Repair Of UV-induced DNA Damage Research Articles

Bombyx mori RPL13 participates in UV-induced DNA damage repair of B. mori nucleopolyhedrovirus through interaction with Bm65.

Ribosomal protein L13 (RPL13) is highly conserved in evolution. At present, the properties and functions of RPL13 have not been characterised in insects. In this study, Bombyx mori RPL13 (BmRPL13) was first found to be specifically recruited to the sites of ultraviolet (UV)-induced DNA damage and contributed to UV damage repair. Escherichia coli expressing BmRPL13 showed better resistance to UV radiation. After knocking down the expression of BmRPL13 in BmN cells, the repair speed of UV-damaged DNA slowed down. The further results showed that BmRPL13 interacted with B. mori nucleopolyhedrovirus (BmNPV) ORF65 (Bm65) protein to locate at the UV-induced DNA damage sites of BmNPV and helped repair UV-damaged viral DNA.

One More for Light-triggered Conformational Changes in Cryptochromes: CryP from Phaeodactylum tricornutum

Cryptochromes are a ubiquitously occurring class of photoreceptors. Together with photolyases, they form the Photolyase Cryptochrome Superfamily (PCSf) by sharing a common protein architecture and binding mode of the FAD chromophore. Despite these similarities, PCSf members exert different functions. Photolyases repair UV-induced DNA damage by photocatalytically driven electron transfer between FADH¯ and the DNA lesion, whereas cryptochromes are light-dependent signaling molecules and trigger various biological processes by photoconversion of their FAD redox and charge states. Given that most cryptochromes possess a C-terminal extension (CTE) of varying length, the functions of their CTE have not yet been fully elucidated and are hence highly debated. In this study, the role of the CTE was investigated for a novel subclass of the PCSf, the CryP-like cryptochromes, by hydrogen/deuterium exchange and mass-spectrometric analysis. Striking differences in the relative deuterium uptake were observed in different redox states of CryP from the diatom Phaeodactylum tricornutum. Based on these measurements we propose a model for light-triggered conformational changes in CryP-like cryptochromes that differs from other known cryptochrome families like the insect or plant cryptochromes.

Molluscan Genomes Reveal Extensive Differences in Photopigment Evolution Across the Phylum.

In animals, opsins and cryptochromes are major protein families that transduce light signals when bound to light-absorbing chromophores. Opsins are involved in various light-dependent processes, like vision, and have been co-opted for light-independent sensory modalities. Cryptochromes are important photoreceptors in animals, generally regulating circadian rhythm, they belong to a larger protein family with photolyases, which repair UV-induced DNA damage. Mollusks are great animals to explore questions about light sensing as eyes have evolved multiple times across, and within, taxonomic classes. We used molluscan genome assemblies from 80 species to predict protein sequences and examine gene family evolution using phylogenetic approaches. We found extensive opsin family expansion and contraction, particularly in bivalve xenopsins and gastropod Go-opsins, while other opsins, like retinochrome, rarely duplicate. Bivalve and gastropod lineages exhibit fluctuations in opsin repertoire, with cephalopods having the fewest number of opsins and loss of at least 2 major opsin types. Interestingly, opsin expansions are not limited to eyed species, and the highest opsin content was seen in eyeless bivalves. The dynamic nature of opsin evolution is quite contrary to the general lack of diversification in mollusk cryptochromes, though some taxa, including cephalopods and terrestrial gastropods, have reduced repertoires of both protein families. We also found complete loss of opsins and cryptochromes in multiple, but not all, deep-sea species. These results help set the stage for connecting genomic changes, including opsin family expansion and contraction, with differences in environmental, and biological features across Mollusca.

Cross-species investigation into the requirement of XPA for nucleotide excision repair.

After reconstitution of nucleotide excision repair (excision repair) with XPA, RPA, XPC, TFIIH, XPF-ERCC1and XPG, it was concluded that these six factors are the minimal essential components of the excision repair machinery. All six factors are highly conserved across diverse organisms spanning yeast to humans, yet no identifiable homolog of the XPA gene exists in many eukaryotes including green plants. Nevertheless, excision repair is reported to be robust in the XPA-lacking organism, Arabidopsis thaliana, which raises a fundamental question of whether excision repair could occur without XPA in other organisms. Here, we performed a phylogenetic analysis of XPA across all species with annotated genomes and then quantitatively measured excision repair in the absence of XPA using the sensitive whole-genome qXR-Seq method in human cell lines and two model organisms, Caenorhabditis elegans and Drosophila melanogaster. We find that although the absence of XPA results in inefficient excision repair and UV-sensitivity in humans, flies, and worms, excision repair of UV-induced DNA damage is detectable over background. These studies have yielded a significant discovery regarding the evolution of XPA protein and its mechanistic role in nucleotide excision repair.

Unbalanced Growth, the DNA Replication Cycle and Discovery of Repair Replication.

This article recounts my graduate research at Yale University (1954-1958) on unbalanced growth in Eschericia coli during thymine deprivation or following ultraviolet (UV) irradiation, with early evidence for the repair of UV-induced DNA damage. Follow-up studies in Copenhagen (1958-1960) in the laboratory of Ole Maaløe led to my discovery that the DNA replication cycle can be synchronized by inhibiting protein and RNA syntheses and that an RNA synthesis step is essential for initiation of the cycle, but not for its completion. This work set the stage for my subsequent research at Stanford University, where the repair replication of damaged DNA was documented, to provide compelling evidence for an excision-repair pathway. That universal pathway validates the requirement for the redundant information in the complementary strands of duplex DNA to ensure genomic stability.

Bombyx mori Flap endonuclease 1 correlates with the repair of ultraviolet-induced DNA damage

Solar ultraviolet radiation (UV) can cause DNA damage in microorganisms. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and plays important roles in DNA replication and repair. At present, the properties and functions of FEN1 have not been characterized in detail in invertebrates such as Bombyx mori. In this study, Bombyx mori FEN1 (BmFEN1) was expressed in E. coli, and was shown to have nuclease activity that nonspecifically cleaved DNA in vitro. However, inside the cell, BmFEN1 did not cleave DNA randomly. Truncated BmFEN1 missing the nuclear localization signal (346–380 aa) still had the nuclease activity, but was no longer precisely localized to the sites of UV-induced DNA damage. It was further found that BmFEN1 favored the faster repair of UV-damaged DNA. The present study will provide a reference for further understanding the functions of BmFEN1 and UV-induced DNA damage repair mechanisms in insects.

604 RNA methylation facilitates the repair of UV-induced DNA damage and suppresses photocarcinogenesis

UV-induced DNA damage is repaired by nucleotide excision repair (NER) that corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here we show that METTL14 (methyltransferase-like 14), a critical component of the m6A RNA methyltransferase complex, promotes the repair of UV-induced DNA damage through regulating m6A mRNA methylation-mediated translation of the NER factor DDB2 and suppresses UV-induced skin tumorigenesis. Ultraviolet irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy.

BmNPV Orf 65 (Bm65) Is Identified as an Endonuclease Directly Facilitating UV-Induced DNA Damage Repair.

Baculoviruses have been used as biopesticides for the control of Lepidoptera larvae. However, solar UV radiation reduces the activity of baculovirus. In this study, an UV endonuclease, Bm65, was found encoded in the genome of Bombyx mori nuclear polyhedrosis virus (BmNPV). Bm65 (the ortholog of AcMNPV orf79) was guided by a key nuclear localization signal to enter the nucleus and accumulated at UV-induced DNA damage sites. Subsequent results further showed that Bm65-mediated DNA damage repair was not the only UV damage repair pathway of BmNPV. BmNPV also used host DNA repair proteins to repair UV-induced DNA damage. In summary, these results revealed that Bm65 was very important in UV-induced DNA damage repair of BmNPV, and BmNPV repaired UV-damaged DNA through a variety of ways. IMPORTANCE Baculovirus biopesticides are environmentally friendly insecticides and specifically infect invertebrates. UV radiation from the sunlight greatly reduces the activity of baculovirus biopesticides. However, the molecular mechanisms of most baculoviruses to repair UV-induced DNA damage remain unclear. Nucleotide excision repair (NER) is a major DNA repair pathway that removes UV-induced DNA lesions. At present, there are few reports about the nucleotide excision repair pathway in viruses. Here, we showed for the first time that the baculovirus Bm65 endonuclease actually cleaved UV-damaged DNA. Meanwhile, we found that BmNPV used both viral-encoded enzymes and host DNA damage repair proteins to reverse UV-induced DNA damage. These results will provide a reference for the research of UV damage repair of other viruses.

BAP1 promotes the repair of UV-induced DNA damage via PARP1-mediated recruitment to damage sites and control of activity and stability.

BRCA1-associated protein-1 (BAP1) is a ubiquitin C-terminal hydrolase domain-containing deubiquitinase with tumor suppressor activity. The gene encoding BAP1 is mutated in various human cancers, with particularly high frequency in kidney and skin cancers, and BAP1 is involved in many cancer-related cellular functions, such as DNA repair and genome stability. Although BAP1 stimulates DNA double-strand break repair, whether it functions in nucleotide excision repair (NER) is unknown. Here, we show that BAP1 promotes the repair of ultraviolet (UV)-induced DNA damage via its deubiquitination activity in various cell types, including primary melanocytes. Poly(ADP-ribose) polymerase 1 (PARP1) interacts with and recruits BAP1 to damage sites, with BAP1 recruitment peaking after the DDB2 and XPC damage sensors. BAP1 recruitment also requires histone H2A monoubiquitinated at Lys119, which accumulates at damage sites. PARP1 transiently poly(ADP-ribosyl)ates (PARylates) BAP1 at multiple sites after UV damage and stimulates the deubiquitination activity of BAP1 both intrinsically and via PARylation. PARP1 also promotes BAP1 stability via crosstalk between PARylation and ubiquitination. Many PARylation sites in BAP1 are mutated in various human cancers, among which the glutamic acid (Glu) residue at position 31, with particularly frequent mutation in kidney cancer, plays a critical role in BAP1 stabilization and promotes UV-induced DNA damage repair. Glu31 also participates in reducing the viability of kidney cancer cells. This study therefore reveals that BAP1 functions in the NER pathway and that PARP1 plays a role as a novel factor that regulates BAP1 enzymatic activity, protein stability, and recruitment to damage sites. This activity of BAP1 in NER, along with its cancer cell viability-reducing activity, may account for its tumor suppressor function.

Camptothecin compromises transcription recovery and cell survival against cisplatin and ultraviolet irradiation regardless of transcription-coupled nucleotide excision repair

DNA-damaging anti-cancer drugs are used clinically to induce cell death by causing DNA strand breaks or DNA replication stress. Camptothecin (CPT) and cisplatin are commonly used anti-cancer drugs, and their combined use enhances the anti-tumour effects. However, the mechanism underlying this enhanced effect has not been well studied. In this study, we analysed the combined effect of CPT and cisplatin or ultraviolet (UV) and found that CPT suppresses transcription recovery after UV damage and induces the disappearance of the Cockayne syndrome group B (CSB) protein, a transcription-coupled nucleotide excision repair (TC-NER) factor. This CPT-induced disappearance of CSB expression was suppressed by proteasome and transcription inhibitors. Moreover, CSB ubiquitination was detected after CPT treatment in a transcription-dependent manner, suggesting that the transcription stress caused by CPT induces CSB ubiquitination, resulting in CSB undetectability. However, Cockayne syndrome group A (CSA) and CUL4A were not involved in the CPT-induced CSB undetectability, suggesting that CSB ubiquitination caused by CPT is regulated differently from the UV response. However, cisplatin or UV sensitivity was enhanced by CPT even in CSB- or CSA-knockout cells. Furthermore, the excessive CSB expression, which suppressed CSB ubiquitination, did not cancel the combined effect of CPT. These results suggest that CPT blocks the repair of cisplatin or UV-induced DNA damage regardless of TC-NER status. CPT possibly compromised the alternative repair pathways other than TC-NER, leading to the suppression of transcription recovery and enhancement of cell killing.

Heterogeneous clinical features in Cockayne syndrome patients and siblings carrying the same CSA mutations

BackgroundCockayne syndrome (CS) is a rare autosomal recessive disorder caused by mutations in ERCC6/CSB or ERCC8/CSA that participate in the transcription-coupled nucleotide excision repair (TC-NER) of UV-induced DNA damage. CS patients display a large heterogeneity of clinical symptoms and severities, the reason of which is not fully understood, and that cannot be anticipated in the diagnostic phase. In addition, little data is available for affected siblings, and this disease is largely undiagnosed in North Africa.MethodsWe report here the clinical description as well as genetic and functional characterization of eight Tunisian CS patients, including siblings. These patients, who belonged to six unrelated families, underwent complete clinical examination and biochemical analyses. Sanger sequencing was performed for the recurrent mutation in five families, and targeted gene sequencing was done for one patient of the sixth family. We also performed Recovery RNA Synthesis (RRS) to confirm the functional impairment of DNA repair in patient-derived fibroblasts.ResultsSix out of eight patients carried a homozygous indel mutation (c.598_600delinsAA) in exon 7 of ERCC8, and displayed a variable clinical spectrum including between siblings sharing the same mutation. The other two patients were siblings who carried a homozygous splice-site variant in ERCC8 (c.843+1G>C). This last pair presented more severe clinical manifestations, which are rarely associated with CSA mutations, leading to gastrostomy and hepatic damage. Impaired TC-NER was confirmed by RRS in six tested patients.ConclusionsThis study provides the first deep characterization of case series of CS patients carrying CSA mutations in North Africa. These mutations have been described only in this region and in the Middle-East. We also provide the largest characterization of multiple unrelated patients, as well as siblings, carrying the same mutation, providing a framework for dissecting elusive genotype–phenotype correlations in CS.

DTL facilitates the Fanconi anemia pathway for ultraviolet-induced DNA repair in retinal pigment epithelial cells

The excessive energy of light, especially the invisible rays with lower wavelength, is basically absorbed by retinal pigment epithelium (RPE) and usually causes DNA damage. The molecular mechanism behind DNA damage repair response to this frequent stress in RPE is not clearly understood. In this study, we determined that the Fanconi anemia (FA) pathway was activated in human RPE ARPE-19 cells after ultraviolet (UV) B and C treatment. Moreover, immunoprecipitation (IP) of FANCD2 indicated that denticleless E3 ubiquitin protein ligase homolog (DTL) closely interacted with FANCD2. Knockdown of DTL weakened the activity of the FA pathway in ARPE-19 cells responding to UV treatment. Finally, the DTL promoter was incubated with a biotin-labeled probe and pulled down by streptavidin beads followed by the genomic DNA sonication. p53 was indicated by mass spectrum and further determined by chromatin IP assay. Taken together, our results demonstrated that DTL regulated by p53 could activate the FA pathway for UV-induced DNA damage repair in retinal pigment epithelial cells.

DTL facilitates the Fanconi anemia pathway for ultraviolet-induced DNA repair in retinal pigment epithelial cells

The excessive energy of light, especially the invisible rays with lower wavelength, is basically absorbed by retinal pigment epithelium (RPE) and usually causes DNA damage. The molecular mechanism behind DNA damage repair response to this frequent stress in RPE is not clearly understood. In this study, we determined that the Fanconi anemia (FA) pathway was activated in human RPE ARPE-19 cells after ultraviolet (UV) B and C treatment. Moreover, immunoprecipitation (IP) of FANCD2 indicated that denticleless E3 ubiquitin protein ligase homolog (DTL) closely interacted with FANCD2. Knockdown of DTL weakened the activity of the FA pathway in ARPE-19 cells responding to UV treatment. Finally, the DTL promoter was incubated with a biotin-labeled probe and pulled down by streptavidin beads followed by the genomic DNA sonication. p53 was indicated by mass spectrum and further determined by chromatin IP assay. Taken together, our results demonstrated that DTL regulated by p53 could activate the FA pathway for UV-induced DNA damage repair in retinal pigment epithelial cells.

SerpinB10, a Serine Protease Inhibitor, Is Implicated in UV-Induced Cellular Response.

UV-induced DNA damage response and repair are extensively studied processes, as any malfunction in these pathways contributes to the activation of tumorigenesis. Although several proteins involved in these cellular mechanisms have been described, the entire repair cascade has remained unexplored. To identify new players in UV-induced repair, we performed a microarray screen, in which we found SerpinB10 (SPB10, Bomapin) as one of the most dramatically upregulated genes following UV irradiation. Here, we demonstrated that an increased mRNA level of SPB10 is a general cellular response following UV irradiation regardless of the cell type. We showed that although SPB10 is implicated in the UV-induced cellular response, it has no indispensable function in cell survival upon UV irradiation. Nonetheless, we revealed that SPB10 might be involved in delaying the duration of DNA repair in interphase and also in S-phase cells. Additionally, we also highlighted the interaction between SPB10 and H3. Based on our results, it seems that SPB10 protein is implicated in UV-induced stress as a “quality control protein”, presumably by slowing down the repair process.

Vitamin D Receptor Promotes Global Nucleotide Excision Repair by Facilitating XPC Dissociation from Damaged DNA

Vitamin D receptor (VDR) is important for normal DNA repair, although the mechanism by which it acts is unclear. After focal UV irradiation to create subnuclear spots of DNA damage, epidermal keratinocytes from VDR-null mice as well as human epidermal keratinocytes depleted of VDR with small interfering RNA removed pyrimidine-pyrimidone (6-4) photoproducts more slowly than control cells. Costaining with antibodies to XPC, the DNA damage recognition sensor that initiates nucleotide excision repair, showed that XPC rapidly accumulated at spots of damage and gradually faded in control human keratinocytes. In VDR-depleted keratinocytes, XPC associated with DNA damage with comparable efficiency; however, XPC's dissociation dynamics were altered so that significantly more XPC was bound and retained over time than in control cells. The XPF endonuclease, which acts subsequently in nucleotide excision repair, bound and dissociated with comparable kinetics in control and VDR-depleted cells, but the extent of binding was reduced in the latter. These results as well as kinetic modeling of the data suggest that VDR's importance in the repair of UV-induced DNA damage is mediated in part by its ability to facilitate the dissociation of XPC from damaged DNA for the normal recruitment and assembly of other repair proteins to proceed.

The UVSSA complex alleviates MYC-driven transcription stress.

Cancer cells develop strong genetic dependencies, enabling survival under oncogenic stress. MYC is a key oncogene activated across most cancers, and identifying associated synthetic lethality or sickness can provide important clues about its activity and potential therapeutic strategies. On the basis of previously conducted genome-wide screenings in MCF10A cells expressing MYC fused to an estrogen receptor fragment, we identified UVSSA, a gene involved in transcription-coupled repair, whose knockdown or knockout decreased cell viability when combined with MYC expression. Synthetic sick interactions between MYC expression and UVSSA down-regulation correlated with ATM/CHK2 activation, suggesting increased genome instability. We show that the synthetic sick interaction is diminished by attenuating RNA polymerase II (RNAPII) activity; yet, it is independent of UV-induced damage repair, suggesting that UVSSA has a critical function in regulating RNAPII in the absence of exogenous DNA damage. Supporting this hypothesis, RNAPII ChIP-seq revealed that MYC-dependent increases in RNAPII promoter occupancy are reduced or abrogated by UVSSA knockdown, suggesting that UVSSA influences RNAPII dynamics during MYC-dependent transcription. Taken together, our data show that the UVSSA complex has a significant function in supporting MYC-dependent RNAPII dynamics and maintaining cell survival during MYC addiction. While the role of UVSSA in regulating RNAPII has been documented thus far only in the context of UV-induced DNA damage repair, we propose that its activity is also required to cope with transcriptional changes induced by oncogene activation.

Super hotspots and super coldspots in the repair of UV-induced DNA damage in the human genome

Super hotspots and super coldspots in the repair of UV-induced DNA damage in the human genome

Super hotspots and super coldspots in the repair of UV-induced DNA damage in the human genome

The formation of UV-induced DNA damage and its repair are influenced by many factors that modulate lesion formation and the accessibility of repair machinery. However, it remains unknown which genomic sites are prioritized for immediate repair after UV damage induction, and whether these prioritized sites overlap with hotspots of UV damage. We identified the super hotspots subject to the earliest repair for (6-4) pyrimidine–pyrimidone photoproduct by using the eXcision Repair-sequencing (XR-seq) method. We further identified super coldspots for (6-4) pyrimidine–pyrimidone photoproduct repair and super hotspots for cyclobutane pyrimidine dimer repair by analyzing available XR-seq time-course data. By integrating datasets of XR-seq, Damage-seq, adductSeq, and cyclobutane pyrimidine dimer-seq, we show that neither repair super hotspots nor repair super coldspots overlap hotspots of UV damage. Furthermore, we demonstrate that repair super hotspots are significantly enriched in frequently interacting regions and superenhancers. Finally, we report our discovery of an enrichment of cytosine in repair super hotspots and super coldspots. These findings suggest that local DNA features together with large-scale chromatin features contribute to the orders of magnitude variability in the rates of UV damage repair.

A topologically distinct class of photolyases specific for UV lesions within single-stranded DNA.

Photolyases are ubiquitously occurring flavoproteins for catalyzing photo repair of UV-induced DNA damages. All photolyases described so far have a bilobal architecture with a C-terminal domain comprising flavin adenine dinucleotide (FAD) as catalytic cofactor and an N-terminal domain capable of harboring an additional antenna chromophore. Using sequence-similarity network analysis we discovered a novel subgroup of the photolyase/cryptochrome superfamily (PCSf), the NewPHLs. NewPHL occur in bacteria and have an inverted topology with an N-terminal catalytic domain and a C-terminal domain for sealing the FAD binding site from solvent access. By characterizing two NewPHL we show a photochemistry characteristic of other PCSf members as well as light-dependent repair of CPD lesions. Given their common specificity towards single-stranded DNA many bacterial species use NewPHL as a substitute for DASH-type photolyases. Given their simplified architecture and function we suggest that NewPHL are close to the evolutionary origin of the PCSf.

All You Need Is Light. Photorepair of UV-Induced Pyrimidine Dimers.

Although solar light is indispensable for the functioning of plants, this environmental factor may also cause damage to living cells. Apart from the visible range, including wavelengths used in photosynthesis, the ultraviolet (UV) light present in solar irradiation reaches the Earth’s surface. The high energy of UV causes damage to many cellular components, with DNA as one of the targets. Putting together the puzzle-like elements responsible for the repair of UV-induced DNA damage is of special importance in understanding how plants ensure the stability of their genomes between generations. In this review, we have presented the information on DNA damage produced under UV with a special focus on the pyrimidine dimers formed between the neighboring pyrimidines in a DNA strand. These dimers are highly mutagenic and cytotoxic, thus their repair is essential for the maintenance of suitable genetic information. In prokaryotic and eukaryotic cells, with the exception of placental mammals, this is achieved by means of highly efficient photorepair, dependent on blue/UVA light, which is performed by specialized enzymes known as photolyases. Photolyase properties, as well as their structure, specificity and action mechanism, have been briefly discussed in this paper. Additionally, the main gaps in our knowledge on the functioning of light repair in plant organelles, its regulation and its interaction between different DNA repair systems in plants have been highlighted.