UV-damaged DNA-binding Protein Research Articles

Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair

UV light-induced photoproducts are recognized and removed by the nucleotide-excision repair (NER) pathway. In humans, the UV-damaged DNA-binding protein (UV-DDB) is part of a ubiquitin E3 ligase complex (DDB1-CUL4A(DDB2)) that initiates NER by recognizing damaged chromatin with concomitant ubiquitination of core histones at the lesion. We report the X-ray crystal structure of the human UV-DDB in a complex with damaged DNA and show that the N-terminal domain of DDB2 makes critical contacts with two molecules of DNA, driving N-terminal-domain folding and promoting UV-DDB dimerization. The functional significance of the dimeric UV-DDB [(DDB1-DDB2)(2)], in a complex with damaged DNA, is validated by electron microscopy, atomic force microscopy, solution biophysical, and functional analyses. We propose that the binding of UV-damaged DNA results in conformational changes in the N-terminal domain of DDB2, inducing helical folding in the context of the bound DNA and inducing dimerization as a function of nucleotide binding. The temporal and spatial interplay between domain ordering and dimerization provides an elegant molecular rationale for the unprecedented binding affinities and selectivities exhibited by UV-DDB for UV-damaged DNA. Modeling the DDB1-CUL4A(DDB2) complex according to the dimeric UV-DDB-AP24 architecture results in a mechanistically consistent alignment of the E3 ligase bound to a nucleosome harboring damaged DNA. Our findings provide unique structural and conformational insights into the molecular architecture of the DDB1-CUL4A(DDB2) E3 ligase, with significant implications for the regulation and overall organization of the proteins responsible for initiation of NER in the context of chromatin and for the consequent maintenance of genomic integrity.

Characterisation of alleles of tomato light signalling genes generated by TILLING

Targeting Induced Local Lesions IN Genomes (TILLING) combines chemical mutagenesis with high throughput screening to allow the generation of alleles of selected genes. In this study, TILLING has been applied to produce a series of mutations in genes encoding essential components of the tomato light signal transduction pathway in an attempt to enhance fruit nutritional quality. Point mutations to DEETIOLATED1 (DET1), which is responsible for the high pigment2 (hp2) tomato mutant, resulted in elevated levels of both carotenoid and phenylpropanoid phytonutrients in ripe fruit, whilst immature fruit showed increased chlorophyll content, photosynthetic capacity and altered fruit morphology. Furthermore, genotypes with mutations to the UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), COP1 and COP1like were also characterised. These genotypes largely did not display phenotypes characteristic of mutation to light signalling components but their characterisation has enabled interrogation of structure function relationships of the mutated genes.

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.

Regulation of Nucleotide Excision Repair by UV-DDB: Prioritization of Damage Recognition to Internucleosomal DNA

Author SummaryLike all molecules in living organisms, DNA undergoes spontaneous decay and is constantly under attack by endogenous and environmental agents. Unlike other molecules, however, DNA—the blueprint of heredity—cannot be re-created de novo; it can only be copied. The original blueprint must therefore remain pristine. All kinds of DNA damage pose a health hazard. DNA lesions induced by the ultraviolet (UV) component of sunlight, for example, can lead to skin aging and skin cancer. A repair process known as nucleotide excision repair (NER) is dedicated to correcting this UV damage. Although the enzymatic steps of this repair process are known in detail, we still do not understand how it copes with the native situation in the cell, where the DNA is tightly wrapped around protein spools called nucleosomes. Our study has revealed the molecular mechanism by which an enigmatic component of NER called UV-DDB stimulates excision of UV-induced lesions in the landscape of nucleosome-packaged DNA in human skin cells. In particular, we describe how this accessory protein prioritizes, in space and time, which UV lesions in packaged DNA to target for repair by NER complexes, thus optimizing the repair process.

Overexpression of UV-DAMAGED DNA BINDING PROTEIN 1 links plant development and phytonutrient accumulation in high pigment-1 tomato

Fruits of tomato plants carrying the high pigment-1 mutations hp-1 and hp-1w are characterized by an increased number of plastids coupled with enhanced levels of functional metabolites. Unfortunately, hp-1 mutant plants are also typified by light-dependent retardation in seedling and whole-plant growth and development, which limits their cultivation. These mutations were mapped to the gene encoding UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1) and, recently, fruit-specific RNA interference studies have demonstrated an increased number of plastids and enhanced carotenoid accumulation in the transgenic tomato fruits. However, whole-plant overexpression of DDB1, required to substantiate its effects on seedling and plant development and to couple them with fruit phenotypes, has heretofore been unsuccessful. In this study, five transgenic lines constitutively overexpressing normal DDB1 in hp-1 mutant plants were analysed. Eleven-day-old seedlings, representing these lines, displayed up to ∼73- and ∼221-fold overexpression of the gene in hypocotyls and cotyledons, respectively. This overexpression resulted in statistically significant reversion to the non-mutant developmental phenotypes, including more than a full quantitative reversion. This reversion of phenotypes was generally accompanied by correlated responses in chlorophyll accumulation and altered expression of selected light signalling genes: PHYTOCHROME A, CRYPTOCHROME 1, ELONGATED HYPOCOTYL 5, and the gene encoding CHLOROPHYLL A/B-BINDING PROTEIN 4. Cumulatively, these results provide the missing link between DDB1 and its effects on tomato plant development.

Effect of overexpression of Arabidopsis Damaged DNA-binding protein 1A on De-etiolated 1

In Arabidopsis thaliana, de-etiolated 1 mutants (det1) grown in the dark resemble light-grown wild-type seedlings. Arabidopsis DET1 encodes a 62 kD protein, which is a negative regulator of light signaling. UV-damaged DNA-binding protein 1 (DDB1) was initially identified due to its role in human DNA damage repair. Arabidopsis has two DDB1 homologs: DDB1A and DDB1B. DDB1A mutation enhances det1 mutant phenotypes. In this study, we generated Arabidopsis lines that overexpress DDB1A-3HA in wild-type, det1, as well as Myc-DET1 or GFP-DET1 rescued genetic backgrounds. DDB1A-3HA overexpression resulted in decreased apical hook formation in wild-type dark-grown seedlings, and enhanced det1 small rosette and early flowering time phenotypes. In the Myc-DET1 background, DDB1A-3HA overexpression resulted in decreased rescue of dark- and light-grown hypocotyl length, light-grown anthocyanin and chlorophyll levels, adult height and stem number phenotypes. This result is consistent with the decreased levels of Myc-DET1 protein detected in the DDB1A-3HA overexpression line. The GFP-DET1 DDB1A-3HA double overexpression line exhibited increased rescue of dark and light-grown hypocotyl length and light-grown chlorophyll level phenotypes relative to GFP-DET1 alone, despite the fact that GFP-DET1 protein also decreased in the double overexpression line. In addition, increased DET1 resulted in decreased DDB1A-3HA levels due to proteasomal degradation. Overall, DDB1A-3HA overexpression affected phenotypes in a variety of DET1 backgrounds, reduced epitope-tagged DET1 levels, and, correlatively, in general dampened the rescue of det1 mutants by the DET1-DDB1A complex.

UV-DDB: A molecular machine linking DNA repair with ubiquitination

UV-damaged DNA-binding protein (UV-DDB) is characterized by its very high affinity and specificity for UV-damaged DNA. Although precise roles for UV-DDB have been quite enigmatic since its discovery, accumulating evidence indicates that it promotes recognition of and protein assembly on UV photolesions in the global genome nucleotide excision repair pathway. The recently solved crystal structure of UV-DDB bound to DNA containing a (6-4) photoproduct has revealed that the DDB2/XPE subunit is responsible for the interaction, which induces flipping out of the two affected bases into a binding pocket, indicating that UV-DDB has evolved especially to recognize dinucleotide lesions, like UV photolesions. Taken together with the previously solved structure of the DDB1-CUL4A E3 ligase, this study has also novel insights into how this factor coordinates ubiquitination of various protein substrates around the site of DNA damage.

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.

Schizosaccharomyces pombe Ddb1 Recruits Substrate-Specific Adaptor Proteins through a Novel Protein Motif, the DDB-Box

DDB1 was isolated as a UV-damaged DNA-binding protein, but recent studies established that it plays a role as a component of cullin 4A ubiquitin ligases. Cullin-RING complexes are the largest known ubiquitin ligase family, with hundreds of substrate-specific adaptor subunits and which are defined by characteristic motifs. A common motif for DDB1/cullin 4 ubiquitin ligases, a WDXR motif, was recently reported. Here, we show that Schizosaccharomyces pombe Ddb1 associates with several WD40 repeat proteins that share a novel protein motif designated the DDB-box, a motif essential for interaction with Ddb1 and independent of WD40 repeats, unlike the WDXR motif. We also show that ddb1(+) and the putative CSA homolog ckn1(+) are involved in transcription-coupled nucleotide excision repair and that the DDB-box is essential for the ckn1(+) function in vivo. These data indicate that the DDB-box is another common motif which defines adaptor proteins for DDB1/cullin 4 ubiquitin ligases.

Hepatitis B virus X protein affects S phase progression leading to chromosome segregation defects by binding to damaged DNA binding protein 1

Chronic hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC), but its role in the transformation process remains unclear. HBV encodes a small protein, known as HBx, which is required for infection and has been implicated in hepatocarcinogenesis. Here we show that HBx induces lagging chromosomes during mitosis, which in turn leads to formation of aberrant mitotic spindles and multinucleated cells. These effects require the binding of HBx to UV-damaged DNA binding protein 1 (DDB1), a protein involved in DNA repair and cell cycle regulation, and are unexpectedly attributable to HBx interfering with S-phase progression and not directly with mitotic events. HBx also affects S-phase and induces lagging chromosomes when expressed from its natural viral context and, consequently, exhibits deleterious activities in dividing, but not quiescent, hepatoma cells. In addition to its reported role in promoting HBV replication, the binding of HBx to DDB1 may induce genetic instability in regenerating hepatocytes and thereby contribute to HCC development, thus making this HBV-host protein interaction an attractive target for new therapeutic intervention.

The Cullin 4B–Based UV-Damaged DNA-Binding Protein Ligase Binds to UV-Damaged Chromatin and Ubiquitinates Histone H2A

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

Genetic analysis of the Neurospora crassa RAD14 homolog mus-43 and the RAD10 homolog mus-44 reveals that they belong to the mus-38 pathway of two nucleotide excision repair systems

Saccharomyces cerevisiae Rad14 and Rad10 proteins are essential for nucleotide excision repair (NER). Rad14 is a UV-damaged DNA binding protein and Rad10 is a structure-specific endonuclease that functions in a complex with Rad1. In this study, we identified and characterized the RAD14 and RAD10 homolog genes in Neurospora crassa, which we named mus-43 and mus-44, respectively. Disruption of mus-43 and mus-44 conferred sensitivity to UV and 4-nitroquinoline 1-oxide, but not to methyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, camptothecin, hydroxyurea, or bleomycin. The mus-44 mutant was more sensitive to UV than the mus-43 mutant. Genetic analysis indicated that mus-43 and mus-44 are epistatic to mus-38 which is a homolog of the S. cerevisiae RAD1, but not to mus-18 which belongs to a second excision repair pathway. Immunological assays demonstrated that both mus-43 and mus-44 retained the ability to excise UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts, but that excision ability was completely abolished in the mus-43 mus-18 and mus-44 mus-18 double mutants. These double mutants exhibited extremely high sensitivity to UV. In mus-43 and mus-44 mutants, the UV-induced mutation frequency increased compared to that of the wild-type. The mus-44 mutants also exhibited a partial photoreactivation defect phenotype similar to mus-38. These results suggest that both mus-43 and mus-44 function in the mus-38 NER pathway, but not in the mus-18 excision repair pathway.

In Vivo Destabilization and Functional Defects of the Xeroderma Pigmentosum C Protein Caused by a Pathogenic Missense Mutation

Xeroderma pigmentosum group C (XPC) protein plays an essential role in DNA damage recognition in mammalian global genome nucleotide excision repair (NER). Here, we analyze the functional basis of NER inactivation caused by a single amino acid substitution (Trp to Ser at position 690) in XPC, previously identified in the XPC patient XP13PV. The Trp690Ser change dramatically affects the in vivo stability of the XPC protein, thereby causing a significant reduction of its steady-state level in XP13PV fibroblasts. Despite normal heterotrimeric complex formation and physical interactions with other NER factors, the mutant XPC protein lacks binding affinity for both undamaged and damaged DNA. Thus, this single amino acid substitution is sufficient to compromise XPC function through both quantitative and qualitative alterations of the protein. Although the mutant XPC fails to recognize damaged DNA, it is still capable of accumulating in a UV-damaged DNA-binding protein (UV-DDB)-dependent manner to UV-damaged subnuclear domains. However, the NER factors transcription factor IIH and XPA failed to colocalize stably with the mutant XPC. As well as highlighting the importance of UV-DDB in recruiting XPC to UV-damaged sites, these findings demonstrate the role of DNA binding by XPC in the assembly of subsequent NER intermediate complexes.

DDB1 is essential for genomic stability in developing epidermis

The mammalian epidermis is maintained by proliferation and differentiation of epidermal progenitor cells in a stereotyped developmental program. Here we report that tissue-specific deletion of the UV-damaged DNA-binding protein 1 (DDB1) in mouse epidermis led to dramatic accumulation of c-Jun and p21Cip1, arrest of cell cycle at G(2)/M, selective apoptosis of proliferating cells, and as a result, a nearly complete loss of the epidermis and hair follicles. Deletion of the p53 tumor suppressor gene partially rescued the epithelial progenitor cells from death and allowed for the accumulation of aneuploid cells in the epidermis. Our results suggest that DDB1 plays an important role in development by controlling levels of cell cycle regulators and thereby maintaining genomic stability.

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.

Characterization of T-DNA Insertion Mutants and RNAi Silenced Plants of Arabidopsis thaliana UV-damaged DNA Binding Protein 2 (AtUV-DDB2)

The human UV-damaged DNA binding protein (UV-DDB), a heterodimeric protein composed of 127 kDa (UV-DDB1) and 48 kDa (UV-DDB2) subunits, has been shown to be involved in DNA repair. To elucidate the in vivo function of plant UV-DDB2, we have analyzed T-DNA insertion mutants of the Arabidopsis thaliana UV-DDB2 subunit (atuv-ddb2 mutants) and AtUV-DDB2 RNAi silenced plants (atuv-ddb2 silenced plants). atuv-ddb2 mutants and atuv-ddb2 silenced plants were both viable, suggesting that AtUV-DDB2 is not essential for survival. Interestingly, both plant types showed a dwarf phenotype, implying impaired growth of the meristem. To the best of our knowledge, this is the first occasion that a dwarf phenotype has been found to be associated with a UV-DDB2 mutation in either plants or animals. The mutants also demonstrated increased sensitivity to UV irradiation, methyl methanesulfonate and hydrogen peroxide treatment, indicating that AtUV-DDB2 is also involved in DNA repair. Our results lead us to suggest that not only does AtUV-DDB2 function in DNA repair, it also has a direct or indirect influence on cell proliferation in the plant meristem.

The DDB1-CUL4A DDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites

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

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.

Ddb1 controls genome stability and meiosis in fission yeast

The human UV-damaged DNA-binding protein Ddb1 associates with cullin 4 ubiquitin ligases implicated in nucleotide excision repair (NER). These complexes also contain the signalosome (CSN), but NER-relevant ubiquitination targets have not yet been identified. We report that fission yeast Ddb1, Cullin 4 (Pcu4), and CSN subunits Csn1 and Csn2 are required for degradation of the ribonucleotide reductase (RNR) inhibitor protein Spd1. Ddb1-deficient cells have >20-fold increased spontaneous mutation rate. This is partly dependent on the error-prone translesion DNA polymerases. Spd1 deletion substantially reduced the mutation rate, suggesting that insufficient RNR activity accounts for approximately 50% of observed mutations. Epistasis analysis indicated that Ddb1 contributed to mutation avoidance and tolerance to DNA damage in a pathway distinct from NER. Finally, we show that Ddb1/Csn1/Cullin 4-mediated Spd1 degradation becomes essential when cells differentiate into meiosis. These results suggest that Ddb1, along with Cullin 4 and the signalosome, constitute a major pathway controlling genome stability, repair, and differentiation via RNR regulation.

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.