast, overexpressing DDB2 in DDB2-depleted cells with recombinant adenovirus, which carries DDB2 cDNA, markedly inhibited the extent of UV-induced activation of DNA fragmentation factor, and caspase-3. Interestingly, a mutated form of DDB2, which is defective in interacting with DDB1 and binding to UV-damaged DNA, also markedly inhibited the activation of apoptosis executioners. These results indicate that DDB2 is a modulator of UV-induced apoptosis, and that UV resistance can be overcome by inhibition of DDB2. The findings also suggest that modulation of UV-induced apoptosis by DDB2 may be independent of DNA repair.

Qien Wang - One of the best experts on this subject based on the ideXlab platform.

  • Abstract B62: DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1
    Genetics and Molecular Drivers, 2018
    Co-Authors: Tiantian Cui, Altaf A. Wani, Xiaoli Zhang, Amit K. Srivastava, Chunhua Han, Qien Wang
    Abstract:

    Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that DNA damage-binding protein 2 (DDB2) is able to reduce the abundance of CSCs in the bulk ovarian cancer cells, providing a novel mechanism to explain the DDB2-mediated suppression of tumorigenicity and metastasis, and also suggesting that low expression of DDB2 is required for the maintenance of CSCs. However, the underlying mechanisms still remain unclear. By using the Tet-On DDB2 modulation system, we have confirmed our previous finding that downregulation of DDB2 expands the CSC population in the 2008 ovarian cancer cell line. We also found that DDB2 is able to suppress non-CSC-to-CSC conversions in this cancer cell line. DDB2 has been recognized as a transcriptional regulator. Our microarray analysis has identified ALDH1A1 to be targeted and regulated by DDB2. Downregulation of ALDH1A1 expression by DDB2 at both mRNA and protein levels has been validated in various ovarian cancer cell lines. The mechanistic investigation demonstrated that DDB2 can bind to the promoter region of the ALDH1A1 gene, facilitating the enrichment of histone H3K27me3 by recruiting EZH2 to the promoter region, eventually inhibiting the promoter activity of the ALDH1A1 gene. In addition, we also found that DDB2 competes with transcription factor C/EBPβ for binding to the ALDH1A1 promoter, indirectly inhibiting the ALDH1A1 promoter activity. Finally, we knocked down the expression of DDB2 and ALDH1A1 individually or simultaneously in the 2008 ovarian cancer cell line, and analyzed their tumorigenicity. We found that downregulation of ALDH1A1 is able to block DDB2 silencing-induced expansion of the CSC population, indicating that ALDH1A1 plays a critical role in DDB2-mediated suppression of the CSC population in ovarian cancer cells. We further showed that treatment with a selective ALDH1A1 inhibitor blocked DDB2 silencing-induced expansion of CSCs, and halted orthotopic xenograft tumor growth. In summary, our data demonstrated that DDB2, functioning as a transcription repressor, is able to abrogate ovarian CSC properties by downregulating ALDH1A1 expression. This study provides a novel mechanism underlying the regulation of the CSC population, and would facilitate the development of efficient strategies for eliminating CSCs to prevent tumor relapse and metastasis in ovarian cancers. (Supported by NIH R01CA151248, R01CA211175.) Citation Format: Tiantian Cui, Amit Kumar Srivastava, Chunhua Han, Xiaoli Zhang, Altaf A. Wani, Qi-En Wang. DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B62.

  • UV radiation-induced SUMOylation of DDB2 regulates nucleotide excision repair.
    Carcinogenesis, 2017
    Co-Authors: Chunhua Han, Qien Wang, Ran Zhao, Gulzar Wani, John Kroger, Altaf A. Wani
    Abstract:

    Abstract Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.

  • Abstract 4784: Regulation of ovarian cancer stem cell population by DDB2
    Tumor Biology, 2017
    Co-Authors: Tiantian Cui, Altaf A. Wani, Xiaoli Zhang, Amit K. Srivastava, Chunhua Han, Zhiqin Gao, Qien Wang
    Abstract:

    Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that DNA damage-binding protein 2 (DDB2) is able to reduce the abundance of CSCs in the bulk ovarian cancer cells, providing a novel mechanism to explain the DDB2-mediated suppression of tumorigenicity and metastasis, and also suggesting that low expression of DDB2 is required for the maintenance of CSCs. However, the underlying mechanisms still remain unclear. By using the Tet-On DDB2 modulation system, we have confirmed our previous finding that downregulation of DDB2 expands the CSC population in the 2008 ovarian cancer cell line. We also found that DDB2 is able to suppress non-CSC-to-CSC conversions in this cancer cell line. DDB2 has been recognized as a transcriptional regulator. Our microarray analysis has identified ALDH1A1 to be targeted and regulated by DDB2. The downregulation of ALDH1A1 expression by DDB2 at both mRNA and protein levels has been validated in various ovarian cancer cell lines. The mechanistic investigation demonstrated that DDB2 can bind to the promoter region of the ALDH1A1 gene, facilitating the enrichment of histone H3K27me3 by recruiting EZH2 to the promoter region, eventually inhibiting the promoter activity of the ALDH1A1 gene. In addition, we also found that DDB2 competes with transcription factor C/EBPβ for binding to the ALDH1A1 promoter, indirectly inhibiting the ALDH1A1 promoter activity. Finally, we knocked down the expression of DDB2 and ALDH1A1 individually or simultaneously in the 2008 ovarian cancer cell line, and analyzed their tumorigenicity. We found that downregulation of ALDH1A1 is able to block DDB2 silencing-induced expansion of the CSC population, indicating that ALDH1A1 plays a critical role in DDB2-mediated suppression of the CSC population in ovarian cancer cells. In summary, our data demonstrated that DDB2, functioning as a transcription repressor, is able to abrogate ovarian CSC properties by downregulating ALDH1A1 expression. This study provides a novel mechanism underlying the regulation of the CSC population, and would facilitate the development of efficient strategies for eliminating CSCs to prevent tumor relapse and metastasis in ovarian cancers. (Supported by NIH R01CA151248) Citation Format: Tiantian Cui, Amit Kumar Srivastava, Chunhua Han, Zhiqin Gao, Xiaoli Zhang, Altaf A. Wani, Qi-En Wang. Regulation of ovarian cancer stem cell population by DDB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4784. doi:10.1158/1538-7445.AM2017-4784

  • DDB2 increases radioresistance of NSCLC cells by enhancing DNA damage responses
    Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 2016
    Co-Authors: Ning Zou, Tiantian Cui, Guozhen Xie, Amit Kumar Srivastava, Linlin Yang, Shaozhong Wei, Yanfang Zheng, Qien Wang
    Abstract:

    Radiotherapy resistance is one of the major factors limiting the efficacy of radiotherapy in lung cancer patients. The extensive investigations indicate the diversity in the mechanisms underlying radioresistance. Here, we revealed that DNA damage binding protein 2 (DDB2) is a potential regulator in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. DDB2, originally identified as a DNA damage recognition factor in the nucleotide excision repair, promotes the survival and inhibits the apoptosis of NSCLC cell lines upon ionizing radiation (IR). Mechanistic investigations demonstrated that DDB2 is able to facilitate IR-induced phosphorylation of Chk1, which plays a critical role in the cell cycle arrest and DNA repair in response to IR-induced DNA double-strand breaks (DSBs). Indeed, knockdown of DDB2 compromised the G2 arrest in the p53-proficient A549 cell line and reduced the efficiency of homologous recombination (HR) repair. Taken together, our data indicate that the expression of DDB2 in NSCLC could be used as a biomarker to predict radiosensitivity of the patients. Targeting Chk1 can be used to increase the efficacy of radiotherapy in patients of NSCLC possessing high levels of DDB2.

  • DDB2 modulates tgf β signal transduction in human ovarian cancer cells by downregulating nedd4l
    Nucleic Acids Research, 2015
    Co-Authors: Ran Zhao, Altaf A. Wani, Jianhua Yu, Qien Wang, Jinshan He, Amit Srivastava, Xiaoli Zhang
    Abstract:

    The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor β signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-β signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-β signal transduction and increases the responsiveness of ovarian cancer cells to TGF-β-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-β signaling and its downstream effects that impinge upon tumor growth in ovarian cancers.

Altaf A. Wani - One of the best experts on this subject based on the ideXlab platform.

  • Abstract B62: DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1
    Genetics and Molecular Drivers, 2018
    Co-Authors: Tiantian Cui, Altaf A. Wani, Xiaoli Zhang, Amit K. Srivastava, Chunhua Han, Qien Wang
    Abstract:

    Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that DNA damage-binding protein 2 (DDB2) is able to reduce the abundance of CSCs in the bulk ovarian cancer cells, providing a novel mechanism to explain the DDB2-mediated suppression of tumorigenicity and metastasis, and also suggesting that low expression of DDB2 is required for the maintenance of CSCs. However, the underlying mechanisms still remain unclear. By using the Tet-On DDB2 modulation system, we have confirmed our previous finding that downregulation of DDB2 expands the CSC population in the 2008 ovarian cancer cell line. We also found that DDB2 is able to suppress non-CSC-to-CSC conversions in this cancer cell line. DDB2 has been recognized as a transcriptional regulator. Our microarray analysis has identified ALDH1A1 to be targeted and regulated by DDB2. Downregulation of ALDH1A1 expression by DDB2 at both mRNA and protein levels has been validated in various ovarian cancer cell lines. The mechanistic investigation demonstrated that DDB2 can bind to the promoter region of the ALDH1A1 gene, facilitating the enrichment of histone H3K27me3 by recruiting EZH2 to the promoter region, eventually inhibiting the promoter activity of the ALDH1A1 gene. In addition, we also found that DDB2 competes with transcription factor C/EBPβ for binding to the ALDH1A1 promoter, indirectly inhibiting the ALDH1A1 promoter activity. Finally, we knocked down the expression of DDB2 and ALDH1A1 individually or simultaneously in the 2008 ovarian cancer cell line, and analyzed their tumorigenicity. We found that downregulation of ALDH1A1 is able to block DDB2 silencing-induced expansion of the CSC population, indicating that ALDH1A1 plays a critical role in DDB2-mediated suppression of the CSC population in ovarian cancer cells. We further showed that treatment with a selective ALDH1A1 inhibitor blocked DDB2 silencing-induced expansion of CSCs, and halted orthotopic xenograft tumor growth. In summary, our data demonstrated that DDB2, functioning as a transcription repressor, is able to abrogate ovarian CSC properties by downregulating ALDH1A1 expression. This study provides a novel mechanism underlying the regulation of the CSC population, and would facilitate the development of efficient strategies for eliminating CSCs to prevent tumor relapse and metastasis in ovarian cancers. (Supported by NIH R01CA151248, R01CA211175.) Citation Format: Tiantian Cui, Amit Kumar Srivastava, Chunhua Han, Xiaoli Zhang, Altaf A. Wani, Qi-En Wang. DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B62.

  • UV radiation-induced SUMOylation of DDB2 regulates nucleotide excision repair.
    Carcinogenesis, 2017
    Co-Authors: Chunhua Han, Qien Wang, Ran Zhao, Gulzar Wani, John Kroger, Altaf A. Wani
    Abstract:

    Abstract Subunit 2 of DNA damage-binding protein complex (DDB2) is an early sensor of nucleotide excision repair (NER) pathway for eliminating DNA damage induced by UV radiation (UVR) and cisplatin treatments of mammalian cells. DDB2 is modified by ubiquitin and poly(ADP-ribose) (PAR) in response to UVR, and these modifications play a crucial role in regulating NER. Here, using immuno-analysis of irradiated cell extracts, we have identified multiple post-irradiation modifications of DDB2 protein. Interestingly, although the DNA lesions induced by both UVR and cisplatin are corrected by NER, only the UV irradiation, but not the cisplatin treatment, induces any discernable DDB2 modifications. We, for the first time, show that the appearance of UVR-induced DDB2 modifications depend on the binding of DDB2 to the damaged chromatin and the participation of functionally active 26S proteasome. The in vitro and in vivo analysis revealed that SUMO-1 conjugations comprise a significant portion of these UVR-induced DDB2 modifications. Mapping of SUMO-modified sites demonstrated that UVR-induced SUMOylation occurs on Lys-309 residue of DDB2 protein. Mutation of Lys-309 to Arg-309 diminished the DDB2 SUMOylation observable both in vitro and in vivo. Moreover, K309R mutated DDB2 lost its function of recruiting XPC to the DNA damage sites, as well as the ability to repair cyclobutane pyrimidine dimers following cellular UV irradiation. Taken together, our results indicate that DDB2 is modified by SUMOylation upon UV irradiation, and this post-translational modification plays an important role in the initial recognition and processing of UVR-induced DNA damage occurring within the context of chromatin.

  • Abstract 4784: Regulation of ovarian cancer stem cell population by DDB2
    Tumor Biology, 2017
    Co-Authors: Tiantian Cui, Altaf A. Wani, Xiaoli Zhang, Amit K. Srivastava, Chunhua Han, Zhiqin Gao, Qien Wang
    Abstract:

    Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that DNA damage-binding protein 2 (DDB2) is able to reduce the abundance of CSCs in the bulk ovarian cancer cells, providing a novel mechanism to explain the DDB2-mediated suppression of tumorigenicity and metastasis, and also suggesting that low expression of DDB2 is required for the maintenance of CSCs. However, the underlying mechanisms still remain unclear. By using the Tet-On DDB2 modulation system, we have confirmed our previous finding that downregulation of DDB2 expands the CSC population in the 2008 ovarian cancer cell line. We also found that DDB2 is able to suppress non-CSC-to-CSC conversions in this cancer cell line. DDB2 has been recognized as a transcriptional regulator. Our microarray analysis has identified ALDH1A1 to be targeted and regulated by DDB2. The downregulation of ALDH1A1 expression by DDB2 at both mRNA and protein levels has been validated in various ovarian cancer cell lines. The mechanistic investigation demonstrated that DDB2 can bind to the promoter region of the ALDH1A1 gene, facilitating the enrichment of histone H3K27me3 by recruiting EZH2 to the promoter region, eventually inhibiting the promoter activity of the ALDH1A1 gene. In addition, we also found that DDB2 competes with transcription factor C/EBPβ for binding to the ALDH1A1 promoter, indirectly inhibiting the ALDH1A1 promoter activity. Finally, we knocked down the expression of DDB2 and ALDH1A1 individually or simultaneously in the 2008 ovarian cancer cell line, and analyzed their tumorigenicity. We found that downregulation of ALDH1A1 is able to block DDB2 silencing-induced expansion of the CSC population, indicating that ALDH1A1 plays a critical role in DDB2-mediated suppression of the CSC population in ovarian cancer cells. In summary, our data demonstrated that DDB2, functioning as a transcription repressor, is able to abrogate ovarian CSC properties by downregulating ALDH1A1 expression. This study provides a novel mechanism underlying the regulation of the CSC population, and would facilitate the development of efficient strategies for eliminating CSCs to prevent tumor relapse and metastasis in ovarian cancers. (Supported by NIH R01CA151248) Citation Format: Tiantian Cui, Amit Kumar Srivastava, Chunhua Han, Zhiqin Gao, Xiaoli Zhang, Altaf A. Wani, Qi-En Wang. Regulation of ovarian cancer stem cell population by DDB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4784. doi:10.1158/1538-7445.AM2017-4784

  • DDB2 modulates tgf β signal transduction in human ovarian cancer cells by downregulating nedd4l
    Nucleic Acids Research, 2015
    Co-Authors: Ran Zhao, Altaf A. Wani, Jianhua Yu, Qien Wang, Jinshan He, Amit Srivastava, Xiaoli Zhang
    Abstract:

    The expression of DNA damage-binding protein 2 (DDB2) has been linked to the prognosis of ovarian cancer and its underlying transcription regulatory function was proposed to contribute to the favorable treatment outcome. By applying gene microarray analysis, we discovered neural precursor cell expressed, developmentally downregulated 4-Like (NEDD4L) as a previously unidentified downstream gene regulated by DDB2. Mechanistic investigation demonstrated that DDB2 can bind to the promoter region of NEDD4L and recruit enhancer of zeste homolog 2 histone methyltransferase to repress NEDD4L transcription by enhancing histone H3 lysine 27 trimethylation (H3K27me3) at the NEDD4L promoter. Given that NEDD4L plays an important role in constraining transforming growth factor β signaling by targeting activated Smad2/Smad3 for degradation, we investigated the role of DDB2 in the regulation of TGF-β signaling in ovarian cancer cells. Our data indicate that DDB2 enhances TGF-β signal transduction and increases the responsiveness of ovarian cancer cells to TGF-β-induced growth inhibition. The study has uncovered an unappreciated regulatory mode that hinges on the interaction between DDB2 and NEDD4L in human ovarian cancer cells. The novel mechanism proposes the DDB2-mediated fine-tuning of TGF-β signaling and its downstream effects that impinge upon tumor growth in ovarian cancers.

  • Overexpression of DDB2 enhances the sensitivity of human ovarian cancer cells to cisplatin by augmenting cellular apoptosis.
    International journal of cancer, 2010
    Co-Authors: Qien Wang, Mohamed A. El-mahdy, Gulzar Wani, Chunhua Han, Bassant M. Barakat, Keisha Milum, De-tao Yin, Qun Zhao, El-shaimaa A. Arafa, Altaf A. Wani
    Abstract:

    Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin-induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin-induced DNA damage, but can be induced by cisplatin treatment. DDB2-deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin-resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin-sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin-induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl-2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl-2 expression through decreasing Bcl-2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.

Pradip Raychaudhuri - One of the best experts on this subject based on the ideXlab platform.

  • DDB2 regulates epithelial to mesenchymal transition emt in oral head and neck squamous cell carcinoma
    Oncotarget, 2018
    Co-Authors: Pradip Raychaudhuri, Prashant V Bommi, Sriram Ravindran, Srilata Bagchi
    Abstract:

    DDB2 is a sensor of DNA damage and it plays an important role in Global Genomic Repair (GG-NER). Our previous studies show that DDB2 is involved in the regulation of metastasis in colon adenocarcinoma. Squamous Cell Carcinomas in the Oral/Head & Neck region (HNSCC) are particularly aggressive due to high incidence of recurrence and distant metastasis. In this study, we show that DDB2 expression is downregulated in advanced HNSCCs and loss of DDB2 expression coincides with reduced survival. Recent meta-analysis of gene expression data characterized the mesenchymal-type (EMT-type) as one most aggressive cancer cluster in HNSCC. Here, we report that DDB2 constitutively represses mRNA expression of the EMT- regulatory transcription factors SNAIL, ZEB1, and angiogenic factor VEGF in HNSCC cells. As a result, re-expression of DDB2 in metastatic cells reversed EMT with transcriptional upregulation of epithelial marker E-cadherin, and downregulation of mesenchymal markers N-cadherin, Vimentin, and Fibronectin. Interestingly, in a reverse assay, depletion of DDB2 in non-metastatic cells induced expression of the same EMT-regulatory transcription factors. TGFβs are major regulators of Snail and Zeb1, and we observed that DDB2 transcriptionally regulates expression of TGFB2 in HNSCC cells. Re-expression of DDB2 in mouse embryonic fibroblasts (MEFs) isolated from DDB2 (-/-) knockout-mice resulted in repression of EMT-regulatory factors Zeb1, Snail and Tgfb2. Taken together, these results support the active role of DDB2 as a candidate suppressor of the EMT-process in HNSCC. Early detection leads to significantly higher survival in HNSCC and DDB2 expression in tumors can be a predictor of EMT progression.

  • DDB2 Is a Novel Regulator of Wnt Signaling in Colon Cancer
    Cancer research, 2017
    Co-Authors: Shuo Huang, Srilata Bagchi, Damiano Fantini, Bradley J. Merrill, Grace Guzman, Pradip Raychaudhuri
    Abstract:

    Deregulation of the Wnt/β-catenin signaling pathway drives the development of colorectal cancer, but understanding of this pathway remains incomplete. Here, we report that the damage-specific DNA-binding protein DDB2 is critical for β-catenin–mediated activation of RNF43, which restricts Wnt signaling by removing Wnt receptors from the cell surface. Reduced expression of DDB2 and RNF43 was observed in human hyperplastic colonic foci. DDB2 recruited EZH2 and β-catenin at an upstream site in the Rnf43 gene, enabling functional interaction with distant TCF4/β-catenin–binding sites in the intron of Rnf43 . This novel activity of DDB2 was required for RNF43 function as a negative feedback regulator of Wnt signaling. Mice genetically deficient in DDB2 exhibited increased susceptibility to colon tumor development in a manner associated with higher abundance of the Wnt receptor–expressing cells and greater activation of the downstream Wnt pathway. Our results identify DDB2 as both a partner and regulator of Wnt signaling, with an important role in suppressing colon cancer development. Cancer Res; 77(23); 6562–75. ©2017 AACR .

  • Accumulation of Reactive Oxygen Species and Induction of Premature Senescence: Role of DDB2
    Tumor Dormancy Quiescence and Senescence Volume 1, 2013
    Co-Authors: Nilotpal Roy, Pradip Raychaudhuri
    Abstract:

    Oxidative stress results from an imbalance between free radical formation and anti-oxidant defense. Reactive Oxygen Species (ROS) have been implicated in several oxidative stress related pathological disorders such as aging, cancer and neurological abnormalities. Thenucleotide excision repair protein DDB2 plays an important role in the regulation of ROS. DDB2 deficient human or murine cells fail to accumulate ROS following DNA damage. The lack of ROS accumulation in DDB2 deficiency results from high-level expression of the anti-oxidant genes MnSOD and Catalase. DDB2 represses expression of these anti-oxidant genes by recruiting Cul4a and Suv39h and by increasing Histone3K9 tri-methylation in the promoter region. Moreover, expression of DDB2 is induced by ROS. Thus, upon oxidative stress, DDB2 functions in a positive feedback loop by repressing the ROS scavenger genes to cause a persistent accumulation of ROS. DDB2 mediated ROS-accumulation is related to premature senescence and its role in inhibiting skin tumorigenesis. Following UV damage, DDB2 induces ROS accumulation in the skin, triggering a senescence response that contributes to suppression of skin cancer. Thus, in addition to its role in NER, DDB2 plays an important role in the DNA damage induced ROS accumulation and ROS induced premature senescence, which are significant in the suppression of skin cancer.

  • Tumor regression by phenethyl isothiocyanate involves DDB2.
    Cancer biology & therapy, 2012
    Co-Authors: Nilotpal Roy, Indira Elangovan, Srilata Bagchi, Pradip Raychaudhuri
    Abstract:

    Phenethyl isothiocyanate (PEITC) is a promising cancer chemopreventive agent commonly found in edible cruciferous vegetables. It has been implicated also for therapy, and is in clinical trial for lung cancer. Here, we provide evidence that the tumor suppressive effect of PEITC is related to its ability to induce expression of damaged DNA binding protein 2 (DDB2), a DNA repair protein involved also in apoptosis and premature senescence. DDB2 expression is attenuated in a wide variety of cancers including the aggressive colon cancers. We show that, in colon cancer cells, reactive oxygen species, which are induced by PEITC, augment expression of DDB2 through the p38MAPK/JNK pathway, independently of p53. PEITC-induced expression of DDB2 is critical for inhibition of tumor progression by PEITC. Tumors derived from DDB2-deficient colon cancer cells are refractory to PEITC-treatments, resulting from deficiencies in apoptosis and senescence. The DDB2-proficient tumors, on the other hand, respond effectively to P...

  • Abstract 2410: Reduced expression of DDB2 promotes epithelial to mesenchymal transition in colon cancer
    Tumor Biology, 2012
    Co-Authors: Nilotpal Roy, Pradip Raychaudhuri
    Abstract:

    Invasion and metastasis are the hallmarks of malignant tumor progression and the principal cause of death related to solid tumors. Epithelial to mesenchymal transition (EMT) is intricately associated with cancer progression, as it confers cancer cells the ability to invade and metastasize to a distant organ. Here we identify a novel function of nucleotide excision repair protein DDB2 as an important antagonist of the EMT in colon carcinoma through its maintenance of epithelial phenotype. In human colon carcinoma samples, loss of DDB2 expression is highly correlated with the aggressiveness of the disease. Moreover, stimuli (TGF-beta and hypoxia) that induce EMT inhibit expression of DDB2; and loss of DDB2 is sufficient to promote EMT of colon cancer cells. The mechanism involves DDB2 mediated transcriptional repression of VEGF and MMP3. DDB2 represses these genes by histone deacetylation. We also observed that loss of DDB2 results in resistance to anoikis, an important barrier to metastasis. The lack of anoikis response is associated with increased Akt and ERK activity. Furthermore, treatment with Phenethyl Isothiocyanate (PEITC), a promising cancer chemopreventive component, augmented E-cadherin expression that is related to an increase in DDB2 expression. The present study suggests that anti-metastatic effect of PEITC involves important role of DDB2. Together, our results show that loss of DDB2 expression is both necessary and sufficient for EMT during colon carcinoma progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2410. doi:1538-7445.AM2012-2410

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  • wt uv ddb performs a 3d search on dna whereas the xp e mutant k244e DDB2 mutant slides
    Biophysical Journal, 2013
    Co-Authors: Harshad Ghodke, Ching L Hsieh, Vesna Rapicotrin, Arthur S. Levine, Hong Wang, Gregory A Gibson, Simon C Watkins, Bennett Van Houten
    Abstract:

    The DNA damage binding protein complex (UV-DDB) recognizes ultraviolet light (UV) induced lesions such as 6-4 photoproducts (6-4PPs) and cyclobutane pyrimidine dimers (CPDs) in DNA and initiates human nucleotide excision repair in chromatin. Crystallographic studies have revealed that UV-DDB binds to damaged DNA as a heterodimer of DDB1 and DDB2 on short DNA substrates; however, its oligomeric state on longer, physiologically relevant substrates and on nucleosomes remains undetermined. Additionally, the question of how UV-DDB searches a sea of undamaged chromatin for UV-induced lesions remains unresolved. We assayed purified UV-DDB for binding to a 517 bp UV-irradiated, PCR fragment using atomic force microscopy (AFM). Volume analysis revealed that UV-DDB binds primarily as a dimer of heterodimers to UV damaged DNA, with 19% of these UV-DDB dimers binding simultaneously to two DNA molecules In order to study damage recognition in real time, we used a His-tag on UV-DDB (either DDB1 or DDB2) to conjugate quantum dots(QDs). QD-UV-DDB retained DNA damage binding activity, as assayed by electrophoretic mobility shift assays. To identify the search mode, we have employed an oblique angle fluorescence microscopy setup to track single molecules of QD tagged UV-DDB on UV-damaged DNA tightropes. We have identified that WT UV-DDB employs a 3D search to identify DNA damage, with a long residence time when bound to sites of damage. Consistent with several salt-bridges observed in the co-crystal structure, we have found that the mobility of UV-DDB on DNA is salt dependent. Further, we have assayed the disease causing K244E mutant of DDB2 and observed that the mutant retains DNA binding activity, but slides on DNA compared to the WT. Our results reveal the stoichiometry and search mechanism of UV-DDB in damage surveillance.

  • monoubiquitinated histone h2a destabilizes photolesion containing nucleosomes with concomitant release of uv damaged dna binding protein e3 ligase
    Journal of Biological Chemistry, 2012
    Co-Authors: Satoshi Nakajima, Maria G Kapetanaki, Ching L Hsieh, Matthew V Fagerburg, Karen Thickman, Pedro Rodriguezcollazo, Sanford H Leuba, Arthur S. Levine, Vesna Rapicotrin
    Abstract:

    How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4BDDB2. We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4BDDB2 E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4BDDB2 from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.

  • the cullin 4b based uv damaged dna binding protein ligase binds to uv damaged chromatin and ubiquitinates histone h2a
    Cancer Research, 2008
    Co-Authors: Jennifer Guerrerosantoro, Maria G Kapetanaki, Ching L Hsieh, Arthur S. Levine, Ilya Gorbachinsky, Vesna Rapicotrin
    Abstract:

    By removing UV-induced lesions from DNA, the nucleotide excision repair (NER) pathway preserves the integrity of the genome. The UV-damaged DNA-binding (UV-DDB) protein complex is involved in the recognition of chromatin-embedded UV-damaged DNA, which is the least understood step of NER. UV-DDB consists of DDB1 and DDB2, and it is a component of the cullin 4A (CUL4A)–based ubiquitin ligase, DDB1-CUL4ADDB2. We previously showed that DDB1-CUL4ADDB2 ubiquitinates histone H2A at the sites of UV lesions in a DDB2-dependent manner. Mutations in DDB2 cause a cancer prone syndrome, xeroderma pigmentosum group E (XP-E). CUL4A and its paralog, cullin 4B (CUL4B), copurify with the UV-DDB complex, but it is unclear whether CUL4B has a role in NER as a separate E3 ubiquitin ligase. Here, we present evidence that CUL4A and CUL4B form two individual E3 ligases, DDB1-CUL4ADDB2 and DDB1-CUL4BDDB2. To investigate CUL4B9s possible role in NER, we examined its subcellular localization in unirradiated and irradiated cells. CUL4B colocalizes with DDB2 at UV-damaged DNA sites. Furthermore, CUL4B binds to UV-damaged chromatin as a part of the DDB1-CUL4BDDB2 E3 ligase in the presence of functional DDB2. In contrast to CUL4A, CUL4B is localized in the nucleus and facilitates the transfer of DDB1 into the nucleus independently of DDB2. Importantly, DDB1-CUL4BDDB2 is more efficient than DDB1-CUL4ADDB2 in monoubiquitinating histone H2A in vitro. Overall, this study suggests that DDB1-CUL4BDDB2 E3 ligase may have a distinctive function in modifying the chromatin structure at the site of UV lesions to promote efficient NER. [Cancer Res 2008;68(13):5014–22]

  • the ddb1 cul4aDDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group e and targets histone h2a at uv damaged dna sites
    Proceedings of the National Academy of Sciences of the United States of America, 2006
    Co-Authors: Maria G Kapetanaki, Ching L Hsieh, Vesna Rapicotrin, Jennifer Guerrerosantoro, Dawn C Bisi, Arthur S. Levine
    Abstract:

    Xeroderma pigmentosum (XP) is a heritable human disorder characterized by defects in nucleotide excision repair (NER) and the development of skin cancer. Cells from XP group E (XP-E) patients have a defect in the UV-damaged DNA-binding protein complex (UV-DDB), involved in the damage recognition step of NER. UV-DDB comprises two subunits, products of the DDB1 and DDB2 genes, respectively. Mutations in the DDB2 gene account for the underlying defect in XP-E. The UV-DDB complex is a component of the newly identified cullin 4A-based ubiquitin E3 ligase, DDB1-CUL4ADDB2. The E3 ubiquitin ligases recognize specific substrates and mediate their ubiquitination to regulate protein activity or target proteins for degradation by the proteasomal pathway. In this study, we have addressed the role of the UV-DDB-based E3 in NER and sought a physiological substrate. We demonstrate that monoubiquitinated histone H2A in native chromatin coimmunoprecipitates with the endogenous DDB1-CUL4ADDB2 complex in response to UV irradiation. Further, mutations in DDB2 alter the formation and binding activity of the DDB1-CUL4ADDB2 ligase, accompanied by impaired monoubiquitination of H2A after UV treatment of XP-E cells, compared with repair-proficient cells. This finding indicates that DDB2, as the substrate receptor of the DDB1-CUL4A-based ligase, specifically targets histone H2A for monoubiquitination in a photolesion-binding-dependent manner. Given that the loss of monoubiquitinated histone H2A at the sites of UV-damaged DNA is associated with decreased global genome repair in XP-E cells, this study suggests that histone modification, mediated by the XPE factor, facilitates the initiation of NER.

  • true xp group e patients have a defective uv damaged dna binding protein complex and mutations in DDB2 which reveal the functional domains of its p48 product
    Human Molecular Genetics, 2003
    Co-Authors: Vesna Rapicotrin, Arthur S. Levine, Dawn C Bisi, Tiziana Nardo, Valentina Navazza, Elena Botta, Mary P Mclenigan, Miria Stefanini
    Abstract:

    Xeroderma pigmentosum (XP) is a skin cancer-prone autosomal recessive disease characterized by inability to repair UV-induced DNA damage. The major form of XP is defective in nucleotide excision repair (NER) and comprises seven complementation groups (A–G). The genes defective in all groups have been identified unambiguously with the exception of group E. The cells of some XP-E patients are deficient in a protein complex (consisting of two subunits: p127/DDBI and p48/DDB2) which binds to UV-damaged DNA (UV-DDB) and is specifically involved in the removal of photoproducts from the non-transcribed regions of the genome. However, other XP-E patients have been reported not to lack UV-damaged DNA binding activity (DDB þ ). Here we describe several genetically unrelated XP-E patients, not previously analyzed in depth, each carrying two mutated alleles for DDB2, causing either a single amino acid change or a protein truncation or internal deletion. These defects result in a severe decrease of detectable p48 protein, abolish interaction with the p127 subunit, and produce a deficiency in UV-DDB binding activity (DDB � ). The role of p48 in the repair defect of these patients was demonstrated in vivo and in vitro. Investigation of four DDB þ cell strains from patients previously assigned to XP-E, allowed us to reclassify all of them into other groups and to show that they do not share the molecular and biochemical features typical for XP-E. Besides confirming that the true XP-E phenotype is DDB � , resulting from defects in a single gene, DDB2, our results identify the functional domains of the corresponding p48 protein.