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Mol Cell Biol. 2010 June; 30(11): 2708–2723.
Published online 2010 April 5. doi:  10.1128/MCB.01460-09
PMCID: PMC2876528

DDB2 Complex-Mediated Ubiquitylation around DNA Damage Is Oppositely Regulated by XPC and Ku and Contributes to the Recruitment of XPA[down-pointing small open triangle]

Abstract

UV-damaged-DNA-binding protein (UV-DDB) is a heterodimer comprised of DDB1 and DDB2 and integrated in a complex that includes a ubiquitin ligase component, cullin 4A, and Roc1. Here we show that the ubiquitin ligase activity of the DDB2 complex is required for efficient global genome nucleotide excision repair (GG-NER) in chromatin. Mutant DDB2 proteins derived from xeroderma pigmentosum group E patients are not able to mediate ubiquitylation around damaged sites in chromatin. We also found that CSN, a negative regulator of cullin-based ubiquitin ligases, dissociates from the DDB2 complex when the complex binds to damaged DNA and that XPC and Ku oppositely regulate the ubiquitin ligase activity, especially around damaged sites. Furthermore, the DDB2 complex-mediated ubiquitylation plays a role in recruiting XPA to damaged sites. These findings shed some light on the early stages of GG-NER.

Nucleotide excision repair (NER) is a principal pathway that removes a variety of helix-distorting DNA lesions, such as the cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine-pyrimidone photoproducts (6-4PPs) generated by UV light (14). In addition, NER can repair with different efficiencies the lesions formed by cisplatin, polycyclic carcinogens, psoralens, and other chemical compounds (46, 60). There are two subpathways of NER: global genome NER (GG-NER) and transcription-coupled NER (TC-NER). GG-NER eliminates DNA lesions over the entire genome, whereas TC-NER rapidly removes lesions on the transcribed strands of transcriptionally active genes (18, 51).

Various hereditary diseases involve defects in NER, including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy (TTD), and UV-sensitive syndrome (UVsS) (20, 30). XP is characterized by hypersensitivity to sunlight and an increased incidence of sunlight-induced skin cancers (30). CS is also characterized by photosensitivity of the skin, but CS patients have no predisposition to UV-induced skin cancers. Instead, they exhibit severe developmental and neurological abnormalities, as well as premature aging (37). Genetic complementation analysis has defined seven complementation groups in XP (XP-A to XP-G) and two in CS (CS-A and CS-B) (61). It is worth noting that some XP patients exhibit features of CS in addition to symptoms of XP (XP-B/CS, XP-D/CS, and XP-G/CS) and that all gene products responsible for these disorders are integrated in the XPG-transcription factor IIH (TFIIH) complex, suggesting a close relationship between transcription and NER (24).

The two pathways differ primarily in how damaged DNA is first recognized (14). In GG-NER, the XPC-HR23B-centrin2 complex and UV-damaged-DNA-binding protein (UV-DDB), and in TC-NER, the RNA polymerase II stalled at a lesion on the transcribed strand, play a role in the initial recognition process (45, 49, 55). These are followed by NER reactions common to both pathways. TFIIH, XPG, XPA, and replication protein A (RPA) are recruited to the lesion, leading to the local unwinding of the DNA double helix. Then, the nucleases XPF-ERCC1 and XPG cleave the damaged strand on the 5′ and 3′ sides of the lesion, respectively, leading to the excision of an approximately 27- to 32-nucleotide single-stranded DNA (ssDNA) fragment containing the lesion. Finally, gap filling by DNA synthesis and ligation restore the original duplex.

UV-DDB is a heterodimer comprised of DDB1 (p127) and DDB2 (p48) (27) and identified biochemically as a protein factor that binds robustly and specifically to UV-irradiated DNA (7, 12, 17, 27). These observations suggest that UV-DDB initiates GG-NER cooperatively with the XPC complex (7, 49, 57). Although UV-DDB is not required for cell-free NER (1, 3, 36), mutations in the DDB2 gene are responsible for XP-E, a relatively mild form of XP uncomplicated by CS or TTD, and cells from XP-E patients lack the ability to bind UV-damaged DNA (7, 23, 25, 38, 42). In addition, these cells show moderate sensitivity to UV and reduced levels (50 to 80% of normal) of UV-induced unscheduled DNA synthesis (UV-UDS) (28, 40). Electrophoretic mobility shift assays (EMSAs) using purified proteins revealed that UV-DDB exhibits the highest affinity for 6-4PP and also binds moderately to CPD (5, 15, 44, 50, 53, 59). In XP-E cells, XPC still localizes to 6-4PPs and, to a lesser extent, to CPDs, with substantially delayed kinetics (35), and the elimination of CPDs is significantly impaired, whereas that of 6-4PPs is only slightly affected (22). Moreover, the translocation of UV-DDB to damaged sites was not affected by the absence of XPC, indicating that UV-DDB functions at an earlier stage of GG-NER than XPC (31, 56). Structural analysis revealed that DDB2 interacts extensively with DNA containing a lesion, while DDB1 stabilizes DDB2 but does not bind to DNA. DDB2 unwinds and kinks the DNA, and the lesion is consequently flipped out and partially held in a shallow binding pocket of DDB2 (8, 47).

The finding that UV-DDB is part of a cullin-containing ubiquitin ligase (E3) complex has shed some light on the role of UV-DDB-mediated damage recognition (16). One physiological substrate of this complex is DDB2 itself. Analysis using the recombinant UV-DDB-cullin 4A (Cul4A)-Roc1 complex revealed that DDB2 is ubiquitylated in vitro and that the ubiquitylated DDB2 appears to lose its affinity for lesions (34, 50). These results are consistent with findings that DDB2 is degraded in UV-irradiated cells (13, 41, 50). XPC was also ubiquitylated by the UV-DDB-Cul4A-Roc1 complex in a cell-free system and in response to UV irradiation in cells (50). Moreover, the ubiquitylation did not trigger degradation, though the ubiquitylated XPC exhibited stronger DNA-binding activity than the unmodified form. These results indicate that the ubiquitylation of these two proteins functions in the hand-off from UV-DDB to the XPC complex at the damaged site. The UV-DDB-Cul4A-Roc1 complex also mediates histone ubiquitylation, which may affect the structure of the nucleosome and help the NER factors to access lesions in nucleosomal DNA (26, 58).

The E3 activity of UV-DDB-Cul4A-Roc1 is subjected to diverse forms of regulation (16). For example, CSN, which is composed of CSN1 to CSN8 and appears to act as a negative regulator of cullin-based ubiquitin ligases, was coimmunoprecipitated with the DDB2 E3 complex from untreated cells. It has been reported that CSN promotes the removal of a ubiquitin-like protein, NEDD8, from cullin and consequently abrogates the stimulating effect of neddylation on cullin-based ubiquitin ligases (9, 32). Indeed, neddylated Cul4A was not detected in this complex. In contrast, after UV irradiation, the DDB2 E3 complex was found in the chromatin-bound fraction. Under these conditions, CSN was not coimmunoprecipitated with this E3 complex and neddylated Cul4A was detected. These results suggest that the E3 activity of the UV-DDB-Cul4A-Roc1 complex is triggered in response to UV irradiation.

The emerging model suggests that the UV-DDB-Cul4A-Roc1 ubiquitin ligase functions in the recognition of damage for GG-NER within the chromatin. However, the mechanism involved and its significance have not been fully elucidated. Here, we provide further evidence for the involvement of the UV-DDB-Cul4A-Roc1 complex in damage recognition. An analysis of mutant DDB2 proteins revealed that both DNA-binding and E3 activities of the UV-DDB-Cul4A-Roc1 complex are required for efficient GG-NER. Furthermore, the E3 activity of the DDB2 complex is regulated by XPC and Ku, as well as CSN, and plays a role in the recruitment of other NER factors to the damaged sites in chromatin.

MATERIALS AND METHODS

Plasmid constructs for DDB2 expression.

For the generation of a FLAG epitope-tagged DDB2 expression construct, the BamHI-NotI fragment containing the full-length DDB2-coding region from pcDNA3.1-HA-DDB2 was exchanged with the full-length fragment of DDB1 in pcDNA3-FLAG-DDB1, resulting in pcDNA3-FLAG-DDB2. pcDNA3.1-HA-DDB2 and pcDNA3-FLAG-DDB1 were provided by T. Chiba, Tsukuba University. Point mutations identified in XP-E patient cell lines (XP82TO, XP2RO, XP3RO, and Ops1) were introduced into pcDNA3-FLAG-DDB2 using a QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. DNA fragments containing the mutations were isolated by digestion with EcoRV-EcoRI and exchanged with the corresponding part in pcDNA3-FLAG-DDB2. DNA sequencing excluded additional mutations introduced elsewhere in the mutant cDNA.

For the generation of a FLAG-hemagglutinin (HA) epitope-tagged DDB2 expression construct, DDB2 cDNA without a start codon was amplified by PCR from pcDNA3-FLAG-DDB2 with 5′-CGGCTCGAGGCTCCCAAGAAACGCCCAGAAACCC-3′ (forward; DDB2 cDNA flanked by an XhoI site) and 5′-TCGAGCGGCCGCTCGAGTCACTTCCG-3′ (reverse; DDB2 cDNA flanked by an XhoI site) as primers. The DDB2 cDNA regions are underlined.

The PCR product was digested with XhoI and cloned in-frame and downstream from the sequence encoding a FLAG epitope, followed by a HA epitope in pOZ-N (provided by Y. Nakatani), resulting in pOZ-N-DDB2. The BglII-NotI fragment containing the full-length DDB2-coding region with the epitope tags from pOZ-N-DDB2 was blunt ended and ligated at the blunt-ended XhoI site in pCAGGS.

Isolation of stable transfectants.

HeLa cells were transfected with the FLAG-HA epitope-tagged DDB2 expression constructs using Effectene transfection reagent (Qiagen) according to the manufacturer's recommendations. Stable transfectants were selected in the presence of G418 (500 μg/ml).

Purification of protein factors.

FLAG epitope-tagged wild-type and mutant DDB2 proteins were expressed transiently in HeLa cells. Cells were extracted in NETN buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM dithiothreitol [DTT], and 0.25 mM phenylmethylsulfonyl fluoride [PMSF]) at 4°C for 30 min. The cell lysate was clarified by centrifugation at 17,600 × g for 10 min and incubated at 4°C for 1 h with protein G Sepharose (GE Healthcare). The supernatant, obtained by centrifugation at 3,800 × g for 1 min, was incubated with anti-FLAG M2 antibody-conjugated agarose (Sigma) at 4°C for 2 h. The bound proteins were eluted with FLAG peptide.

FLAG-HA epitope-tagged wild-type and K244E DDB2 complexes were purified from the stable transfectants. The proteins eluted from anti-FLAG M2 antibody-conjugated agarose were further incubated with anti-HA antibody-conjugated agarose (Sigma) at 4°C for 2 h. The bound proteins were eluted with HA peptide. The XPG complex was purified from HEK293 cells stably expressing FLAG-V5-6×His epitope-tagged XPG (24).

XPA was purified from HEK293 cells stably expressing 3×FLAG-His-V5 epitope-tagged XPA. The XPC-HR23B heterodimer was provided by K. Sugasawa, Kobe University, and the Ku proteins were purchased from Vaxron.

Preparation of nuclear extract and solubilized chromatin fraction.

Cells were irradiated with 20 J/m2 of UV-C (peak 254 nm), incubated for the periods indicated below, and harvested by trypsinization. After washing with buffer A (20 mM Tris-HCl [pH 7.5], 3 mM MgCl2, 1 mM EDTA, 0.5 mM PMSF, and 2 mM 2-mercaptoethanol), cells were extracted in buffer A containing 0.025% Triton X-100 at 4°C for 20 min. The (crude) nuclei were separated by centrifugation at 900 × g for 5 min and washed with buffer A. The nuclear pellet was extracted with buffer B (20 mM Tris-HCl [pH 7.3], 300 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 10 mM 2-mercaptoethanol, and 0.25 mM PMSF) at 4°C for 30 min and centrifuged at 17,600 × g for 30 min. The supernatant was combined with an equal volume of low-salt buffer (20 mM Tris-HCl [pH 7.3], 20 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 10 mM 2-mercaptoethanol, and 0.25 mM PMSF) and designated the nuclear extract. The pellet was then washed with micrococcal nuclease (MNase) buffer (20 mM Tris-HCl [pH 7.5], 100 mM KCl, 2 mM MgCl2, 1 mM CaCl2, 300 mM sucrose, 0.1% Triton X-100, 1 mM DTT, and a complete protease inhibitor cocktail [Roche]) and incubated with 15 U/ml of micrococcal nuclease in MNase buffer at room temperature for 25 min. The reaction was terminated by adding EDTA to 5 mM, and the reaction mixture centrifuged at 3,800 × g for 5 min at 4°C. The pellet was washed with MNase buffer. The supernatants were combined and used as the solubilized chromatin fraction.

MiniQ column chromatography.

The affinity-purified DDB2 complex was loaded onto a MiniQ column (GE Healthcare) equilibrated with 150 mM NaCl in buffer M (20 mM Tris-HCl [pH 7.8], 10% glycerol, 0.1% Tween 20, 10 mM 2-mercaptoethanol). The column was washed with 6 bed volumes of the same buffer and eluted with 10 bed volumes of a linear gradient of NaCl from 150 mM to 1 M in buffer M. The fractions were subjected to immunoblotting.

Local UV irradiation.

For localized UV irradiation through micropores, cells grown on glass coverslips were washed with phosphate-buffered saline (PBS) and covered with an Isopore polycarbonate filter containing pores 5 μm in diameter (Millipore). The filter-covered cells were irradiated with 100 J/m2 of UV-C. The filter was then gently removed, and the cells were processed immediately.

Immunofluorescence microscopy.

Immunofluorescence staining of the cells was carried out as described previously (58), with some modifications. All procedures were performed at room temperature. The cells irradiated with UV-C were washed with PBS and twice with CSK buffer {100 mM NaCl, 300 mM sucrose, 10 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid); pH 7.0], and 3 mM MgCl2}, and permeabilized with 0.5% Triton X-100 in CSK buffer for 5 min. After two more washes with CSK buffer, the cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, and incubated with blocking buffer (1% bovine serum albumin [BSA] in PBS) for 30 min. The cells were incubated with primary antibodies in blocking buffer for 1 h. After three additional washes with blocking buffer, the cells were incubated with secondary antibodies for 1 h and washed three times with PBS. For nuclear staining, the cells were incubated with To-Pro-3 iodide at a concentration of 0.2 mM in blocking buffer for 5 min. After two washes with PBS, coverslips were mounted on the glass slides with Vectashield mounting media (Vector), and the samples were examined with an MRC-1024 fluorescence microscopy system (Bio-Rad).

Assay of ubiquitin ligase activity in vitro.

The in vitro ubiquitylation assay was conducted essentially as described previously (50). Samples were incubated at 37°C for 1 h in a standard reaction mixture (14 μl) containing 50 mM Tris-HCl (pH 7.6), 10 mM MgCl2, 0.2 mM CaCl2, 4 mM ATP, 1 mM DTT, 0.1 μg of E1, 0.4 μg of E2 (UbcH5a), and 5 μg of ubiquitin or 6×His-ubiquitin (Boston Biochem). Where indicated below, the reaction mixture volume was changed.

Assay of binding to damaged DNA.

The biotinylated DNA (165 bp) was amplified by PCR from the pBluescriptIISK(−) plasmid with a 5′-biotinylated oligonucleotide as an upper primer. The DNA was purified and then irradiated with 10 kJ/m2 of UV-C. Binding reactions were performed at 30°C for 30 min in a mixture (7.5 μl) containing DDB assay buffer (20 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 1 mM EDTA, 150 mM NaCl, 5% glycerol, 0.01% Triton X-100, and 1 mM DTT), 1.5 μg of BSA, 2.5 pmol biotinylated DNA, and the DDB2 complex. Reaction mixtures were then incubated with streptavidin agarose beads (7.5 μl of packed gel volume) at 4°C for 1 h. After the supernatant was saved as the unbound fraction, the beads were washed twice with 300 μl of DDB assay buffer and suspended in SDS sample buffer. Aliquots of the unbound and bound fractions were subjected to SDS-PAGE followed by immunoblotting.

Reconstitution and ubiquitylation of nucleosomes.

A mononucleosome was assembled on the biotinylated DNA (165 bp, 6.06 pmol) using a chromatin assembly kit (Active Motif) and immobilized on streptavidin agarose beads (14 μl of packed gel volume). After the beads were washed twice with DDB assay buffer, binding reactions were performed at 30°C for 30 min in a mixture (28 μl) containing the DDB assay buffer, 3 μg of BSA, the DDB2 complex, and the beads. The supernatant was saved, and the beads were washed twice with 300 μl of DDB assay buffer. The proteins bound to the beads and in the supernatant were subjected to an in vitro ubiquitylation assay (28 μl) as described above. After the proteins released from the beads were saved as the released fraction, the beads were washed twice with DDB assay buffer and suspended in SDS sample buffer as the retained fractions. For the XPA and XPC-HR23B binding assays and in vitro ubiquitylation assay, including XPC-HR23B, 200 ng of XPA and 56 ng of XPC-HR23B were used for each reaction mixture and the reaction mixture volume and the packed gel volume of the beads were reduced to 14 μl and 7 μl, respectively.

Knockdown of Ku86 by siRNA.

The Ku86-targeting and control nontargeting small interfering RNA (siRNA) duplex mixtures were purchased from Santa Cruz Biotechnology and Dharmacon, respectively. The siRNAs were transfected into HeLa cells expressing FLAG-HA epitope-tagged DDB2 using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. The transfection was repeated 24 h later, and the cells were processed for immunoblotting and local UV irradiation followed by immunofluorescence microscopy 72 h after the first transfection.

Antibodies.

The antibodies employed in this study were as follows: anti-DDB1, anti-DDB2 (DDB2N), and anti-Roc1 (Zymed); anti-HA (3F10) (Roche Diagnostics); anti-XPC (C-XPC) (GeneTex); anti-CSN7 (Biomol); anti-Ku70, anti-Ku86, anti-XPA (FL-273), anti-CSN5 (JAB1), and antiubiquitin (FL-76) (Santa Cruz Biotechnology); anti-histone 2A (H2A), anti-H2B, and anti-H4 (T. Ikura, Tohoku University, and Upstate); anti-H3 (Abcam); anti-HR23B (C. Masutani, Osaka University); and Alexa Fluor 488-conjugated anti-rat and Alexa Fluor 568-conjugated anti-rabbit antibodies (Molecular Probes).

RESULTS

The K244E DDB2 protein forms an E3 complex and mediates histone ubiquitylation in vitro.

Mutations in the DDB2 gene, which encodes a protein of 427 amino acids containing seven WD-40 repeat motifs, have been found in XP-E cell lines. To examine whether the mutant DDB2 proteins derived from XP-E patients retain activity to form the E3 complex, a FLAG epitope-tagged wild-type DDB2 and three mutant DDB2 proteins (Fig. (Fig.1A)1A) were expressed transiently in HeLa cells. The K224E mutant contains a single substitution, lysine to glutamate at the 244th amino acid residue in the 4th WD-40 repeat motif, derived from patient XP82TO. The R273H mutant also contains a single substitution, arginine to histidine at the 273rd residue in the 4th WD-40 repeat motif, derived from patients XP2RO and XP3RO. The Δ313-427 mutant has a deletion of 115 amino acids from the 313th to the 427th residue as a result of a nonsense mutation in the 5th WD-40 repeat motif, derived from patient Ops1. Whole-cell lysates prepared from the corresponding cells were immunoprecipitated with anti-FLAG antibody. An immunoblot analysis revealed that the wild-type and K244E DDB2 were coimmunoprecipitated with DDB1, Cul4A, and Roc1 as described previously (34). On the other hand, neither R273H nor Δ313-427 DDB2 was coimmunoprecipitated with these proteins (Fig. (Fig.1B).1B). The precipitated samples with the wild-type and K244E DDB2 had E3 activity, which was shown by polyubiquitin chain formation and shifts in the molecular weight of DDB2 (data not shown). Moreover, these signals were not detected when the samples with R273H and Δ313-427 DDB2 were used. These results indicate that neither R273H nor Δ313-427 DDB2 forms a complex and displays E3 activity in vitro.

FIG. 1.
The K244E DDB2 protein forms the E3 complex, which mediates histone ubiquitylation. (A) Schematic representation of mutant DDB2 proteins. The mutant proteins are derived from the mutations found in XP-E patients. Two DDB2 mutants (K244E and R273H) have ...

Previous studies have shown that histones are targets of Cul4A-DDB-Roc1 E3 activity (26, 58). We therefore examined whether the K244E DDB2 complex can mediate histone ubiquitylation. For more effective purification, we established HeLa cell lines stably expressing FLAG-HA epitope-tagged wild-type and K244E DDB2 proteins, and the DDB2 complexes were prepared from the corresponding cell lines by immunoprecipitation with anti-FLAG and anti-HA antibodies (Fig. (Fig.1C).1C). Proteins associated with the K244E DDB2 were similar to those with wild-type DDB2. We detected DDB1, Cul4A, Roc1, and CSN subunits by immunoblotting (data not shown). When the wild-type DDB2 complex was incubated with histone octamer, the molecular masses of H3 and H4 shifted to higher positions, indicating the ubiquitylation of H3 and H4 (Fig. (Fig.1D,1D, lanes 5 and 6). DDB2, Cul4A, and Roc1 were also ubiquitylated. The ubiquitylation of these proteins was confirmed by 6×His-ubiquitin-mediated shifts of the bands. In addition, these shifted bands were detected with almost the same intensities when the K244E DDB2 complex was used (Fig. (Fig.1D,1D, lanes 9 and 10). These results indicate that both the wild-type and K244E DDB2 complexes retain the E3 activity and ubiquitylate histones H3 and H4 in vitro with similar efficiencies.

The K244E DDB2 complex does not bind to UV-damaged DNA.

The results of the electrophoretic mobility shift assay (EMSA) using highly purified proteins and the results of structural studies revealed that the DNA-binding property of UV-DDB is conferred by the DDB2 subunit (47, 59) and that the activity to bind damaged DNA, as measured by EMSA, is absent or defective in all the XP-E patient cell lines tested (7, 23, 25, 38, 42). First, we examined the DNA-binding activity of the wild-type and K244E DDB2 complexes using a different method. To make a substrate for the assay, a 165-bp DNA duplex was amplified by PCR with a 5′-biotinylated upper primer. The DNA fragment was UV irradiated at 10 kJ/m2, incubated with the wild-type or K244E DDB2 complex, and then immobilized on streptavidin agarose. The unbound and bound fractions were separated and subjected to immunoblotting. When the wild-type DDB2 complex was incubated with UV-irradiated DNA, DDB1 and DDB2 were mostly detected in the bound fraction (Fig. (Fig.2,2, lane 9). Substantial amounts of Cul4A and Roc1 were also detected in the bound fraction, but some were found in the unbound fraction, suggesting a somewhat unstable association of the E3 subunits with the UV-DDB (50). In contrast, only a small amount of the K244E DDB2 complex was detected in the bound fraction (Fig. (Fig.2,2, lane 11). Interestingly, CSN7 was only ever detected in the unbound fraction, suggesting that CSN is released from the core when the DDB2 complex binds to UV-damaged DNA.

FIG. 2.
The K244E DDB2 complex does not bind to UV-damaged DNA in vitro. The biotinylated DNA was irradiated with 10 kJ/m2 of UV or mock irradiated. The wild-type (WT) and K244E DDB2 complexes were incubated with biotinylated DNA, and the biotinylated DNA was ...

Taken together, these results indicate that the K244E DDB2 complex has only residual DNA-binding activity compared with that of the wild-type DDB2 complex.

The K244E DDB2 complex is not associated with UV-damaged sites in chromatin.

To further compare the properties of the wild-type and K244E DDB2 complexes, we purified the proteins from UV-irradiated cells. Immunoblotting and silver staining revealed that the wild-type but not K244E DDB2 was coimmunoprecipitated with core histones from the solubilized chromatin fractions of UV-irradiated cells (Fig. 3A and B). Although the interactions between the wild-type DDB2 complex and histones were detected at low levels in the mock-irradiated cells, the amounts of histones coimmunoprecipitated with wild-type DDB2 were significantly increased in the UV-irradiated cells. As shown in Fig. Fig.3B,3B, the amounts of histones coimmunoprecipitated with the wild-type DDB2 complex increased immediately after UV irradiation and declined thereafter. These results suggest that the interaction between the DDB2 complex and histones is stabilized in the presence of damaged DNA in vivo.

FIG. 3.
The K244E DDB2 complex is not associated with core histones and does not localize to the sites damaged by UV irradiation. (A) Immunopurification of the wild-type (WT) and K244E DDB2 complexes after UV irradiation. HeLa cells stably expressing FLAG-HA ...

Next, to examine the accumulation of the DDB2 complex at UV-damaged sites in vivo, we employed the micropore UV irradiation technique, which can localize damage to a particular area of the nucleus. HeLa cell lines stably expressing FLAG-HA-tagged wild-type and K244E DDB2 proteins as described above were irradiated with 100 J/m2 of UV through a 5-μm-pore Isopore filter. Immunostaining revealed that the wild-type DDB2 was colocalized with the damaged foci, indicated by XPC staining, immediately after UV irradiation (Fig. (Fig.3C).3C). However, the UV-induced recruitment of K244E DDB2 was dramatically inhibited. These results were consistent with the finding that only a small amount of the K244E DDB2 complex was detected in the UV-irradiated DNA-bound fraction (Fig. (Fig.2,2, lane 11).

The wild-type DDB2 complex interacts with Ku and the modified forms of XPC in a UV-dependent manner.

A 75-kDa band was predominantly found in the wild-type DDB2 complex purified from the solubilized chromatin fraction of UV-irradiated cells (data not shown). Mass spectrometric analysis identified Ku70 in this band. Consistent with this, immunoblotting revealed that the amounts of Ku70 and Ku86 in the wild-type DDB2 complex increased markedly immediately after UV irradiation (Fig. (Fig.3A).3A). In contrast to the wild-type DDB2 complex, neither Ku70 nor Ku86 was detected in the K244E DDB2 complex. Additionally, XPC was found in the DDB2 complex regardless of the mutation of DDB2 and UV irradiation, but modified forms of XPC were detected only in the wild-type DDB2 complex in a UV-dependent manner (Fig. (Fig.3A3A).

To further examine the interaction of these proteins, the wild-type DDB2 complex purified from the solubilized chromatin fractions of UV or mock-treated cells was fractionated on a MiniQ anion exchange column. The immunoblot analysis showed that the DDB2 complex was eluted from the column with two peaks (Fig. (Fig.3D,3D, UV−/UV+, fractions 2 to 5 and fraction 11) and the amounts of the DDB2 complex, XPC, and histones detected in fraction 11 were significantly increased in a UV irradiation-dependent manner (Fig. (Fig.3D,3D, UV+, fraction 11). The modified forms of XPC were also coeluted with the DDB2 complex and histones in the same fraction. In contrast, CSN was detected at low levels in the fraction, suggesting that the DDB2 complex in the fraction is bound to the damaged sites in chromatin. Interestingly, modified histones H2B and H3 were also detected in the fraction. The shifts in molecular weight of these histones matched the changes observed in the in vitro ubiquitylation assay (compare Fig. Fig.3D3D with Fig. Fig.1D).1D). These results suggest that XPC and histones are modified around the damaged sites in chromatin and that these modifications are related to the function of the DDB2 E3 complex.

The wild-type but not K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in nucleosomes in vitro.

To further examine the precise roles of the DDB2 complex around the lesions in chromatin, we employed an in vitro assay, depicted in Fig. Fig.4A.4A. Mononucleosomes, bearing mock- or UV-irradiated DNA, were immobilized on streptavidin beads and incubated with the wild-type or K244E DDB2 complex. The proteins were separated into unbound (supernatant) and bound (beads) fractions, both of which were subjected to ubiquitylation. When the bound fraction was used, the proteins released from or retained by the nucleosomes were separated after the ubiquitylation reaction.

FIG. 4.
The wild-type (WT) but not the K244E DDB2 complex can mediate histone ubiquitylation around damaged sites in nucleosomes in vitro. (A) Schematic representation of the strategy used to determine the effects of E3 activity of the DDB2 complex around the ...

When the K244E DDB2 complex was used, most of the complex was detected in the flowthrough fraction, regardless of UV damage (Fig. (Fig.4B,4B, lanes 5 and 6). These results indicate that the K244E DDB2 complex has only residual activity to bind damaged sites in nucleosomes, as well as in naked DNA. In the same reaction mixtures, no ubiquitylated histones were detected (Fig. (Fig.4B,4B, lanes 17 and 18), although the autoubiquitylation of K244E DDB2 and Cul4A was detected (Fig. (Fig.4B,4B, lanes 5 and 6), suggesting that not only the E3 activity but also the DNA-binding activity of the DDB2 complex was required for histone ubiquitylation in the nucleosomes bearing UV-damaged DNA.

In contrast to the K244E DDB2 complex, little of the wild-type DDB2 complex except CSN7 was detected in the flowthrough fraction when the nucleosomes bearing UV-irradiated DNA were used (Fig. (Fig.4B,4B, lane 4). These results suggest that the wild-type DDB2 core complex binds to the damaged sites in nucleosomes more stably than in naked DNA (compare with Fig. Fig.2,2, lane 9) and that the CSN complex is released when the DDB2 complex binds to the nucleosomes. Shifts in the molecular mass of DDB2 were much more pronounced in the bound fraction than in the flowthrough fraction (Fig. (Fig.4B,4B, compare lanes 10 and 16 with 3), indicating that the dissociation of CSN from the DDB2 complex stimulates the E3 activity of the complex (16). In addition, a significant amount of DDB2 remained unmodified or moderately ubiquitylated in the retained fraction (Fig. (Fig.4B,4B, lane 16), but only extensively ubiquitylated DDB2 (>250 kDa) was detected in the released fraction (Fig. (Fig.4B,4B, lane 10). Correspondingly, some DDB1 was detected in the released fraction. These results indicate that UV-DDB loses its DNA-binding activity through extensive ubiquitylation of DDB2. In contrast to UV-DDB, a large amount of ubiquitylated Cul4A and almost all of the Roc1 were detected in the released fraction. These results suggest that autoubiquitylation destabilizes the E3 complex. To our surprise, histones H2B and H3 were not detected in the flowthrough or released fraction, although they were ubiquitylated in the retained fraction, suggesting that histone ubiquitylation alone is not sufficient for the release of histones from nucleosomes.

Taken together, these results indicate that the wild-type DDB2 complex mediates histone ubiquitylation specifically in nucleosomes bearing UV-damaged DNA.

The DDB2 complex-mediated ubiquitylation around damaged sites in nucleosomes facilitates the recruitment of XPA.

Local UV irradiation experiments revealed that the ectopically expressed DDB2 was colocalized with endogenous XPA in vivo (Fig. (Fig.5A).5A). The results shown in Fig. Fig.4B4B and and5A5A raised the possibility that the DDB2 complex-mediated ubiquitylation around the damaged sites serves as a signal for activating the recruitment of other NER factors. To explore this possibility, we examined the interaction of XPA and XPC-HR23B with nucleosomes in several different states. The nucleosomes described in Fig. Fig.4A4A (I to III) were incubated with XPA or XPC-HR23B, and the unbound and bound fractions were separated (Fig. (Fig.5B).5B). Interestingly, more XPA was bound to the ubiquitylated nucleosomes than to the others (Fig. (Fig.5C,5C, lane 8). In contrast, the binding of XPC was affected neither by UV-damaged DNA nor by histone ubiquitylation (Fig. (Fig.5D,5D, lanes 5 to 8). In addition, histones H2B and H3 were not dissociated from the nucleosomes by the binding of XPC (Fig. (Fig.5D)5D) and XPA (data not shown). These results suggest that the DDB2 complex-mediated ubiquitylation around the damaged sites in nucleosomes promotes the recruitment of XPA.

FIG. 5.
The functional DDB2 complex facilitates the recruitment of XPA to the damaged sites. (A) The ectopically expressed wild-type (WT) DDB2 was colocalized with endogenous XPA. Local UV irradiation experiments were carried out as described in the Fig. ...

XPC and Ku regulate the E3 activity of the DDB2 complex differently.

The results in Fig. Fig.3A3A showing that the DDB2 complex interacts with XPC and Ku in a UV-dependent manner prompted us to investigate whether the E3 activity of the DDB2 complex is affected by the interaction with XPC and Ku. As summarized in Fig. Fig.6A,6A, the nucleosomes bearing UV-damaged DNA were immobilized on streptavidin beads and incubated with the DDB2 complex either alone or, subsequently, with XPC-HR23B. Then, the proteins bound to the nucleosomes were subjected to ubiquitylation in the presence or absence of Ku. The released and retained fractions were separated and analyzed by immunoblotting. Additionally, the DDB2 complex was incubated with XPC-HR23B and/or Ku in the absence of the nucleosomes bearing UV-damaged DNA (nucleosome free) and the ubiquitylation reaction was performed.

FIG. 6.
The E3 activity of the DDB2 complex is stimulated by binding to the UV-damaged sites and by XPC-HR23B but suppressed by Ku. (A) Schematic representation of the strategy used to determine the effects of XPC-HR23B and Ku on the E3 activity of the DDB2 complex. ...

When the DDB2 complex bound to the nucleosomes was subjected to the ubiquitylation reaction, a significant amount of DDB2 was shifted to higher-molecular-weight regions compared with the amount in these regions in the nucleosome-free conditions, indicating that DDB2 was extensively ubiquitylated when it bound to the nucleosome bearing UV-damaged DNA (Fig. (Fig.6B,6B, compare lane 8 with lane 14 in the immunoblot showing the results of the 60-min reaction). Ubiquitylation of Cul4A was also stimulated in the presence of the nucleosomes (Fig. (Fig.6B,6B, compare lane 8 with lane 14). These results suggest that the E3 activity of the DDB2 complex is stimulated by its binding to nucleosomes. The activity seemed to be further stimulated in the presence of XPC (Fig. (Fig.6B,6B, compare lane 8 with lane 11 in the immunoblots showing the results of Cul4A and DDB2 in the 30-min reaction), although this stimulatory effect was not observed in the nucleosome-free conditions (Fig. (Fig.6B,6B, compare lane 14 with lane 17 in the immunoblots showing the results of Cul4A and DDB2 in the 60-min reaction). Correspondingly, the ubiquitylation of XPC was also stimulated in the presence of the nucleosomes (Fig. (Fig.6B,6B, compare lane 11 with lane 17). Moreover, histone ubiquitylation was slightly stimulated in the presence of XPC (Fig. (Fig.6B,6B, compare lane 8 with lane 11).

It was notable that more UV-DDB was dissociated from the nucleosomes as the ubiquitylation of DDB2 was stimulated by XPC (Fig. (Fig.6B,6B, compare lanes 2 and 8 with lanes 5 and 11 in the immunoblot showing the results of DDB1; cartoon models of the results are shown in Fig. Fig.8A).8A). A very small amount of ubiquitylated XPC was also detected in the released fractions (Fig. (Fig.6B,6B, lane 5 in the results of XPC). On the other hand, most of the Roc1 was released from the nucleosomes in the absence of XPC, but some still remained in the retained fraction in the presence of XPC (Fig. (Fig.6B,6B, compare lane 8 with lane 11). These results suggest that XPC stabilizes the DDB2 complex around the damaged sites in nucleosomes and, consequently, stimulates the E3 activity of the DDB2 complex (see Fig. Fig.8A).8A). In addition, histones were not detected in the released fractions even when histone ubiquitylation was stimulated in the presence of XPC (Fig. (Fig.6B,6B, lanes 2 and 5), indicating that the histone ubiquitylation did not affect the composition of nucleosomes.

FIG. 8.
Models of the mechanism of damage recognition in GG-NER. (A) Cartoon models of the results shown in Fig. Fig.6B.6B. Each panel (a to c) corresponds to characters (a to c) in the dotted box at the top of Fig. Fig.6B.6B. (B) A model of the ...

In contrast to XPC, Ku decreased the E3 activity of the DDB2 complex regardless of the presence or absence of the nucleosomes. The amounts of ubiquitylated DDB2, Cul4A, and histones were decreased in the presence of Ku (Fig. (Fig.6B,6B, compare lane 8 with lane 9 and lane 14 with lane 15; a greater effect was detected in the results of DDB2 in the 60-min reaction). The inhibitory effect by Ku was more prominent in the presence of the nucleosomes. The stimulatory effect of XPC on the E3 activity was also inhibited by Ku (Fig. (Fig.6B,6B, compare lane 11 with lane 12 and lane 17 with lane 18), and consequently, the ubiquitylated XPC itself was decreased. Correspondingly, the amount of UV-DDB released from the nucleosomes was reduced (Fig. (Fig.6B,6B, compare lane 2 with lane 3 and lane 5 with lane 6 in the immunoblot showing the results of DDB1). In contrast, the amount of Roc1 found in the released fraction was not decreased. These results suggest that Ku may affect the stability of the DDB2 E3 complex (see Fig. Fig.8A).8A). Taken together, these results indicate that the E3 activity of the DDB2 complex was stimulated in the presence of the nucleosomes and XPC and suppressed by Ku.

Ku physically interacts with the DDB2 complex and acts as a negative regulator for the E3 activity.

We further examined whether Ku physically interacts with the DDB2 complex and directly affects the E3 activity. The DDB2 complex immobilized on agarose beads was subjected to ubiquitylation in the presence or absence of Ku. After the reaction, the released and retained fractions were separated.

Consistent with previous results, the ubiquitylation of Cul4A and DDB2 was suppressed in the presence of Ku (Fig. (Fig.6C,6C, compare lane 7 with lane 8). To our surprise, the majority of Ku86, a subunit of Ku, was detected in the retained fraction (Fig. (Fig.6C,6C, lanes 6 and 8) although Ku70, another subunit of Ku, was detected in the released fraction (Fig. (Fig.6C,6C, lanes 2 and 4). These results indicate that Ku86 alone binds directly with the DDB2 complex. Interestingly, little of the Roc1 was detected in the retained fraction in the presence of Ku even when ubiquitylation did not occur, while almost all the UV-DDB was detected in the retained fraction (Fig. (Fig.6C,6C, compare lane 5 with lane 6). These results indicate that Ku interacts physically with the DDB2 complex, destabilizes the E3 complex, and consequently, inhibits the E3 activity.

Ku specifically interacts with the DDB2 complex and affects the stability of the binding of DDB2 to the damaged sites in vivo.

To confirm the interaction between the DDB2 complex and Ku, we examined whether Ku interacts specifically with the DDB2 complex (Fig. (Fig.7A).7A). The DDB2 and XPG complexes were purified from the solubilized chromatin fraction prepared by the same method described above. HeLa cells stably expressing FLAG-HA epitope-tagged DDB2 were irradiated with 20 J/m2 of UV. Immediately after the irradiation, the DDB2 complex was immunopurified with anti-FLAG and anti-HA antibodies from the solubilized chromatin fraction of the cells. HEK293 cells stably expressing FLAG-V5-6×His epitope-tagged XPG were irradiated with 20 J/m2 of UV and incubated for 30 min. Then, the XPG complex was immunopurified with anti-FLAG antibody from the solubilized chromatin fraction of the cells. Immunoblot analysis revealed that histone H3 was coimmunoprecipitated with both the DDB2 and XPG complexes, suggesting that the XPG complex also interacts with damaged sites induced by UV irradiation in chromatin. Importantly, Ku was coimmunoprecipitated with the DDB2 complex but not with the XPG complex under these conditions. These results clearly rule out the possibility that the DDB2 complex interacts with Ku in a UV-dependent manner via nonspecific or spurious binding of Ku to chromatin and strongly suggest the specific interaction of the DDB2 complex with Ku.

FIG. 7.
Ku interacts specifically with the DDB2 complex and affects the accumulation of DDB2 at the damaged sites in vivo. (A) Immunopurification of the DDB2 and XPG complexes after UV irradiation. HeLa cells stably expressing FLAG-HA epitope-tagged DDB2 were ...

Then, we explored a functional role of Ku in the recognition step of GG-NER in vivo. The results shown in Fig. Fig.6B6B imply that Ku stabilizes the binding of DDB2 to the damaged sites in chromatin. Therefore, we examined this possibility using the local-UV-irradiation technique (Fig. (Fig.7C).7C). The Ku86-depleted HeLa cells stably expressing FLAG-HA epitope-tagged DDB2 by siRNA (Fig. (Fig.7B)7B) were irradiated with 100 J/m2 of UV through a 5-μm-pore Isopore filter. After the irradiation, the accumulation of DDB2 at the damaged sites was visualized by fluorescent immunostaining using anti-HA antibody (Fig. (Fig.7C,7C, green). UV-irradiated sites were visualized using anti-XPC antibody (Fig. (Fig.7C,7C, red). Immunostaining revealed that DDB2 was colocalized with the damaged DNA spots in the control nontargeting siRNA-transfected cells immediately after UV irradiation and that the DDB2 signals were slightly attenuated 15 min after UV irradiation. These results were consistent with the results shown in Fig. 3B and C. In contrast, the fluorescence signals of DDB2 detected in Ku86-depleted cells immediately after UV irradiation were much weaker than those in the control siRNA-transfected cells and were no longer distinguishable 15 min after UV irradiation. These results indicate that depletion of Ku86 reduced the accumulation of DDB2 at the damaged sites in chromatin.

Taken together, Ku is considered to stabilize the binding of the DDB2 complex to damaged sites.

DISCUSSION

In this study, we provided further evidence for the properties and functions of the DDB2 E3 complex in the recognition step of GG-NER. Experiments with DDB2 mutants from XP-E patients indicated that both the DNA-binding and E3 activity of the DDB2 complex play an important role in the process of GG-NER within the chromatin. The E3 activity of the DDB2 complex is diversely regulated, e.g., by binding to the lesion, CSN, XPC, and Ku, and the DDB2 complex-mediated ubiquitylation around the damaged sites in chromatin contributes to the recruitment of other NER factors to the lesions.

Both the DNA-binding and E3 activity of the DDB2 complex are required to promote GG-NER in chromatin.

Several studies have established that mutations in DDB2 are responsible for XP-E (7, 23, 25, 38, 42). To explore the precise role of DDB2 in GG-NER, we analyzed the function of a mutant DDB2 derived from XP-E patients in the context of a DDB2 complex with activity to bind UV-damaged DNA and the activity of a ubiquitin ligase. The results of an epitope-tagging method indicated that neither R273H nor Δ313-427 DDB2 was able to form the cullin-based E3 complex. In contrast, K244E DDB2 was able to form the complex. Furthermore, the K244E DDB2 complex displayed E3 activity and mediated the ubiquitylation of DDB2, Cul4A, Roc1, and core histones in vitro (Fig. (Fig.1)1) (26, 58). However, the K244E DDB2 complex was neither distributed to UV-damaged sites in vivo nor bound to nucleosomes in the cells in response to UV irradiation (Fig. (Fig.3).3). Experiments in vitro showed that the K244E DDB2 complex did not bind to the UV-damaged DNA (Fig. (Fig.2)2) or the nucleosomes bearing the UV-irradiated DNA and, consequently, was not able to mediate the ubiquitylation of the nucleosomes assembled on UV-damaged DNA (Fig. (Fig.4).4). Structural analysis revealed that the K244 residue of DDB2 is in direct contact with the phosphodiester backbone of the DNA via charge-stabilized hydrogen bonds. R273 contributes to the intermolecular interactions and affects the structure of the DNA-binding surface of DDB2, although it is not in contact with the DNA directly (47). These results indicate that both the DNA-binding and E3 activity of the DDB2 complex are required for promoting GG-NER in chromatin.

Various regulatory mechanisms for the E3 activity of the DDB2 complex.

It has been reported that CSN is a component of the DDB2 E3 complex and negatively regulates the E3 activity in vitro. CSN was not present in the DDB2 complex prepared from the chromatin-bound fraction derived from UV-irradiated cells, indicating that the E3 activity of the DDB2 complex is stimulated in response to UV irradiation in vivo (16). However, it remains unclear how CSN dissociates from the DDB2 complex. Our experiments in vitro revealed that CSN is released from the DDB2 complex when the complex binds to lesions (Fig. (Fig.22 and and4B).4B). Correspondingly, the E3 activity of the DDB2 complex was stimulated (Fig. (Fig.4B).4B). In addition, an in vitro assay using the recombinant DDB2 core complex revealed that the binding of the DDB2 complex to the lesion per se facilitates the E3 activity (50). These results suggest that the binding of the DDB2 complex to the lesion alters its configuration, thereby inducing the dissociation of CSN, and consequently stimulates the E3 activity of the DDB2 complex.

The E3 activity of the DDB2 complex is further regulated by XPC around the damaged sites. The present results clearly showed that XPC is not only a substrate of the DDB2 E3 complex but also a stimulator of the E3 activity of the DDB2 complex, especially when the complex is bound to damaged sites in nucleosomes (Fig. (Fig.6B).6B). This stimulatory effect is at least partly due to the XPC-mediated stabilization of the DDB2 complex around the damaged sites. The DDB2 complex bound to the nucleosomes with XPC retained Roc1 more stably than that bound alone after the ubiquitylation reaction (Fig. (Fig.6B).6B). However, this stimulatory effect of XPC was not observed when the ubiquitylation was carried out in nucleosome-free conditions. Similarly, immunoprecipitation experiments revealed that the modified forms of XPC were coimmunoprecipitated with the wild-type but not K244E DDB2 complex in a UV-dependent manner, while the interaction between the DDB2 complex and XPC was detected regardless of the mutation of DDB2 and UV irradiation (Fig. 3A and D). These results indicate that XPC can affect the stability of the DDB2 complex only when the complex binds to the damaged sites and that the inhibitory effect of CSN on the E3 activity of the DDB2 complex predominates over the stimulatory effect of XPC.

We also found that Ku was specifically coimmunoprecipitated with the wild-type DDB2 complex in a UV-dependent manner (Fig. (Fig.3A3A and and7A).7A). Ku is a heterodimer of two subunits, Ku70 and Ku86, and a component of the nonhomologous end-joining (NHEJ) pathway (10, 14). Ku is involved not only in the binding of DNA ends and repair of double-strand breaks (DSBs) but also in other functions, such as chromatin compaction, transcriptional silencing, and prevention of telomeric fusion (4, 6, 21, 29, 33, 43, 52, 54). Furthermore, it has been shown recently that Ku70 can mediate deubiquitylation (2). Consistent with this report, we showed here that Ku negatively regulates the E3 activity of the DDB2 complex by destabilizing it (Fig. (Fig.6).6). Similarly to CSN, the negative effect of Ku was predominant over the stimulatory effect of XPC. Although this negative regulation was observed in vitro regardless of whether the DDB2 complex bound to the damaged sites or not, the interaction of Ku with the DDB2 complex was detected only when the complex was immunoprecipitated from the solubilized chromatin fraction of the UV-irradiated cells (Fig. (Fig.3A).3A). These results indicate that Ku acts on the DDB2 complex specifically around damaged sites. It is worth noting that Ku inhibited but did not abrogate the DDB2 complex-mediated ubiquitylation. In addition, this inhibitory effect was much more pronounced on the autoubiquitylation of DDB2 than on the ubiquitylation of other proteins, e.g., XPC and histones (Fig. (Fig.6B).6B). The results shown in Fig. Fig.6C6C revealed that Ku86 but not Ku70 interacted with the DDB2 complex, suggesting that Ku86 mainly contributes to the destabilization of the E3 complex. Furthermore, the Ku86 depletion resulted in the reduction of the accumulation of DDB2 at the damaged sites in chromatin in vivo (Fig. (Fig.7C).7C). Thus, we conclude that Ku particularly inhibits the extensive autoubiquitylation of DDB2 and, consequently, prevents the dissociation of the DDB2 complex from damaged sites.

Implications for the DDB2 complex-mediated ubiquitylation in GG-NER.

Plausible implications for the DDB2 complex-mediated ubiquitylation in GG-NER have been discussed. First, the ubiquitylation of DDB2 and XPC around damaged sites may contribute to the hand-off of the lesion from DDB2 to XPC (50). In line with this idea, our results showed that extensively ubiquitylated DDB2 lost activity to bind to nucleosomes bearing UV-irradiated DNA (Fig. (Fig.4B4B and and6B).6B). On the other hand, the polyubiquitylation of XPC was less extensive than that of DDB2 (Fig. (Fig.6B).6B). However, the biological significance of autoubiquitylation of the DDB2 complex and polyubiquitylation of XPC around damaged sites remains to be elucidated. Several studies using quantitative fluorescence microscopy have revealed that the accumulation of NER factors at damaged sites occurs in seconds (19, 31, 39), while the interaction of the DDB2 complex with histones was detected even 1 h after UV irradiation (Fig. (Fig.3B).3B). Moreover, after local UV irradiation, the ectopically expressed DDB2 was colocalized with endogenous XPA (Fig. (Fig.5A5A).

Second, the DDB2 complex-mediated ubiquitylation of histones may play a role in destabilizing chromatin (58). However, inconsistent with this idea, our results indicated that histone ubiquitylation is not sufficient to destabilize the nucleosome (Fig. (Fig.4B4B and and6B).6B). The release of histones from nucleosomes bearing UV-irradiated DNA was not observed even when histone ubiquitylation was stimulated by XPC (Fig. (Fig.6B6B).

These results imply another possibility, that the DDB2 complex-mediated ubiquitylation around damaged sites in chromatin functions in the signaling pathway during the recognition step in GG-NER. The ubiquitylation-mediated signaling pathway in response to DSBs has been reported (48). This report suggests that RNF8 mediates the ubiquitylation of H2A and H2AX around DSB sites and that ubiquitylated H2A promotes recruitment of the RNF168-associated ubiquitin ligase. The RNF168 complex amplifies H2A ubiquitylation and mediates the ubiquitylation of other proteins around the DSB sites, thereby recruiting RAP80-ABRA1-BRCA1. Similarly, our results suggest that the DDB2 complex-mediated ubiquitylation around damaged sites in nucleosomes enhanced the recruitment of XPA to lesions (Fig. (Fig.5C).5C). Notably, the binding of XPA to the damaged sites did not affect the composition of nucleosomes either.

Furthermore, the DDB2 complex-mediated ubiquitylation around damaged sites in chromatin may also affect the immobilization of NER factors at the damaged sites. Indeed, Ku prevented the dissociation of DDB2 from the damaged sites in chromatin by inhibiting the autoubiquitylation (Fig. (Fig.7C).7C). In contrast, ubiquitylation of XPC potentiates its own damaged-DNA-binding activity (50). Additionally, in CHO cells which lack functional DDB2, a greater degree of XPG immobilization after UV irradiation was detected than in human fibroblasts (62).

Taking into account all of these findings, we proposed a model for the early stages of GG-NER in chromatin (Fig. (Fig.8B).8B). When the DDB2 complex binds to lesions in chromatin, CSN dissociates from the complex, and the E3 activity of the complex is consequently enhanced. Around the damaged sites, XPC and Ku further regulate this activity oppositely. Ku restrains the autoubiquitylation of DDB2 particularly and, consequently, prevents the dissociation of DDB2 from the damaged sites in chromatin. Then, XPC, core histones, and subunits of the DDB2 complex except DDB1 are ubiquitylated. This ubiquitylation mediates the dissociation of the DDB2 complex from damaged sites and, importantly, the recruitment of other NER factors to lesions. In contrast, the K244E DDB2 complex is not able to bind to damaged sites although it displays E3 activity. Consequently, the DDB2 complex-mediated ubiquitylation around damaged sites in chromatin does not occur, and this loss is the likely cause of the phenotypes associated with XP-E.

Acknowledgments

We thank I. Kuraoka and T. Ikura for helpful suggestions.

This work was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) and by the Solution Oriented Research for Science and Technology program of the Japan Science and Technology Agency.

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 April 2010.

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