UV-damaged DNA-binding Protein Research Articles

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage.

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.

The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the high pigment-1 mutant phenotype.

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 ( hp-1) and the high pigment-1(w) ( hp-1(w)) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 ( hp-2), high pigment-2(j) ( hp-2(j)) and dark green ( dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1(w) mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A(931)-to-T(931) base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1(w) mutant, on the other hand, a single G(2392)-to-A(2392) transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F(2) population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25<LOD score<26, R(2)=62.8%). These results strongly support the hypothesis that DDB1 is the gene encoding the hp-1 and hp-1(w) mutant phenotypes.

True XP group E patients have a defective UV-damaged DNA binding protein complex and mutations in DDB2 which reveal the functional domains of its p48 product.

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

Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities.

The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.

Rice UV-damaged DNA binding protein homologues are most abundant in proliferating tissues

Ultraviolet-damaged DNA binding protein (UV-DDB) is an important factor involved in DNA repair. To study the role of UV-DDB, we attempted to obtain the cDNA and the protein of a plant UV-DDB. We succeeded in isolating both genes for UV-DDB subunits from rice ( Oryza sativa cv. Nipponbare), designated as OsUV-DDB1 and OsUV-DDB2. OsUV-DDB2 (65 kDa) was much larger than human UV-DDB2, but immunoprecipitation and gel mobility shift assay suggested that OsUV-DDB2 is a plant counterpart of UV-DDB2. The transcripts were expressed in proliferating tissues such as the meristem, but were detected at only low levels in the mature leaves, although the leaves are strongly exposed to UV. These transcripts were induced in the meristem after UV-irradiation. The expression levels of OsUV-DDB were significantly reduced when cell proliferation was temporarily halted. These results indicated that the level of OsUV-DDB expression is correlated with cell proliferation, and its expression may be required mostly for DNA repair in DNA replication.

De-Etiolated 1 and Damaged DNA Binding Protein 1 Interact to Regulate Arabidopsis Photomorphogenesis

Background: Plant development is exquisitely sensitive to light. Seedlings grown in the dark have a developmentally arrested etiolated phenotype, whereas in the light they develop leaves and complete their life cycle. Arabidopsis de-etiolated 1 (det1) mutants develop like light-grown seedlings even when grown in the dark. DET1 encodes a nuclear protein that appears to act downstream from multiple photoreceptors to regulate morphogenesis and gene expression in response to light. However, its function has remained unknown.Results: We used microarrays to examine defects in transcription in dark-grown det1 seedlings. We found extensive changes in gene expression, including many of the transcriptional responses observed in light-treated wild-type seedlings. We used an epitope-tagging approach to determine the basis of DET1 function. GFP-DET1 rescues the det1 phenotype, is localized to the nucleus, and forms an approximately 350 kDa complex, which is required for full DET1 activity. We affinity-purified the DET1 complex and identified an approximately 120 kDa copurifying protein that is the plant homolog of UV-Damaged DNA Binding Protein 1 (DDB1), a protein implicated in the human disease xeroderma pigmentosa. A null mutation in Arabidopsis DDB1A results in no obvious phenotype on its own, yet it enhances the phenotype of a weak det1 allele.Conclusions: DET1 and DDB1 interact both biochemically and genetically. In animal cells, DDB1 interacts with histone acetyltransferase complexes. The DET1/DDB1 complex may regulate gene expression in response to light via recruitment of HAT activity. Thus, DET1, whose sequence is conserved in both animals and plants, may play a direct role in the regulation of many genes.

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.

Hepatitis B virus X protein interferes with cell viability through interaction with the p127-kDa UV-damaged DNA-binding protein.

The hepatitis B virus X protein (HBx) is essential for establishing natural viral infection and has been implicated in the development of liver cancer associated with chronic infection. The basis for HBx function in either process is not understood. In cell culture, HBx exhibits pleiotropic activities affecting transcription, DNA repair, cell growth, and apoptotic cell death. Numerous cellular proteins including the p127-kDa subunit of UV-damaged DNA-binding activity have been reported to interact with HBx but the functional significance of these interactions remains unclear. Here we show that the binding of HBx to p127 interferes with cell viability. Mutational analysis reveals that HBx contacts p127 via a region to which no function has been assigned previously. An HBx variant bearing a single-charge reversal substitution within this region loses p127 binding and concomitant cytotoxicity. This mutant regains activity when directly fused to p127. These studies confirm that p127 is an important cellular target of HBx, and they indicate that HBx does not exert its effect by sequestering p127, and thereby preventing its normal function, but instead by conferring to p127 a deleterious activity.

UV-damaged DNA-binding protein in the TFTC complex links DNA damage recognition to nucleosome acetylation.

Initiation of transcription of protein-encoding genes by RNA polymerase II (Pol II) was thought to require transcription factor TFIID, a complex comprised of the TATA box-binding protein (TBP) and TBP-associated factors (TAF(II)s). In the presence of TBP-free TAF(II) complex (TFTC), initiation of Pol II transcription can occur in the absence of TFIID. TFTC containing the GCN5 acetyltransferase acetylates histone H3 in a nucleosomal context. We have identified a 130 kDa subunit of TFTC (SAP130) that shares homology with the large subunit of UV-damaged DNA-binding factor. TFTC preferentially binds UV-irradiated DNA, UV-damaged DNA inhibits TFTC-mediated Pol II transcription and TFTC is recruited in parallel with the nucleotide excision repair protein XP-A to UV-damaged DNA. TFTC preferentially acetylates histone H3 in nucleosomes assembled on UV-damaged DNA. In agreement with this, strong histone H3 acetylation occurs in intact cells after UV irradiation. These results suggest that the access of DNA repair machinery to lesions within chromatin may be facilitated by TFTC via covalent modification of chromatin. Thus, our experiments reveal a molecular link between DNA damage recognition and chromatin modification.

PSI-BLAST searches using hidden markov models of structural repeats: prediction of an unusual sliding DNA clamp and of beta-propellers in UV-damaged DNA-binding protein.

We have designed hidden Markov models (HMMs) of structurally conserved repeats that, based on pairwise comparisons, are unconserved at the sequence level. To model secondary structure features these HMMs assign higher probabilities of transition to insert or delete states within sequence regions predicted to form loops. HMMs were optimized using a sampling procedure based on the degree of statistical uncertainty associated with parameter estimates. A PSI-BLAST search initialized using a checkpoint-recovered profile derived from simulated sequences emitted by such a HMM can reveal distant structural relationships with, in certain instances, substantially greater sensitivity than a normal PSI-BLAST search. This is illustrated using two examples involving DNA- and RNA-associated proteins with structurally conserved repeats. In the first example a putative sliding DNA clamp protein was detected in the thermophilic bacterium Thermotoga maritima. This protein appears to have arisen by way of a duplicated beta-clamp gene that then acquired features of a PCNA-like clamp, perhaps to perform a PCNA-related function in association with one or more of the many archaeal-like proteins present in this organism. In the second example, beta-propeller domains were predicted in the large subunit of UV-damaged DNA-binding protein and in related proteins, including the large subunit of cleavage-polyadenylation specificity factor, the yeast Rse1p and human SAP130 pre-mRNA splicing factors and the fission yeast Rik1p gene silencing protein.

Cullin 4A Associates with the UV-damaged DNA-binding Protein DDB

The damaged DNA-binding protein (DDB) is believed to be involved in DNA repair, and it has been linked to the repair deficiency disease xeroderma pigmentosum. DDB also exhibits transcriptional activities. DDB binds to the activation domain of E2F1 and stimulates E2F1-activated transcription. Here we provide evidence that DDB or DDB-associated proteins are targets of cullin 4A (CUL-4A). CUL-4A is a member of the cullin family of proteins, which are believed to be ubiquitin-protein isopeptide ligases (type E3). The CUL-4A gene has been shown to be amplified and up-regulated in breast carcinomas. In this study, we identify CUL-4A as one of the DDB-associated proteins. CUL-4A co-immunoprecipitates with DDB, but not with a naturally occurring mutant of DDB. Moreover, CUL-4A in HeLa nuclear extracts co-purifies with DDB, suggesting they are parts of the same complex. The observation provides insights how CUL-4A, through an interaction with DDB, might be playing a role in the development of breast carcinomas.

DNA structures recognized by the human UV-DDB protein.

DNA recognition by the human UV-damaged DNA-binding (UV-DDB) protein was characterized. By circular permutation analyses, DNA duplexes containing the (6-4) photoproduct and the abasic site analog were found to be bent at angles of 54 degrees and 57 degrees, respectively, when they formed a complex with this protein. Although kinked NMR structures have been reported, fluorescence resonance energy transfer experiments revealed that these duplexes had no intrinsic bend. These results suggest that the UV-DDB protein binds DNA that can be bent easily at the above angle.

Purification and biochemical characterization of interchromatin granule clusters.

Components of the pre-mRNA splicing machinery are localized in interchromatin granule clusters (IGCs) and perichromatin fibrils (PFs). Here we report the biochemical purification of IGCs. Approximately 75 enriched proteins were present in the IGC fraction. Protein identification employing a novel mass spectrometry strategy and peptide microsequencing identified 33 known proteins, many of which have been linked to pre-mRNA splicing, as well as numerous uncharacterized proteins. Thus far, three new protein constituents of the IGCs have been identified. One of these, a 137 kDa protein, has a striking sequence similarity over its entire length to UV-damaged DNA-binding protein, a protein associated with the hereditary disease xeroderma pigmentosum group E, and to the 160 kDa subunit of cleavage polyadenylation specificity factor. Overall, these results provide a key framework that will enable the biological functions associated with the IGCs to be elucidated.

Characterization of DNA Recognition by the Human UV-damaged DNA-binding Protein

The UV-damaged DNA-binding (UV-DDB) protein is the major factor that binds DNA containing damage caused by UV radiation in mammalian cells. We have investigated the DNA recognition by this protein in vitro, using synthetic oligonucleotide duplexes and the protein purified from a HeLa cell extract. When a 32P-labeled 30-mer duplex containing the (6-4) photoproduct at a single site was used as a probe, only a single complex was detected in an electrophoretic mobility shift assay. It was demonstrated by Western blotting that both of the subunits (p48 and p127) were present in this complex. Electrophoretic mobility shift assays using various duplexes showed that the UV-DDB protein formed a specific, high affinity complex with the duplex containing an abasic site analog, in addition to the (6-4) photoproduct. By circular permutation analyses, these DNA duplexes were found to be bent at angles of 54 degrees and 57 degrees in the complexes with this protein. From the previously reported NMR studies and the fluorescence resonance energy transfer experiments in the present study, it can be concluded that the UV-DDB protein binds DNA that can be bent easily at the above angle.

A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein.

Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids. It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease. To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol. Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa). UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates. APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody. These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain. In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP.

CDNA cloning, tissue expression, and chromosomal assignment of a mouse gene, encoding a 127 kDa UV-damaged DNA binding protein which is defective in XPE cells.

A subset of xeroderma pigmentosum (XP) group E cells lack a factor of the UV-damaged DNA binding activity. Both 127 kDa and 48 kDa proteins have been reported to be responsible for the binding activity. A cDNA for the 127 kDa UV-damaged DNA-binding protein (p127-Ddb1) was isolated from a mouse fetal brain full length-enriched cDNA library, and an open reading frame of 1140 amino acids was identified. Reverse transcription-coupled polymerase chain reaction (RT-PCR) showed that mouse Ddb1 messenger is ubiquitously expressed in adult tissues as well as in embryo's. The gene was mapped to near the public locus D19Mit22 region of mouse chromosome 19.

The V Protein of the Paramyxovirus SV5 Interacts with Damage-Specific DNA Binding Protein

The simian parainfluenza virus 5 (SV5) V/P gene encodes two proteins: V and the phosphoprotein P. The V and P proteins are amino coterminal for 164 residues, but they have unique carboxyl termini. The unique carboxyl terminus of V contains seven cysteine residues, resembles a zinc finger, and binds two atoms of zinc. In a glutathione-S-transferase (GST)-fusion protein selection of cell lysate assay, the GST-V protein was found to interact with the 127-kDa subunit (DDB1) of the damage-specific DNA binding protein (DDB) [also known as UV-damaged DNA binding protein (UV-DDB), xeroderma pigmentosum group E binding factor (XPE-BF), and the hepatitis B virus X-associated protein 1 (XAP-1)]. A reciprocal GST-DDB1 fusion protein selection assay of SV5-infected cell lysates showed that DDB1 and V interact, and it was found that V and DDB1 could be coimmunoprecipitated from SV5-infected cells or from cells expressing V and DDB1 using the vaccinia virus T7 expression system. The interaction of V and DDB1 involves the carboxyl-terminal domain of V in that either deletion of the V carboxyl-terminal domain or substitution of the cysteine residues (C189, C193, C205, C207, C210, C214, and C217) in the zinc-binding domain with alanine was able to disrupt binding to DDB1. The V proteins of the mumps virus, human parainfluenza virus 2 (hPIV2), and measles virus have also been found to interact with DDB1 in GST-fusion protein selection assays usingin vitrotranscribed and translated DDB1.

Refined mapping of the gene encoding the p127 kDa UV-damaged DNA-binding protein (DDB1) within 11q12-q13.1 and its exclusion in Best's vitelliform macular dystrophy.

Best's vitelliform macular dystrophy (Best's disease) is an autosomal dominant disorder of unknown causes and is typically characterised by an accumulation of lipofuscin-like material in the subretinal space of the macula. The disease gene has been localised to chromosome 11q12-13.1 within a 1.4 Mbp interval flanked by markers at D11S1765 and uteroglobin (UGB). Here we report the refined mapping of the gene encoding the p127 kDa subunit (DDB1) of a UV damage-specific DNA binding protein within the D11S1765-UGB region. Northern blot analysis demonstrates an abundant expression of the DDB1 transcript in the retina suggesting a functional role for DDB1 in this tissue. These considerations together with the chromosomal localisation have led us to evaluate the possible involvement of DDB1 in the pathogenesis of Best's disease.

Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.

Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB- XP-E cell extracts, but microinjection of the protein into DDB- XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.

A novel UV-damaged DNA binding protein emerges during the chromatin-eliminating cleavage period in Ascaris suum.

During the early cleavage period of Ascaris suum , chromatin diminution takes place in the somatic founder cells. In the process of chromatin diminution numerous heterochromatic blocks, consisting predominantly of highly repeated DNA, are discarded during mitotic anaphase and are later on digested in the cytoplasm. Very little is known about proteins that are involved in chromatin diminution. We have detected a nuclear protein and purified it to near homogeneity by its preferential binding to UV-damaged DNA. We termed this protein chromatin diminution associated factor 1 (CDAF1), because maximum binding activity per nucleus was observed to develop in 4-8-cell stages, when chromatin diminution occurs for the first time. CDAF1 recognizes cyclobutane pyrimidine dimers in UV-damaged double-stranded DNA. Its binding properties identify CDAF1 as a novel kind of damaged-DNA binding protein. CDAF1 activity is almost not detectable in 1-celled embryos. It increases dramatically during formation of somatic founder cells and persists up to the first larval stage. However, CDAF1 is absent in tissues of adults. These findings led us to suggest that CDAF1 plays a dual role: during the early segregative cleavage period it might be involved in chromatin diminution as a transfactor and act in nucleotide excision repair as an accessory factor throughout embryogenesis.