DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair Through DDB2 DNA-Binding and Cullin 4A Ubiquitin Ligase Activity

Research Article

DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair through DDB2 DNA-binding and Cullin 4A Ubiquitin Ligase Activity

Jinyou Li,1 Qi-En Wang,1 Qianzheng Zhu,1 Mohamed A. El-Mahdy,1 Gulzar Wani,1 Mette Prætorius-Ibba,1 and Altaf A. Wani1,2,3

Departments of 1Radiology and 2Molecular and Cellular Biochemistry, and 3Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio

Abstract global genomic repair, which removes the lesions in the whole Functional defect in DNA damage binding (DDB) activity has genome, and transcription-coupled repair, which eliminates the a direct relationship to decreased nucleotide excision repair lesions located within the transcribed strands of transcriptionally (NER) and increased susceptibility to cancer. DDB forms a active genes (1). Basic NER contains three distinguishable seg- complex with cullin 4A (Cul4A), which is now known to ments—recognition of damage within the compact chromatin, ubiquitylate DDB2, XPC, and histone H2A. However, the exact incision, and excision of damage containing oligonucleotide and role of DDB1 in NER is unclear. In this study, we show that finally the gap-filling DNA synthesis and ligation (2, 3). The DDB1 knockdown in human cells impaired their ability to biological consequences of NER defects are apparent from the efficiently repair UV-induced cyclobutane pyrimidine dimers inherited multisystem disorders, e.g., xeroderma pigmentosum (CPD) but not 6-4 photoproducts (6-4PP). Extensive nuclear (XP), which is characterized by hypersensitivity to sun light and protein fractionation and chromatin association analysis extremely high incidence of cancer. XP-E is one of these XP forms revealed that upon irradiation, DDB1 protein is translocated and the cells from XP-E patients show the mildest deficiency in from a loosely bound to a tightly bound in vivo chromatin NER among all the XP patients. fraction and the DDB1 translocation required the participa- XP-E cells lack UV-DDB activity associated with a damaged tion of functional DDB2 protein. DDB1 knockdown also DNA-binding (DDB) protein complex (4), which comprises two affected the translocation of Cul4A component to the tightly subunits, DDB1 (p127) and DDB2 (p48; refs. 5–8). Despite the bound form in UV-damaged chromatin in vivo as well as its known ability of DDB protein to bind UV-irradiated DNA, the recruitment to the locally damaged nuclear foci in situ. specific role of DDB in NER has only recently begun to take a However, DDB1 knockdown had no effect on DNA damage clearer shape. The role of DDB2 subunit in NER has been binding capacity of DDB2. The data indicated that DDB2 can extensively studied and the activity-altering sequence changes DDB2 bind to damaged DNA in vivo as a monomer, whereas Cul4A have been identified in the gene in eight human XP-E cases recruitment to damage sites depends on the fully assembled thus far confirmed. On the other hand, no sequence changes have DDB1 complex. Our data also showed that DDB1 is required for the been found in gene (9–11). Growing body of evidence has UV-induced DDB2 ubiquitylation and degradation. In summa- confirmed that DDB2 is undeniably involved in global genomic ry, the results suggest that (a) DDB1 is critical for efficient NER repair. For example, several studies showed that the cells from of CPD; (b) DDB1 acts in bridging DDB2 and ubiquitin ligase some XP-E patients have a partial deficiency in NER (9, 12–14). Cul4A; and (c) DDB1 aids in recruiting the ubiquitin ligase Microinjection of the purified DDB complex into XP-E cells activity to the damaged sites for successful commencement reversed the NER defect (11, 15, 16). Some rodent cell lines, such of lesion processing by NER. (Cancer Res 2006; 66(17): 8590-7) as Chinese hamster V79 cells, rarely express endogenous rodent DDB2, and show very slow removal of cyclobutane pyrimidine Introduction dimer (CPD) from the genome. This repair defect can be restored either by reactivating DDB2 gene expression, or by ectopic DNA repair is the critical cellular process for preventing cell expression of human DDB2 in the cells (9, 12, 13). NER can be killing, mutations, replication errors, persistent DNA damage, and reconstituted with purified components and damaged DNA in genomic instability. Abnormalities of DNA repair have been the absence of DDB. Adding DDB to the purified NER system, using implicated in cancer and aging. Nucleotide excision repair (NER) a substrate with a single CPD lesion, has been shown to result in is the most important DNA repair pathway that fixes the majority no stimulation (17), to a few fold stimulation (18), and even a of bulky lesions in DNA. These lesions include UV-induced 17-fold stimulation (14). Therefore, at present, DDB is believed to photoproducts and bulky adducts e.g., those arising from exposure be relevant only to the NER within the chromatin context and it to carcinogenic chemicals. NER includes two distinct subpathways, could be having a minor accessory role in NER in vitro when chromatin-free DNA is the substrate (19–21). DDB1 was first isolated, together with DDB2, as a subunit of a Note: J. Li and Q.E. Wang contributed equally to this work. heterodimeric DDB protein complex that shows a high affinity to Current address for J. Li: Department of Occupational Health, Shanxi Medical University, 030001 Taiyuan, P.R. China. UV-damaged DNA (for a review, see ref. 22). DDB1 is a relatively Requests for reprints: Altaf A. Wani, Department of Radiology, The Ohio State abundant cellular protein and is present in excess over that of University, 2001 Polaris Parkway, Columbus, OH 43240. Phone: 614-293-0865; its critical partner, DDB2, in unirradiated HeLa cells (5, 23). DDB1 Fax: 614-293-0802; E-mail: [email protected]. I2006 American Association for Cancer Research. is vital for normal cell function and evolutionarily conserved in doi:10.1158/0008-5472.CAN-06-1115 mammals, worms, insects, and plants. To date, no deleterious

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Role of DDB1 in Nucleotide Excision Repair naturally occurring mutations have been found in mammalian (Invitrogen, Carlsbad, CA). Cell transfections were conducted with FUGENE DDB1. More recently, DDB1 and DDB2 were found to form a 6 transfection reagent (Roche, Indianapolis, IN). For the establishment complex with cullin 4A (Cul4A), which serves as an E3 ubiquitin of stably transfected cell line, Myc-tagged Cul4A expression construct ligase (24–26). Cul4A is a member of the cullin family of proteins. was introduced into HeLa-DDB2 cells, or pcDNA3.1-V5-DDB2 construct was transfected into 041 cells. The transfectants were selected with G418 It has been proposed that Cul4A proteins function as E3 ligases, (500 Ag/mL) to obtain the stably expressing transfected cell lines. All the which are involved in selecting specific targets for ubiquitylation. transfectants were confirmed for cognate protein expression through However, unlike other ligases, Cul4A cannot bind to substrates Western blot analysis. directly, but requires a specificity factor to recruit target substrates RNA interference. DDB1 siRNA oligonucleotides and nonspecific siRNA to Cul4A. DDB1 is found to associate with Cul4A and considered were synthesized by Dharmacon (Lafayette, CO) in a purified and annealed to serve the function of such substrate-recruiting factor for Cul4A- duplex form. The sequences, targeting four different regions of DDB1 Roc1 ligases (24, 26–28). In combination with Cul4A, DDB1 has gene, were as follows: duplex-1, 5¶-UAACAUGAGAACUCUUGUC-3¶; duplex- been proposed to either directly dock a substrate to the E3 2, 5¶-AUAAACAGCAGGUCCUUGC-3¶; duplex-3, 5¶-CCAUUAUCAAGGUAU- ¶ ¶ ¶ machinery or indirectly recruit a substrate through an additional GUCA-3 ; duplex-4, 5 -UUAUUAUCGCGAUCUAGUG-3 . siRNA transfection adaptor(s) (26). COP9 signalosome has also been found in complex was done with Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. with DDB1-Cul4A-Roc1, regulating the ubiquitin ligase activities of Whole cell extract preparation and fractionation of cellular this complex (25). DDB1 protein has lately attracted considerable proteins. For whole cell extract preparation, the cells were harvested interest because of its multiple functions in cells. However, by trypsinization and lysed by boiling for 10 minutes in a sample buffer the action of DDB1 in the context of DNA repair has received [2% SDS, 10% glycerol, 10 mmol/L DTT, 62 mmol/L Tris-HCl (pH 6.8), very little attention although DDB is now clearly implicated in NER and protease inhibitor cocktail]. Fractionation of cellular proteins was and the role of its DDB2 component is reasonably well established. conducted as described previously (30). Briefly, the cells were trypsinized We hypothesized that DDB1 can contribute to NER through and resuspended in hypotonic buffer [10 mmol/L HEPES (pH 7.9), 10 bridging Cul4A ligase and DDB2. To address this question, we used mmol/L KCl, 1.5 mmol/L MgCl2, and protease inhibitor cocktail] and DDB1 small interfering RNA (siRNA) to abrogate the expression of treated with 0.1% Triton X-100 to get fraction S1, which was further DDB1 in the mammalian cells and investigated the effect of DDB1 centrifuged at high speed to yield the clear supernatant cytoplasmic soluble deficiency on the removal of UV-induced photolesions as well as proteins. The nuclear pellet was then washed twice with isotonic sucrose buffer [50 mmol/L Tris-HCl (pH 7.4), 0.25 mol/L sucrose, 5 mmol/L MgCl , the recruitment of DDB2 and Cul4A to such damage sites. Our 2 and protease inhibitor cocktail] and treated with higher concentration of results showed that DDB1 is indeed required for the efficient NER Triton X-100 (1%) in LS buffer [10 mmol/L Tris-HCl (pH 7.4), 0.2 mmol/L of CPD, but not 6-4PP, through linkage of DDB2 DNA-binding MgCl2, and protease inhibitor cocktail] to remove the nuclear envelope and and Cul4A E3 ligase activities. Surprisingly, we also found that get the fraction of nucleoplasmic soluble proteins. The nuclear pellet was DDB2 can be recruited to the damage sites in vivo in the absence of further extracted consecutively with increasing concentrations (0.3, 0.5, and its heterodimerization with DDB1. 2.0 mol/L) of NaCl in LS buffer to result in supernatant fractions, respectively, designated as 0.3, 0.5, and 2.0. The nuclear residue comprising of DNA and nuclear matrix was dissolved by sonication in LS buffer. Each Materials and Methods protein fraction, corresponding to an equivalent cell number, was loaded for Cell culture and treatments. The normal human skin fibroblasts SDS-PAGE and analyzed by immunoblotting with indicated antibodies. (OSU-2) were established and maintained in culture as described previously In vivo ubiquitylation. HeLa-DCH cells were first transfected with

(29). HeLa cells stably expressing NH2-terminal FLAG-hemagglutinin (HA)– 100 nmol/L DDB1 siRNA for 24 hours and then transfected with 2 Ag tagged DDB2 (HeLa-DDB2) were kindly provided by Dr. Yoshihiro Nakatani HA-ubiquitin–expressing plasmid for another 24 hours. MG132 (10 nmol/L) (Dana-Farber Cancer Institute, Boston, MA). HeLa-DDB2 cells stably was added to the culture 0.5 hours before UV irradiation at 40 J/m2 and expressing Myc-tagged Cul4A (HeLa-DCH) were established in our kept in the same medium for 2 hours. Cells were collected and the nuclei laboratory. Li-Fraumeni syndrome fibroblast 041 cell line (p53-Null, were extracted with NE buffer [20 mmol/L HEPES (pH 7.9), 25% glycerol, harboring a codon 184 frameshift mutation, resulting in premature 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, and protease termination of translation of p53 protein) was provided by Dr. Michael inhibitor cocktail] and immunoprecipitated with anti-FLAG M2 affinity gel Tainsky (M.D. Anderson Cancer Center, Austin, TX). 041-DDB2 cells stably (Sigma, St. Louis, MO). The immunoprecipitates were subjected to Western expressing V5-His-tagged DDB2 were established in our laboratory. XP-E blotting and detected with anti-ubiquitin antibody (Santa Cruz Biotech- cell line (GM01389) was obtained from National Institute of General nology, Santa Cruz, CA). Medical Sciences Human Genetic Cell Repository (Coriell Institute for Western blot analysis of proteins. The proteins were quantified, Medical Research, Camden, NJ). All the cell lines, except XP-E, were grown separated by SDS-PAGE, and the immunoblot analysis was done by using in DMEM supplemented with 10% FCS and antibiotics or 500 Ag/mL G418 chemiluminescent detection. The following antibodies were used: rabbit (only for stably transfected cell lines) at 37jC in a humidified atmosphere of anti-DDB1 and Cul4A antibodies (kindly provided by Dr. Yue Xiong, Â 5% CO2. XP-E cells were grown in MEM with 2 essential amino acids, University of North Carolina, NC; both at 1:1,000), rabbit anti-DDB2 2Â nonessential amino acids, and 2Â vitamin, supplemented with 15% FCS antibody (produced by us as previously described in ref. 31; 1:1,000), rabbit and antibiotics. For overall UV exposure, the cells were washed with PBS, anti-ubiquitin antibody (Santa Cruz Biotechnology, 1:1,000), monoclonal irradiated at varying UV doses, and incubated in suitable medium for the anti-actin (Neomarkers, Fremont, CA, 1:500). desired time period. The irradiation was done with a germicidal lamp at a Localized micropore UV irradiation and immunofluorescent stain- dose rate of 0.8 J/m2/s as measured by a Kettering model 65 radiometer ing. The cells growing on glass coverslips were washed with PBS and UV (Cole Palmer Instrument, Co., Vernon Hill, IL). irradiated as described previously (32). Briefly, an isopore polycarbonate Expression constructs and cell transfection. Myc-tagged Cul4A filter (Millipore, Bedford, MA), containing pores of 5 Am in diameter, was construct was kindly provided by Dr. Yue Xiong (University of North placed on the top of the cell monolayer. The filter-covered coverslips were Carolina, Chapel Hill, NC). HA-tagged ubiquitin construct was obtained irradiated from above with UV-C (254 nm) at 100 J/m2. The filter was then from Dr. Dirk Bohmann (European Molecular Biology Laboratory, gently removed and the cells were incubated in serum-free medium for Heidelberg, Germany). DDB2 cDNA was amplified using pBJ5-DDB2 0.5 hours followed by fixation and permeabilization with 2% paraformal- expression construct (provided by Dr. Gilbert Chu, Stanford University, dehyde and 0.5% Triton X-100 in PBS. The cells were then double stained Stanford, CA) as template and subcloned into pcDNA3.1-V5 Vector with mouse anti-HA (to visualize DDB2; Roche; 1:100) and rabbit anti-CPD www.aacrjournals.org 8591 Cancer Res 2006; 66: (17). September 1, 2006

(UV-2, generated in our lab; 1:1,000), or mouse anti-Myc (to visualize Cul4A; DDB1 protein translocates to damaged chromatin sites in Roche; 1:40) and rabbit anti-CPD (UV-2) as described previously (32). a DDB2-dependent manner. Previous studies have shown that Fluorescence images were obtained with a Nikon Fluorescence Microscope DDB1 is not needed for the repair of damage in the NER system 80i (Nikon, Tokyo, Japan) fitted with appropriate filters for FITC and Texas reconstituted in vitro (19, 20). However, in vivo studies have shown red. The digital images were then captured with a cooled charge coupled that ectopically expressed DDB1 could be recruited to the damage device camera and processed with the help of its SPOT software (Diagnostic Instruments, Sterling Heights, MI). sites following UV irradiation (33), suggesting that DDB1, as a Quantitation of CPD and 6-4PP by immuno-slot-blot assay. The subunit of DDB, is capable of binding to the damaged sites. In this amounts of different photoproducts in DNA were quantified by previously study, we investigated the chromatin binding and UV-induced established noncompetitive immuno-slot-blot assay (29). Briefly, after UV translocation of DDB1 by using cellular fractionation and exposure (20 J/m2) and desired incubation periods, OSU-2 cells were immunoblot analysis. OSU-2 cells, with or without UV irradiation, recovered by trypsinization and immediately lysed for DNA isolation. The were differentially lysed and the lysates were fractionated into same amount of DNA samples were loaded on nitrocellulose membranes six fractions according to their resistance to detergent or salt and the amounts of CPD or 6-4PP were detected with monoclonal anti-CPD extraction. As shown in Fig. 2A, most of the DDB1 protein in OSU-2 antibody (TDM2, 1:2,000) or monoclonal anti-6-4PP (64M-2, 1:2,000) cells resides in the 0.3 mol/L fraction, indicating that DDB1 is antibody (MBL International, Co., Woburn, MA). The intensity of each loosely bound to chromatin. Following UV irradiation, there is a band was determined by laser densitometric scanning and the amount of damage remaining, compared with the initially induced DNA damage, was prominent shift in DDB1 chromatin association as almost the used to calculate the relative repair rates. entire amount of available DDB1 translocates to the 2.0 mol/L fraction, indicating that the protein is avidly immobilized at the UV damage sites in chromatin. It is clear that the DDB1 has nuclear Results localization and its weak association with undamaged chromatin DDB1 is required for efficient NER of CPD, but not of becomes stronger in the presence of UV-induced damage. The fate 6-4PP, in mammalian cells. Unlike DDB2, DDB1 is vital for of DDB1 protein upon irradiation in DDB2-deficient XP-E cells is organism survival as there are no living individuals with a deficiency in this protein. To understand the role of DDB1 in NER, we applied RNA interference technology to knock down the expression of DDB1 in mammalian cells and analyzed the removal of UV-induced CPD and 6-4PP in these cells. First, we checked the knockdown efficiency of siRNA specific for DDB1. Four different duplexes of siRNA, targeting various sequences of DDB1 mRNA, were individually transfected into normal human fibroblast OSU-2 cells at 100 nmol/L for 48 hours. The whole cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-DDB1 antibody. As shown in Fig. 1A, all the DDB1 siRNA duplexes were able to knock down the expression of DDB1, albeit to varying degrees. Among them, duplex-1 siRNA exhibited the highest proficiency (>90%) to deplete the cellular DDB1 protein, and the inhibition of DDB1 expression was sustained up to 72 hours following transfection (data not shown). We also tested the ability of the RNA interference duplexes in HeLa cells and found knockdown efficiency comparable with that of OSU-2 cells (data not shown). Therefore, duplex-1 was used in all the subsequent experiments to determine the effects of DDB1 abrogation on DNA repair. OSU-2 cells were transfected with DDB1 siRNA duplex-1 for 48 hours to achieve the loss of cellular DDB1 protein, followed by UV irradiation at 20 J/m2, and a repair time up to an additional 24 hours. An aliquot (30 ng) of the DNA isolated at varying postrepair times was subject to the analysis of initial and remaining amount of lesion by immuno- slot-blot analysis with cognate anti-CPD or anti-6-4PP antibodies. As shown in Fig. 1B and C, nonspecific siRNA-transfected OSU-2 cells were able to repair >40% CPD at 24 hours following UV irradiation, which was comparable with repair rate in untrans- Figure 1. DDB1 is required for CPD but not 6-4PP removal. Normal human fected cells (data not shown). However, DDB1 siRNA-transfected fibroblast, OSU-2cells, were transfected with 100 nmol/L of four different cells showed only a 25% repair of CPD during the same period. In DDB1-specific siRNAs for 48 hours to knock down the expression of DDB1 protein. A, cell lysates were loaded on 4% to 12% gradient SDS-PAGE. contrast, the repair efficiency of 6-4PP in DDB1 siRNA-transfected After transferring to nitrocellulose membrane, proteins were detected with cells was identical to that in nonspecific siRNA-transfected cells anti-DDB1 and anti-actin antibodies (arrow, actin band). B to E, cells were UV D E irradiated at 20 J/m2 and incubated for the indicated times. Total DNA was (Fig. 1 and ). We also tested the effect of other three siRNA isolated and 30 ng was loaded for slot-blot analysis. The remaining damage, oligos on the removal of CPD and obtained similar results (data CPD (B and C) and 6-4PP (D and E), in the cells was detected with mouse not shown). These results indicate that DDB1 protein is required anti-CPD and mouse anti-6-4PP antibodies, respectively. The intensity of the bands was calculated by densitometric scanning and the repair efficiency was for the efficient repair of CPD, but not of 6-4PP, in mammalian calculated from relative comparisons of initial and remaining DNA damage. cells. Points (C and E), mean percentage repair of three independent measurements.

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and UV-irradiated normal human OSU-2 cells. Figure 2C shows that about half of the cellular Cul4A resides in cytoplasm and major portion of the nuclear Cul4A occurs in the loosely bound form of the chromatin. However, upon UV irradiation of cells, the nuclear Cul4A shows distinct translocation from the 0.3 to 2.0 mol/L fraction, indicating that UV irradiation induces tight binding of Cul4A to damaged chromatin. It is worth mentioning that Cul4A recovered in the 2.0 mol/L fraction in all these experiments represent a modified form as the corresponding bands show an easily discernable slower migration during SDS-PAGE. It is known that Cul4A can be modified by NEDD8 ubiquitin-like protein (neddylation) and this modification is proposed to stimulate the ubiquitin ligase activity (25, 34, 35). We believe that the slow- migrating Cul4A is a neddylated protein and it is this modified form that binds to damaged chromatin tightly. Altogether, these fractionation/association results indicate that DDB1 subunit component, aided strictly by the DDB2 subunit, translocates and binds tightly to UV-damaged sites in chromatin. DDB1 is required for UV-induced Cul4A, but not DDB2 recruitment. It is already known that DDB1, DDB2, and Cul4A form a complex with an inherent E3 ubiquitin ligase activity (25), and three subunits of this complex can be recruited to UV-induced damage sites (14, 33, 36). Nevertheless, the role of DDB1 in the UV-induced recruitment of Cul4A and/or DDB2 is still unclear. To address this question, we first investigated the role of DDB1 in UV-induced translocation of Cul4A and DDB2 by using RNA Figure 2. UV-induced tight binding of DDB1 to damaged chromatin is DDB2- interference and cellular fractionation. OSU-2 cells were trans- dependent. A, normal human fibroblast (OSU-2) cells and DDB2-deficient (XP-E) cells were UV irradiated at 20 J/m2 and incubated for another 30 minutes. fected with DDB1 siRNA or mock-transfected for 48 hours to knock The cells were fractionated as described in Materials and Methods. Individual down the expression of DDB1. The cells were UV irradiated at protein fractions, corresponding to equivalent cell number, were subjected to 2 SDS-PAGE and analyzed by immunoblotting with anti-DDB1 antibody. B, DDB2 20 J/m and further incubated for 30 minutes to enable the and p53-deficient (041) cells, and 041 cells with stable expression of ectopic initiation of damage processing. The cellular fractionation was DDB2, were UV irradiated, fractionated, and analyzed by immunoblotting. done as before and the nuclear fractions [0.3, 0.5, and 2.0 mol/L, The translocation of DDB1 protein was detected with anti-DDB1 antibody. C, OSU-2cells were UV irradiated at 20J/m 2 and processed as in (A and B). and nuclear residue] were subjected to Western blotting. As shown The translocation of Cul4A protein was detected with anti-Cul4A antibody. in Fig. 3, in the absence of DDB1 siRNA transfection, Cul4A S2, cytoplasmic soluble proteins; TW, nucleoplasmic soluble proteins; translocates almost quantitatively from the lower 0.3 to 2.0 mol/L 0.3, proteins binding to chromatin loosely; 0.5, proteins binding to chromatin with intermediate affinity; 2.0, proteins binding to chromatin tightly; NR, nuclear fraction upon UV irradiation. Cul4A translocation is easily seen to residue. be compromised upon DDB1 knockdown as considerable amount of cellular Cul4A still resides in 0.3 mol/L fraction following UV irradiation and only a minute amount associated with 2.0 mol/L shown in Fig. 2A. Unlike DDB2-proficient OSU-2 cells, most of the fraction. These results indicate that DDB1 is required for tight DDB1 protein was localized in the cytoplasmic fraction of XP-E binding of Cul4A to chromatin following UV irradiation. We also cells. This is consistent with the knowledge that DDB2 is required in the transfer of cytoplasmic DDB1 to the nucleus (23). About half of the DDB1 present in the nucleus is in the loosely bound form as it was released into 0.3 mol/L fraction. Moreover, the distribution of this chromatin-bound DDB1 remained unchanged following UV irradiation of XP-E cells. This indicates that the UV-induced tight-binding of DDB1 to damaged chromatin, observed in normal cells, results from the presence of active DDB2 protein. To confirm the active role of DDB2, we investigated the UV-induced redistribution of DDB1 in another DDB2-deficient 041 cell line as well as the 041 cells with stably transfected DDB2 cDNA. 041 cells lack DDB2 because it is a p53-null cell line that fails to induce p53 downstream effector DDB2 protein (12). As shown in Fig. 2B, the DDB2-deficient 041 cells exhibit a DDB1 distribution pattern identical to XP-E cells and it remains unaltered following Figure 3. Knockdown of DDB1 protein compromises UV-induced Cul4A but not DDB2translocation within chromatin. OSU-2cells were transfected with UV irradiation. On the other hand, UV irradiation results in a clear 100 nmol/L siRNA specific for DDB1 or mock transfected for 48 hours. The cells translocation of DDB1 protein to a tight-binding form in 041 cells were UV irradiated at 20 J/m2, further incubated for 30 minutes, and then in which DDB2 expression was ectopically restored. As part of subjected to cellular fractionation as described in Materials and Methods. The fractions obtained from nuclei (0.3, 0.5, 2.0, and nuclear residue) were the protein translocation experiments, we also determined the loaded for SDS-PAGE and analyzed by Western blotting with anti-Cul4A and localization and fate of Cul4A within chromatin in unirradiated anti-DDB2antibodies. www.aacrjournals.org 8593 Cancer Res 2006; 66: (17). September 1, 2006

Figure 4. Knockdown of DDB1 protein blocks UV-induced in situ recruitment of Cul4A but not DDB2to damage sites. HeLa-DCH cells, grown on coverslips, were UV irradiated at 100 J/m2 through a 5 Am isopore filter and maintained in fresh medium for 30 minutes. The cells were fixed and double immunostained with anti-Myc (Cul4A) and anti-CPD antibodies (A) or anti-HA (DDB2) and anti-CPD antibodies (B), as described in Materials and Methods. The total number of Cul4A or DDB2foci and CPD foci were counted from at least five separate fields. The ratios of Cul4A to CPD or DDB2to CPD were calculated from the numbers for independent fluorescent foci.

analyzed the redistribution of DDB2 following UV irradiation in sites requires DDB1 protein. Meanwhile, we detected the expected OSU-2 cells with and without the knockdown of cellular DDB1. As recruitment of DDB2 in HeLa-DCH cells without DDB1 knockdown previously reported (30), UV irradiation induces a prompt and following UV irradiation. As seen in Fig. 4B, DDB2 is readily unambiguous translocation of DDB2, from 0.3 to 2.0 mol/L fraction, detected at the sites of CPD with 90% damage sites having distinct in the cells with normal DDB1 expression. But surprisingly, in the DNA damage–specific DDB2 recruitment under conditions of absence of DDB1 expression in cells, UV-induced DDB2 transloca- normal DDB1 expression. Consistent with chromatin association tion from loosely bound to tightly bound form within chromatin results above, eliminating DDB1 from cells had no perceivable was completely unaffected. This response, which is in obvious effect on in situ recruitment of DDB2 to damage sites. Greater than contrast to the movement of Cul4A component of the complex, 90% damage-specific DDB2 sites were seen in the absence or indicates that DDB2 can bind tightly to the damaged chromatin as presence of DDB1 knockdown. The data reinforced the contention a monomer and that the participation of partner DDB1 protein is that UV-induced recruitment of DDB2 to the damage sites does not dispensable in this process. To confirm this unique and interesting require DDB1. In essence, these results help conclude that DDB2 observation, we conducted micropore local UV irradiation can be recruited to UV-induced damage sites as an independent combined with in situ immunofluorescent staining to track the component, whereas Cul4A recruitment occurs only in the movement of Cul4A as well as DDB2 within chromatin in the native presence of DDB1, most likely as part of the complex. cellular environment. HeLa-DCH cells with overexpressed Myc- DDB1 participates in NER through the ubiquitylation and tagged Cul4A and FLAG-HA-tagged DDB2 were first transfected degradation of DDB2. It has been shown that DDB2 can be with DDB1 siRNA or mock-transfected for 48 hours. The cells ubiquitylated by DDB-Cul4A E3 ubiquitin ligase in vivo and in vitro growing on coverslips were UV irradiated at 100 J/m2 through (27, 28, 37, 38). Besides, it is also known that Cul4A itself has E3 a filter with pores of 5 Am diameter and then incubated for another activity and that DDB1 acts as an adaptor for Cul4A to ubiquitylate 30 minutes to allow movement of the involved proteins. its substrates, e.g., CDT1 (26). However, the role of DDB1 in the UV-irradiated cells were processed for visualization by double ubiquitylation and degradation of DDB2 has not been established. immunostaining with anti-Myc (Cul4A) and anti-CPD or anti-HA To address this question, we first investigated the UV-induced (DDB2) and anti-CPD antibody combinations. As shown in Fig. 4A, DDB2 degradation in the absence of DDB1 protein. As shown in Cul4A is readily detected at the CPD DNA damage sites following Fig. 5A, knockdown of DDB1 expression by its specific siRNA UV irradiation in the cells with normal expression of DDB1. abolished the key UV-induced early response, i.e., prominent Greater than 90% damage sites exhibited the concomitant Cul4A degradation of DDB2 protein. This indicated that the presence recruitment. However, the UV-induced recruitment of Cul4A was of DDB1 is a key requirement for UV-induced DDB2 degradation dramatically inhibited upon DDB1 knockdown in HeLa-DCH cells in irradiated cells. Next, we analyzed the effect of DDB1 on UV- as <10% damage sites showed detectable Cul4A recruitment. This induced DDB2 ubiquitylation in vivo. HeLa-DCH cells were result confirmed that UV-induced Cul4A recruitment to damage transfected with DDB1 siRNA or mock-transfected for 24 hours

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Role of DDB1 in Nucleotide Excision Repair and then transfected with HA-tagged ubiquitin expression functionally similar to that of DDB2 (12, 30, 39). This is not construct for another 24 hours. Proteasome inhibitor MG132 was surprising as the underlying mechanism to initiate productive added to the cultures 1 hour before UV irradiation and kept in the repair is based on the binding of the heterodimeric protein to DNA cultures during the period of repair to ensure the detection of damage. Earlier studies have reported that DDB2 is required for ubiquitylated proteins. The cell lysates was immunoprecipitated the recruitment of exogenously transfected DDB1 to the local with anti-FLAG M2 affinity gel and the immunoprecipitates were UV-induced damage sites (33). Here, we tested the chromatin detected by Western blotting with anti-ubiquitin antibody. As binding of endogenous DDB1 protein following UV irradiation and shown in Fig. 5B, UV irradiation enhanced the overall cellular show that almost all the native DDB1 in the nuclei translocates ubiquitylation of DDB2 in the cells normally expressing DDB1 from loosely bound to a tightly bound chromatin form upon UV protein (lane 3 versus lane 2). However, upon DDB1 knockdown, irradiation. The DDB1 translocation mimics the UV-induced not only the physiologic, but also the UV-induced, ubiquitylation of response observed with native cellular DDB2 (30). Nevertheless, DDB2 was significantly inhibited (lane 4 versus lane 2 and lane 5 DDB1 translocation to damage is strictly dependent on the versus lane 3). These data indicated that DDB1 is an important presence of functional DDB2 protein. For example, UV-induced adaptor component for Cul4A to execute its E3 ligase activity to translocation of DDB1 failed to occur in two different DDB2- ubiquitylate the target substrate, DDB2. deficient XP-E and 041 cells. However, restoration of DDB2 in 041 cells, by the ectopic expression, also restored the normal Discussion translocation of DDB1. In essence, the data suggest that DDB1 is able to functionally participate in NER only upon its intimate Presence of DDB2 protein is critical for DDB1 to help physical interaction with DDB2 protein component. initiate NER. In this study, we have provided evidence for the DDB and Cul4A cooperate in initiating NER. Several studies direct participation of DDB1 in NER of CPD, which is known to have shown that restoration of DDB2 in DDB2-deficient cells repair at a slower rate in different kinds of mammalian cells. enhanced the efficiency of CPD removal (14, 30, 39). In addition, However, DDB1 was not needed for the removal of another major our recent work has shown that Cul4A is required for global photolesion, 6-4PP, which is rapidly eliminated from DNA of living genomic repair of CPD but not of 6-4PP (40). In the present work, organisms. This differential lesion-specific role of DDB1 in NER is we show that DDB1, as a subunit of DDB-Cul4A E3 ligase, inde- pendently contributes to the efficient global genomic repair of CPD but not of 6-4PP. It is established that a defect in any one of three major subunits of DDB-Cul4A complex could impair its E3 ligase activity (26, 36). Therefore, the requirement of DDB-Cul4A E3 ligase activity for initiating efficient NER could be affected by selective abrogation of any of these components as DDB1, DDB2, and Cul4A must work cooperatively to participate in NER. To date, the known NER-related substrates of DDB-Cul4A E3 ligase include DDB2 itself, XPC, and histone H2A (27, 28, 36–38). DDB2 can be ubiquitylated by DDB-Cul4A E3 ligase in vivo as well as in vitro (27, 28, 37, 38). Upon ubiquitylation, DDB2 is promptly degraded inside the cell via proteasome (39–41). Moreover, evidence is now fast accumulating in support of the critical role of ubiquitylation and degradation of DDB2 for the necessary recruitment of XPC to the damage sites. First, Sugasawa et al. (37) have shown that UV-DDB seems to lose its high binding affinity for 6-4PP when DDB2 is extensively polyubiquitylated in vitro.Second,the inhibition of DDB2 degradation, by treatment with proteasome inhibitor MG132 or by knockdown of Cul4A, severely affects the recruitment of XPC to DNA damage sites in vivo (40, 42). Therefore, after recognition of damaged DNA (e.g., CPD), DDB is thought to act in the physical handover of DNA lesions to the next repair protein, XPC, for assembling the global genomic repair machinery. In the process, DDB2 component is promptly eliminated via proteasomal degradation. Interestingly, following irradiation, XPC too is ubiquitylated upon arrival at the damage sites (31, 37). However, the similar ubiquitylated state of XPC, although mediated Figure 5. Knockdown of DDB1 comprises UV-induced DDB2ubiquitylation and by the same DDB-Cul4A E3 ligase in vitro, does not trigger its degradation. A, HeLa-DCH cells were transfected with DDB1 siRNA or mock degradation. The significance of this differential fate of two closely transfected for 48 hours. The cells were UV irradiated at 20 J/m2 and incubated for another 2hours. The cell lysates were subjected to SDS-PAGE and detected related factors is not entirely clear. It should be noted that with anti-DDB1, anti-DDB2, and anti-actin antibodies. B, HeLa-DCH cells polyubiquitylated XPC in a cell-free system displays a higher DNA- were first transfected with 100 nmol/L DDB1 siRNA or mock-transfected for 24 hours and then transfected with 2 Ag HA-tagged ubiquitin for another binding activity compared with its unmodified form (37). Thus, 24 hours. MG132 (10 nmol/L) was added to the cultures for 30 minutes; the these in vitro binding experiments seem to suggest that XPC cells were UV irradiated at 40 J/m2 and kept in the same medium for 2hours. ubiquitylation could be playing a role in the regulation of factor The cell lysates were immunoprecipitated with anti-FLAG M2affinity gel, the immunoprecipitates were subjected to immunoblotting, and the Western blots assembly at DNA damage sites to achieve the efficient NER. A were detected with anti-ubiquitin antibody. recent study seems to show that DDB-Cul4A E3 ligase actually www.aacrjournals.org 8595 Cancer Res 2006; 66: (17). September 1, 2006

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. Cancer Research targets histone H2A protein for monoubiquitylation at the UV DDB1 could act in NER through alternate mechanisms. In damage sites (36). Given that the ubiquitylation of H2A is related mammalian cells, DDB1 or DDB1-like proteins have been found to the condensation of chromatin (43), this finding suggests that in a significant number of complexes in association with proteins DDB-Cul4A E3 ligase might contribute to NER by opening the other than DDB2. Martinez et al. (48) have reported that DDB1 condensed chromatin for making the buried damage accessible to interacts with STAGA (SPT3-TAFII31-GCN5L acetylase) complex, additional factors needed for completing NER. The emerging model which contains a histone acetyltransferase, GCN5. The interaction indicates a close coordination of the activities of DDB1, DDB2, and of UV-DDB with STAGA might target the nucleosome acetylase Cul4A components to function in NER within the chromatin activity of GCN5L to damaged chromatin sites to facilitate the context. Among the different complex components, DDB2 has assembly and/or function of the NER machinery on nucleosomes, intrinsic DNA-binding activity (44), Cul4A possesses E3 ligase again supporting a role of UV-DDB in DNA repair within activity, whereas DDB1 interacts with both of them to function as chromatin. Another protein, SAP130, with sequence similarity to an essential bridge to link damaged DNA-binding to ubiquitylation DDB1 has been found in the TBP-free TAFII complex (TFTC). activity. In essence, through the DDB1 adaptor molecule, DDB2 TFTC preferentially binds UV-irradiated DNA and preferentially physically recruits the E3 ligase, to the damaged chromatin sites, acetylates histone H3 in nucleosomes assembled on UV-damaged for the ubiquitylation of NER-related proteins, including itself. DNA, suggesting that UV-damaged, DNA-binding protein in the DDB2 can bind to damaged DNA as a monomer. Although TFTC complex links DNA damage recognition to nucleosome DDB has been studied extensively, there is no clear agreement acetylation (49). In addition, Datta et al. (50) have shown that whether both the DDB1 and DDB2 subunit components are DDB associates with the CBP/p300 family of proteins, in vivo and required for DNA binding. Early on, it was suggested that DDB1, in vitro, suggesting that DDB participates in global genomic which might get activated in a catalytic manner (‘‘hit-and-run’’) by repair by recruiting CBP/p300 to the damaged-chromatin. Rapic- DDB2 (45), possesses damage-specific, DNA-binding activity (46). Otrin et al. (41) has further provided evidence about the Another study has indicated that only the DDB1-DDB2 hetero- interaction between p300 and DDB occurring through the dimeric complex could bind to damaged DNA (8). Recently, DDB1 subunit, and independent of DDB2. It is quite possible Kulaksiz et al. (44) tested recombinant DDB1 and DDB2 for binding that the histone acetyltransferase activities of the CBP/p300 activity in vitro and found that the damaged DNA-binding property proteins induce chromatin remodeling at the damaged sites to of DDB is conferred by the DDB2 subunit. However, the results allow recruitment of the repair complexes. Thus, DDB1 is a from Wittschieben et al. (47) showed that binding activity of DDB versatile cellular factor capable of participating in NER, mainly by is reconstituted only when the two subunits are mixed together; bringing together essential components from different complexes both subunits are therefore necessary and sufficient for binding to the sites of damaged chromatin for the purpose of opening to the DNA substrate. To address this question in vivo, we tested the condensed chromatin structure to allow access to NER the recruitment of DDB2 to the damaged chromatin in the absence machinery. of DDB1 by using two different assays, e.g., movement upon chromatin association and recruitment in locally micropore UV-irradiated sites. Both assays were consistent in revealing that Acknowledgments DDB2 can translocate to damage DNA unaided by DDB1. Thus, the Received 3/24/2006; revised 6/16/2006; accepted 6/28/2006. presence of DDB1 does not seem to be a requirement for simple Grant support: NIH grants ES2388, ES12991, and CA93413 (A.A. Wani). The costs of publication of this article were defrayed in part by the payment of page damage-specific binding activity of DDB2 in chromatin. Neverthe- charges. This article must therefore be hereby marked advertisement in accordance less, without DDB1 in the DDB complex, and as discussed above, with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. Yue Xiong for providing Myc-tagged-Cul4A construct and anti-DDB1, E3 ligase cannot locate to damage sites to accomplish the initial anti-Cul4A antibodies; Drs. Michael Tainsky, Gilbert Chu, and Dirk Bohmann for processing steps of global genomic repair. providing valuable cells and reagents.

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2006 American Association for Cancer Research. DNA Damage Binding Protein Component DDB1 Participates in Nucleotide Excision Repair through DDB2 DNA-binding and Cullin 4A Ubiquitin Ligase Activity

Jinyou Li, Qi-En Wang, Qianzheng Zhu, et al.

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