UV-induced ubiquitylation of XPC complex, the UV-DDB-ubiquitin ligase complex, and DNA repair

The DNA nucleotide excision repair (NER) system is our major defense against carcinogenesis. Defects in NER are associated with several human genetic disorders including xeroderma pigmentosum (XP), which is characterized by a marked predisposition to skin cancer. For initiation of the repair reaction at the genome-wide level, a complex containing one of the gene products involved in XP, the XPC protein, must bind to the damaged DNA site. The UV-damaged DNA-binding protein (UV-DDB), which is impaired in XP group E patients, has also been implicated in damage recognition in global genomic NER, but its precise functions and its relationship to the XPC complex have not been elucidated. However, the recent discovery of the association of UV-DDB with a cullin-based ubiquitin ligase has functionally linked the two damage recognition factors and shed light on novel mechanistic and regulatory aspects of global genomic NER. This article summarizes our current knowledge of the properties of the XPC complex and UV-DDB and discusses possible roles for ubiquitylation in the molecular mechanisms that underlie the efficient recognition and repair of DNA damage, particularly that induced by ultraviolet light irradiation, in preventing damage-induced mutagenesis as well as carcinogenesis.     

Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites

Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans with xeroderma pigmentosum group C do not perform NER in the bulk of the genome and are corrected by XPC protein, which forms a complex with hHR23B protein. This complex preferentially binds to some types of damaged DNA, but the extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Electrophoretic mobility-shift competition measurements revealed a 400-fold preference for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This damage preference is much greater than displayed by the XPA protein. The discrimination power is similar to that determined here in parallel for the XP-E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DNA-XPC-hHR23B complexes were stable, with half of the complexes remaining four hours after challenge with excess UV-damaged DNA at 30 degrees C. XPC-hHR23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and alkylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC complex for structures in damaged duplex DNA indicates its importance as a primary damage recognition factor in non-transcribed DNA during human NER.     

In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product

The initial step in mammalian nucleotide excision repair (NER) of the major UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), requires lesion recognition. It is believed that the heterodimeric proteins XPC/hHR23B and UV-DDB (UV-damaged DNA binding factor, composed of the p48 and p127 subunits) perform this function in genomic DNA, but their requirement and lesion specificity in vivo remains unknown. Using repair-deficient xeroderma pigmentosum (XP)-A cells that stably express photoproduct-specific photolyases, we determined the binding characteristics of p48 and XPC to either CPDs or 6-4PPs in vivo. p48 localized to UV-irradiated sites that contained either CPDs or 6-4PPs. However, XPC localized only to UV-irradiated sites that contained 6-4PPs, suggesting that XPC does not efficiently recognize CPDs in vivo. XPC did localize to CPDs when p48 was overexpressed in the same cell, signifying that p48 activates the recruitment of XPC to CPDs and may be the initial recognition factor in the NER pathway.     

UV-induced ubiquitylation of XPC protein mediated by UV-DDB-ubiquitin ligase complex

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.     

An aromatic sensor with aversion to damaged strands confers versatility to DNA repair

It was not known how xeroderma pigmentosum group C (XPC) protein, the primary initiator of global nucleotide excision repair, achieves its outstanding substrate versatility. Here, we analyzed the molecular pathology of a unique Trp690Ser substitution, which is the only reported missense mutation in xeroderma patients mapping to the evolutionary conserved region of XPC protein. The function of this critical residue and neighboring conserved aromatics was tested by site-directed mutagenesis followed by screening for excision activity and DNA binding. This comparison demonstrated that Trp690 and Phe733 drive the preferential recruitment of XPC protein to repair substrates by mediating an exquisite affinity for single-stranded sites. Such a dual deployment of aromatic side chains is the distinctive feature of functional oligonucleotide/oligosaccharide-binding folds and, indeed, sequence homologies with replication protein A and breast cancer susceptibility 2 protein indicate that XPC displays a monomeric variant of this recurrent interaction motif. An aversion to associate with damaged oligonucleotides implies that XPC protein avoids direct contacts with base adducts. These results reveal for the first time, to our knowledge, an entirely inverted mechanism of substrate recognition that relies on the detection of single-stranded configurations in the undamaged complementary sequence of the double helix.     

Centrin 2 stimulates nucleotide excision repair by interacting with xeroderma pigmentosum group C protein

Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed alpha-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.     

Biochemical and structural domain analysis of xeroderma pigmentosum complementation group C protein

XPC is a 940-residue multidomain protein critical for the sensing of aberrant DNA and initiation of global genome nucleotide excision repair. The C-terminal portion of XPC (residues 492-940; XPC-C) has critical interactions with DNA, RAD23B, CETN2, and TFIIH, whereas functional roles have not yet been assigned to the N-terminal portion (residues 1-491; XPC-N). In order to analyze the molecular basis for XPC function and mutational defects associated with xeroderma pigmentosum (XP) disease, a series of stable bacterially expressed N- and C-terminal fragments were designed on the basis of sequence analysis and produced for biochemical characterization. Limited proteolysis experiments combined with mass spectrometry revealed that the full XPC-C is stable but XPC-N is not. However, a previously unrecognized folded helical structural domain was found within XPC-N, XPC(156-325). Pull-down and protease protection assays demonstrated that XPC(156-325) physically interacts with the DNA repair factor XPA, establishing the first functional role for XPC-N. XPC-C exhibits binding characteristics of the full-length protein, including stimulation of DNA binding by physical interaction with RAD23B and CETN2. Analysis of an XPC missense mutation (Trp690Ser) found in certain patients with XP disease revealed that this mutation is associated with a diminished ability to bind DNA. Evidence of contributions to protein interactions from regions in both XPC-N and XPC-C along with recently recognized homologies to yeast PNGase prompted construction of a structural model of a folded XPC core. This model offers key insights into how domains from the two portions of the protein may cooperate in generating specific XPC functions.     

Nucleotide excision repair by mutant xeroderma pigmentosum group A (XPA) proteins with deficiency in interaction with RPA

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.     

Xeroderma pigmentosum complementation group E protein (XPE/DDB2): purification of various complexes of XPE and analyses of their damaged DNA binding and putative DNA repair properties

Xeroderma pigmentosum is characterized by increased sensitivity of the affected individuals to sunlight and light-induced skin cancers and, in some cases, to neurological abnormalities. The disease is caused by a mutation in genes XPA through XPG and the XP variant (XPV) gene. The proteins encoded by the XPA, -B, -C, -D, -F, and -G genes are required for nucleotide excision repair, and the XPV gene encodes DNA polymerase eta, which carries out translesion DNA synthesis. In contrast, the mechanism by which the XPE gene product prevents sunlight-induced cancers is not known. The gene (XPE/DDB2) encodes the small subunit of a heterodimeric DNA binding protein with high affinity to UV-damaged DNA (UV-damaged DNA binding protein [UV-DDB]). The DDB2 protein exists in at least four forms in the cell: monomeric DDB2, DDB1-DDB2 heterodimer (UV-DDB), and as a protein associated with both the Cullin 4A (CUL4A) complex and the COP9 signalosome. To better define the role of DDB2 in the cellular response to DNA damage, we purified all four forms of DDB2 and analyzed their DNA binding properties and their effects on mammalian nucleotide excision repair. We find that DDB2 has an intrinsic damaged DNA binding activity and that under our assay conditions neither DDB2 nor complexes that contain DDB2 (UV-DDB, CUL4A, and COP9) participate in nucleotide excision repair carried out by the six-factor human excision nuclease.     

Nucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex

The XPC protein complex is a DNA damage detector of human nucleotide excision repair (NER). Although the XPC complex specifically binds to certain damaged sites, it also binds to undamaged DNA in a non-specific manner. The addition of a large excess of undamaged naked DNA competitively inhibited the specific binding of the XPC complex to (6-4) photoproducts and the NER dual incision step in cell-free extracts. In contrast, the addition of undamaged nucleosomal DNA as a competitor suppressed both of these inhibitory effects. Although nucleosomes positioned on the damaged site inhibited the binding of the XPC complex, the presence of nucleosomes in undamaged DNA regions may help specific binding of the XPC complex to damaged sites by excluding its non-specific binding to undamaged DNA regions.     

Differential activity of UV-DDB in mouse keratinocytes and fibroblasts: impact on DNA repair and UV-induced skin cancer

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers (CPD) and accelerates repair of 6-4 photoproducts (6-4PP). The high UV-induced skin cancer susceptibility of mice compared to man has been attributed to low expression of the UV-DDB subunit DDB2 in mouse skin cells. However, DDB2 knockout mice exhibit enhanced UVB skin carcinogenesis indicating that DDB2 protects mice against UV-induced skin cancer. To resolve these apparent contradictory findings, we systematically investigated the NER capacity of mouse fibroblasts and keratinocytes. Compared to fibroblasts, keratinocytes exhibited an increased level of UV-DDB activity, contained significantly higher levels of other NER proteins (i.e. XPC and XPB) and displayed efficient repair of CPD. At low UVB dosages, the difference in skin cancer susceptibility between DDB2 KO and wild type mice was even much more pronounced than previously reported with high dose UVB exposures. Hence, our observations show that mouse keratinocytes express sufficient levels of UV-DDB for efficient repair of photolesions and efficient protection against UV-induced skin cancer at physiological relevant UV exposure.     

Sequential binding of UV DNA damage binding factor and degradation of the p48 subunit as early events after UV irradiation

The UV-damaged DNA binding protein complex (UV-DDB) is implicated in global genomic nucleotide excision repair (NER) in mammalian cells. The complex consists of a heterodimer of p127 and p48. UV-DDB is defective in one complementation group (XP-E) of the heritable, skin cancer-prone disorder xeroderma pigmentosum. Upon UV irradiation of primate cells, UV-DDB associates tightly with chromatin, concomitant with the loss of extractable binding activity. We report here that an early event after UV, but not ionizing, radiation is the transient dose-dependent degradation of the small subunit, p48. Treatment of human cells with the proteasomal inhibitor NIP-L₃VS blocks this UV-induced degradation of p48. In XP-E cell lines with impaired UV-DDB binding, p48 is resistant to degradation. UV-mediated degradation of p48 occurs independently of the expression of p53 and the cell’s proficiency for NER, but recovery of p48 levels at later times (12 h and thereafter) is dependent upon the capacity of the cell to repair non-transcribed DNA. In addition, we find that the p127 subunit of UV-DDB binds in vivo to p300, a histone acetyltransferase. The data support a functional connection between UV-DDB binding activity, proteasomal degradation of p48 and chromatin remodeling during early steps of NER.     

The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions

Previous studies point to the XPC-hHR23B complex as the principal initiator of global genome nucleotide excision repair (NER) pathway, responsible for the repair of UV-induced cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) in human cells. However, the UV-damaged DNA binding protein (UV-DDB) has also been proposed as a damage recognition factor involved in repair of UV-photoproducts, especially CPD. Here, we show in human XP-E cells (UV-DDB deficient) that the incision complex formation at UV-induced lesions was severely diminished in locally damaged nuclear spots. Repair kinetics of CPD and 6-4PP in locally and globally UV-irradiated normal human and XP-E cells demonstrate that UV-DDB can mediate efficient targeting of XPC-hHR23B and other NER factors to 6-4PP. The data is consistent with a mechanism in which UV-DDB forms a stable complex when bound to a 6-4PP, allowing subsequent repair proteins--starting with XPC-hHR23B--to accumulate, and verify the lesion, resulting in efficient 6-4PP repair. These findings suggest that (i) UV-DDB accelerates repair of 6-4PP, and at later time points also CPD, (ii) the fraction of 6-4PP that can be bound by UV-DDB is limited due to its low cellular quantity and fast UV dependent degradation, and (iii) in the absence of UV-DDB a slow XPC-hHR23B dependent pathway is capable to repair 6-4PP, and to some extent also CPD.     

Xeroderma pigmentosum complementation group C protein (XPC) serves as a general sensor of damaged DNA

The Xeroderma pigmentosum complementation group C protein (XPC) serves as the primary initiating factor in the global genome nucleotide excision repair pathway (GG-NER). Recent reports suggest XPC also stimulates repair of oxidative lesions by base excision repair. However, whether XPC distinguishes among various types of DNA lesions remains unclear. Although the DNA binding properties of XPC have been studied by several groups, there is a lack of consensus over whether XPC discriminates between DNA damaged by lesions associated with NER activity versus those that are not. In this study we report a high-throughput fluorescence anisotropy assay used to measure the DNA binding affinity of XPC for a panel of DNA substrates containing a range of chemical lesions in a common sequence. Our results demonstrate that while XPC displays a preference for binding damaged DNA, the identity of the lesion has little effect on the binding affinity of XPC. Moreover, XPC was equally capable of binding to DNA substrates containing lesions not repaired by GG-NER. Our results suggest XPC may act as a general sensor of damaged DNA that is capable of recognizing DNA containing lesions not repaired by NER.     

Regulation of nucleotide excision repair by UV-DDB: prioritization of damage recognition to internucleosomal DNA

How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin.     

Monoubiquitinated histone H2A destabilizes photolesion-containing nucleosomes with concomitant release of UV-damaged DNA-binding protein E3 ligase

How the nucleotide excision repair (NER) machinery gains access to damaged chromatinized DNA templates and how the chromatin structure is modified to promote efficient repair of the non-transcribed genome remain poorly understood. The UV-damaged DNA-binding protein complex (UV-DDB, consisting of DDB1 and DDB2, the latter of which is mutated in xeroderma pigmentosum group E patients, is a substrate-recruiting module of the cullin 4B-based E3 ligase complex, DDB1-CUL4B(DDB2). We previously reported that the deficiency of UV-DDB E3 ligases in ubiquitinating histone H2A at UV-damaged DNA sites in the xeroderma pigmentosum group E cells contributes to the faulty NER in these skin cancer-prone patients. Here, we reveal the mechanism by which monoubiquitination of specific H2A lysine residues alters nucleosomal dynamics and subsequently initiates NER. We show that DDB1-CUL4B(DDB2) E3 ligase specifically binds to mononucleosomes assembled with human recombinant histone octamers and nucleosome-positioning DNA containing cyclobutane pyrimidine dimers or 6-4 photoproducts photolesions. We demonstrate functionally that ubiquitination of H2A Lys-119/Lys-120 is necessary for destabilization of nucleosomes and concomitant release of DDB1-CUL4B(DDB2) from photolesion-containing DNA. Nucleosomes in which these lysines are replaced with arginines are resistant to such structural changes, and arginine mutants prevent the eviction of H2A and dissociation of polyubiquitinated DDB2 from UV-damaged nucleosomes. The partial eviction of H3 from the nucleosomes is dependent on ubiquitinated H2A Lys-119/Lys-120. Our results provide mechanistic insight into how post-translational modification of H2A at the site of a photolesion initiates the repair process and directly affects the stability of the human genome.     

Recruitment of the nucleotide excision repair endonuclease XPG to sites of UV-induced dna damage depends on functional TFIIH

The structure-specific endonuclease XPG is an indispensable core protein of the nucleotide excision repair (NER) machinery. XPG cleaves the DNA strand at the 3' side of the DNA damage. XPG binding stabilizes the NER preincision complex and is essential for the 5' incision by the ERCC1/XPF endonuclease. We have studied the dynamic role of XPG in its different cellular functions in living cells. We have created mammalian cell lines that lack functional endogenous XPG and stably express enhanced green fluorescent protein (eGFP)-tagged XPG. Life cell imaging shows that in undamaged cells XPG-eGFP is uniformly distributed throughout the cell nucleus, diffuses freely, and is not stably associated with other nuclear proteins. XPG is recruited to UV-damaged DNA with a half-life of 200 s and is bound for 4 min in NER complexes. Recruitment requires functional TFIIH, although some TFIIH mutants allow slow XPG recruitment. Remarkably, binding of XPG to damaged DNA does not require the DDB2 protein, which is thought to enhance damage recognition by NER factor XPC. Together, our data present a comprehensive view of the in vivo behavior of a protein that is involved in a complex chromatin-associated process.     

A 127 kDa component of a UV-damaged DNA-binding complex, which is defective in some xeroderma pigmentosum group E patients, is homologous to a slime mold protein.

A cDNA which encodes a approximately 127 kDa UV-damaged DNA-binding (UV-DDB) protein with high affinity for (6-4)pyrimidine dimers [Abramic', M., Levine, A.S. & Protic', M., J. Biol. Chem. 266: 22493-22500, 1991] has been isolated from a monkey cell cDNA library. The presence of this protein in complexes bound to UV-damaged DNA was confirmed by immunoblotting. The human cognate of the UV-DDB gene was localized to chromosome 11. UV-DDB mRNA was expressed in all human tissues examined, including cells from two patients with xeroderma pigmentosum (group E) that are deficient in UV-DDB activity, which suggests that the binding defect in these cells may reside in a dysfunctional UV-DDB protein. Database searches have revealed significant homology of the UV-DDB protein sequence with partial sequences of yet uncharacterized proteins from Dictyostelium discoideum (44% identity over 529 amino acids) and Oryza sativa (54% identity over 74 residues). According to our results, the UV-DDB polypeptide belongs to a highly conserved, structurally novel family of proteins that may be involved in the early steps of the UV response, e.g., DNA damage recognition.