UV-mediated DNA damage and repair are important mechanisms in research on UV-induced carcinogenesis. UV-induced DNA-damage and repair can be determined by immunohistochemical staining of photoproduct positive nuclei of keratinocytes in the epidermis. We developed a new method of analysing and quantifying thymine dimer (TT-CPD) positive cells in the epidermis. Normal skin of healthy controls was exposed to UVB ex vivo and in vivo. Skin samples were immunohistochemically stained for TT-CPDs. Digital images of the epidermis were quantified for TT-CPDs both visually and digitally. There was a UVB-dose dependent induction of TT-CPDs present in the ex vivo UVB-irradiated skin samples. The linear measurement range of the digital quantification was increased compared to the manual counting. The average 24-hour repair rate of the initiated TT-CPDs elicited by the UVB irradiation at T=0 of the 8 HCs showed a 34% decrease of TT-CPD photoproducts by the manual counting method and a 51% decrease determined by digital counting. The digital quantification method improves immunohistochemical quantification of DNA photo damage. It is more sensitive in measuring the extent of DNA-damage per nucleus.

Photochemical dimerization of adjacent pyrimidines is fundamental to the creation of mutagenic hotspots caused by ultraviolet light. Distribution of the resulting lesions (cyclobutane pyrimidine dimers, CPDs) is already known to be highly variable in cells, and in vitro models have implicated DNA conformation as a major basis for this observation. Past efforts have primarily focused on mechanisms that influence CPD formation and have rarely considered contributions of CPD reversion. However, reversion is competitive under the standard conditions of 254 nm irradiation as illustrated in this report based on the dynamic response of CPDs to changes in DNA conformation. A periodic profile of CPDs was recreated in DNA held in a bent conformation by λ repressor. After linearization of this DNA, the CPD profile relaxed to its characteristic uniform distribution over a similar time of irradiation to that required to generate the initial profile. Similarly, when a T tract was released from a bent conformation, its CPD profile converted under further irradiation to that consistent with a linear T tract. This interconversion of CPDs indicates that both its formation and reversion exert control on CPD populations long before photo-steady-state conditions are achieved and suggests that the dominant sites of CPDs will evolve as DNA conformation changes in response to natural cellular processes.

Photolyase transgenic mice have opened new avenues to improve our understanding of the cytotoxic effects of ultraviolet (UV) light on skin by providing a means to selectively remove either cyclobutane pyrimidine dimers (CPDs) or pyrimidine (6-4) pyrimidone photoproducts. Here, we have taken a genomics approach to delineate pathways through which CPDs might contribute to the harmful effects of UV exposure. We show that CPDs, rather than other DNA lesions or damaged macromolecules, comprise the principal mediator of the cellular transcriptional response to UV. The most prominent pathway induced by CPDs is that associated with DNA double-strand break (DSB) signalling and repair. Moreover, we show that CPDs provoke accumulation of gamma-H2AX, P53bp1 and Rad51 foci as well as an increase in the amount of DSBs, which coincides with accumulation of cells in S phase. Thus, conversion of unrepaired CPD lesions into DNA breaks during DNA replication may comprise one of the principal instigators of UV-mediated cytotoxicity.

Ultraviolet B radiation (UVB) is an environmental complete carcinogen, which induces and promotes keratinocyte carcinomas, the most common human malignancies. UVB induces the formation of cyclobutane pyrimidine dimers (CPDs). Repairing CPDs through nucleotide excision repair is slow and error-prone in placental mammals. In addition to the mutagenic and malignancy-inducing effects, UVB also elicits poorly understood complex metabolic changes in keratinocytes, possibly through CPDs. To determine the effects of CPDs, CPD-photolyase was overexpressed in keratinocytes using an N1-methyl pseudouridine-containing in vitro-transcribed mRNA. CPD-photolyase, which is normally not present in placental mammals, can efficiently and rapidly repair CPDs to block signaling pathways elicited by CPDs. Keratinocytes surviving UVB irradiation turn hypermetabolic. We show that CPD-evoked mitochondrial reactive oxygen species production, followed by the activation of several energy sensor enzymes, including sirtuins, AMPK, mTORC1, mTORC2, p53, and ATM, is responsible for the compensatory metabolic adaptations in keratinocytes surviving UVB irradiation. Compensatory metabolic changes consist of enhanced glycolytic flux, Szent-Györgyi-Krebs cycle, and terminal oxidation. Furthermore, mitochondrial fusion, mitochondrial biogenesis, and lipophagy characterize compensatory hypermetabolism in UVB-exposed keratinocytes. These properties not only support the survival of keratinocytes, but also contribute to UVB-induced differentiation of keratinocytes. Our results indicate that CPD-dependent signaling acutely maintains skin integrity by supporting cellular energy metabolism.

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO-), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO- scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

Escherichia coli DNA photolyase is an enzyme that repairs the major kind of UV-induced lesions, cyclobutane pyrimidine dimer (CPD) in DNA utilizing 350-450 nm light as energy source. The enzyme has very high photo-repair efficiency (the quantum yield of the reaction is ~0.85), which is significantly greater than many model compounds that mimic photolyase. This suggests that some residues of the protein play important roles in the photo-repair of CPD. In this paper, we have focused on several residues discussed their roles in catalysis by reviewing the existing literature and some hypotheses.

Exposure to sun and especially to ultraviolet radiation (UVR) exerts well known detrimental effects on skin which are implicated in malignancy. UVR induces production of cyclobutane pyrimidine dimers (CPDs), immediately during exposure and even hours after the exposure, these latter being called dark-CPDs, as consequence of the effects of different reactive species that are formed. Fernblock® (FB), an aqueous extract of Polypodium leucotomos, has proven to have photoprotective and antioxidant effects on skin. The aim of our work was to investigate the potential photoprotective effect of FB against dark-CPD formation. Murine melanocytes (B16-F10) were exposed to UVA radiation and the production of dark-CPDs and different reactive oxygen and nitrogen species (ROS and RNS) was measured. Significant dark-CPD formation could be seen at 3 h after UVA irradiation, which was inhibited by the pre-treatment of cells with FB. Formation of nitric oxide, superoxide and peroxynitrite was increased after irradiation, consistent with the increased CPD formation. FB successfully reduced the production of these reactive species. Hence, these results show how dark-CPDs are formed in UVA irradiated melanocytes, and that FB acts as a potential antioxidant and ROS scavenger, preventing the DNA damage induced by sun exposure.

Solar ultraviolet radiation (UVR)-induced DNA damage has acute, and long-term adverse effects in the skin. This damage arises directly by absorption of UVR, and indirectly via photosensitization reactions. The aim of the present study was to assess the effects of vitamin E on UVAI-induced DNA damage in keratinocytes in vitro. Incubation with vitamin E before UVAI exposure decreased the formation of oxidized purines (with a decrease in intracellular oxidizing species), and cyclobutane pyrimidine dimers (CPD). A possible sunscreening effect was excluded when similar results were obtained following vitamin E addition after UVAI exposure. Our data showed that DNA damage by UVA-induced photosensitization reactions can be inhibited by the introduction of vitamin E either pre- or post-irradiation, for both oxidized purines and CPD (including so-called "dark" CPDs). These data validate the evidence that some CPD are induced by UVAI initially via photosensitization, and some via chemoexcitation, and support the evidence that vitamin E can intervene in this pathway to prevent CPD formation in keratinocytes. We propose the inclusion of similar agents into topical sunscreens and aftersun preparations which, for the latter in particular, represents a means to mitigate on-going DNA damage formation, even after sun exposure has ended.

The progression of a normal cell to senescence in vivo and in vitro is accompanied by a reduction in the length of the telomeres, the chromosome capping segments at the end of each linkage group. However, overexpression of the reverse transcriptase subunit (HTERT) of the ribonucleoprotein telomerase restores telomere length and delays cellular senescence. Although some data exist in the literature with respect to survival, no molecular data have shown that DNA repair in telomerase-immortalized cells is normal. Several telomerase-immortalized human skin fibroblast cell lines were constructed from a primary human fibroblast cell line. The primary line and the telomerase-immortalized cell lines were treated with either ultraviolet (UV) radiation or dimethylsulfate (DMS). UV radiation principally produces cyclobutane pyrimidine dimers that are repaired by nucleotide excision repair, whereas DMS introduces mainly N-methylpurines repaired by base excision repair. Here, we show that repair of both types of damage in the telomerase-immortalized human skin fibroblast cell lines is identical to repair observed in normal skin fibroblasts. Thus, telomerase expression and consequent immortalization of skin fibroblasts do not alter nucleotide or base excision repair in human cells.

Sunlight-associated melanomas carry a unique C-to-T mutation signature. UVB radiation induces cyclobutane pyrimidine dimers (CPDs) as the major form of DNA damage, but the mechanism of how CPDs cause mutations is unclear. To map CPDs at single-base resolution genome wide, we developed the circle damage sequencing (circle-damage-seq) method. In human cells, CPDs form preferentially in a tetranucleotide sequence context (5'-Py-T<>Py-T/A), but this alone does not explain the tumor mutation patterns. To test whether mutations arise at CPDs by cytosine deamination, we specifically mapped UVB-induced cytosine-deaminated CPDs. Transcription start sites (TSSs) were protected from CPDs and deaminated CPDs, but both lesions were enriched immediately upstream of the TSS, suggesting a mutation-promoting role of bound transcription factors. Most importantly, the genomic dinucleotide and trinucleotide sequence specificity of deaminated CPDs matched the prominent mutation signature of melanomas. Our data identify the cytosine-deaminated CPD as the leading premutagenic lesion responsible for mutations in melanomas.

The adverse effects of terrestrial solar ultraviolet radiation (UVR) (~295-400 nm) on the skin are well documented, especially in the UVB region (~295-320 nm). The effects of very long-wave UVA (>380 nm) and visible radiation (≥400 nm) are much less known. Sunscreens have been beneficial in inhibiting a wide range of photodamage, however most formulations provide very little protection in the long wave UVA region (380-400 nm) and almost none from shortwave visible wavelengths (400-420 nm). We demonstrate photodamage in this region for a number of different endpoints including cell viability, DNA damage (delayed cyclobutane pyrimidine dimers), differential gene expression (for genes associated with inflammation, oxidative stress and photoageing) and induction of oxidizing species in vitro in HaCaT keratinocytes and in vivo in human volunteers. This work has implications for phototherapy and photoprotection.

Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced.

Nucleotide excision (NER) is generally considered to comprise two partially distinct subpathways. Global genomic repair (GGR) removes damage from the genome overall and transcription-coupled repair (TCR) selectively excises damage from transcribed DNA. Cells from individuals belonging to xeroderma pigmentosum (XP) complementation group C are defective in GGR but retain a functional TCR pathway. DNA mismatch repair (MMR) corrects replication errors but can also process DNA damage. It has been suggested that the essential hMutSalpha and hMutLalpha MMR protein complexes are also required for effective excision of UV-induced cyclobutane pyrimidine dimers (CPD) by TCR. We have combined an MMR and an XPC defect in a human lymphoblastoid cell line. The MMR-defective XPC cells were defective in the hMutSalpha mismatch recognition complex that comprises hMSH2 and hMSH6. They were not detectably more sensitive to killing by UV than their MMR proficient counterparts and were able to excise CPDs from an actively transcribed DNA strand. We conclude efficient TCR does not depend on a functional hMutSalpha complex.

The quest for simple systems achieving the photoreductive splitting of four-membered ring compounds is a matter of interest not only in organic chemistry but also in biochemistry to mimic the activity of DNA photorepair enzymes. In this context, 8-oxoguanine, the main oxidatively generated lesion of guanine, has been shown to act as an intrinsic photoreductant by transferring an electron to bipyrimidine lesions and provoking their cycloreversion. But, in spite of appropriate photoredox properties, the capacity of guanine to repair cyclobutane pyrimidine dimer is not clearly established. Here, dyads containing the cyclobutane thymine dimer and guanine or 8-oxoguanine are synthesized, and their photoreactivities are compared. In both cases, the splitting of the ring takes place, leading to the formation of thymine, with a quantum yield 3.5 times lower than that for the guanine derivative. This result is in agreement with the more favored thermodynamics determined for the oxidized lesion. In addition, quantum chemistry calculations and molecular dynamics simulations are carried out to rationalize the crucial aspects of the overall cyclobutane thymine dimer photoreductive repair triggered by the nucleobase and its main lesion.

The repair of cis-syn cyclobutane pyrimidine dimers (CPDs) can be initiated via the base excision repair (BER) pathway, utilizing pyrimidine dimer-specific DNA glycosylase/lyase enzymes (pdgs). However, prior to incision at lesion sites, these enzymes bind to non-damaged DNAs through charge-charge interactions. Following initial binding to DNA containing multiple lesions, the enzyme incises at most of these sites prior to dissociation. If a subset of these lesions are in close proximity, clustered breaks may be produced that could lead to decreased cell viability or increased mutagenesis. Based on the co-crystal structures of bacteriophage T4-pdg and homology modeling of a related enzyme from Paramecium bursaria Chlorella virus-1, the structure-function basis for the processive incision activity for both enzymes was investigated using site-directed mutagenesis. An assay was developed that quantitatively measured the rates of incision by these enzymes at clustered apurinic/apyrimidinic (AP) sites. Mathematical modeling of random (distributive) versus processive incisions predicted major differences in the rate and extent of the accumulation of singly nicked DNAs between these two mechanisms. Comparisons of these models with biochemical nicking data revealed significant changes in the damage search mechanisms between wild-type pdgs and most of the mutant enzymes. Several conserved arginine residues were shown to be critical for the processivity of the incision activity, without interfering with catalysis at AP sites. Comparable results were measured for incision at clustered CPD sites in plasmid DNAs. These data reveal that pdgs can be rationally engineered to retain full catalytic activity, while dramatically altering mechanisms of target site location.

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.

Sequencing of whole cancer genomes has revealed an abundance of recurrent mutations in gene-regulatory promoter regions, in particular in melanoma where strong mutation hotspots are observed adjacent to ETS-family transcription factor (TF) binding sites. While sometimes interpreted as functional driver events, these mutations are commonly believed to be due to locally inhibited DNA repair. Here, we first show that low-dose UV light induces mutations preferably at a known ETS promoter hotspot in cultured cells even in the absence of global or transcription-coupled nucleotide excision repair (NER). Further, by genome-wide mapping of cyclobutane pyrimidine dimers (CPDs) shortly after UV exposure and thus before DNA repair, we find that ETS-related mutation hotspots exhibit strong increases in CPD formation efficacy in a manner consistent with tumor mutation data at the single-base level. Analysis of a large whole genome cohort illustrates the widespread contribution of this effect to recurrent mutations in melanoma. While inhibited NER underlies a general increase in somatic mutation burden in regulatory elements including ETS sites, our data supports that elevated DNA damage formation at specific genomic bases is at the core of the prominent promoter mutation hotspots seen in skin cancers, thus explaining a key phenomenon in whole-genome cancer analyses.

Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases.

Irradiation of G-quadruplex forming human telomeric DNA with ultraviolet B (UVB) light results in the formation of anti cyclobutane pyrimidine dimers (CPDs) between loop 1 and loop 3 in the presence of potassium ions but not sodium ions. This was unexpected because the sequences involved favor the nonphotoreactive hybrid conformations in K(+) solution, whereas a potentially photoreactive basket conformation is favored in Na(+) solution. To account for these contradictory results, it was proposed that the loops are too far apart in the basket conformation in Na(+) solution but close enough in a two G-tetrad basket-like form 3 conformation that can form in K(+) solution. In the current study, Na(+) was still found to inhibit anti CPD formation in sequences designed to stabilize the form 3 conformation. Furthermore, anti CPD formation in K(+) solution was slower for the sequence previously shown to exist primarily in the proposed photoreactive form 3 conformation than the sequence shown to exist primarily in a nonphotoreactive hybrid conformation. These results suggest that the form 3 conformation is not the principal photoreactive conformation, and that G-quadruplexes in K(+) solution are dynamic and able to access photoreactive conformations more easily than in Na(+) solution.

Octyl methoxycinnamate (OMC) is one of the most widely used sunscreen ingredients. To analyze biological effects of OMC, an in vitro approach was used implying ultraviolet (UV) exposure of two human cell lines, a primary skin fibroblast (GM00498) and a breast cancer (MCF-7) cell lines. End points include cell viability assessment, assay of cyclobutane pyrimidine dimers (CPDs) and oxidated DNA lesions using alkaline elution and lesion-specific enzymes, and gene expression analysis of a panel of 17 DNA damage-responsive genes. We observed that OMC provided protection against CPDs, and the degree of protection correlated with the OMC-mediated reduction in UV dose. No such protection was found with respect to oxidative DNA lesions. Upon UV exposure in the presence of OMC, the gene expression studies showed significant differential changes in some of the genes studied and the expression of p53 protein was also changed. For some genes, the change in expression seemed to be delayed in time by OMC. The experimental approach applied in this study, using a panel of 17 genes in an in vitro cellular system together with genotoxicity assays, may be useful in the initial screening of active ingredients in sunscreens.