Ultraviolet Protection From a Patented Amino Acid Complex Technology.

OBJECTIVE:   This study aimed to investigate the ultraviolet (UV) protection/repair benefits of a patented Amino Acid Complex (AAComplex). METHODS: I) AAComplex was incubated with dermal fibroblasts, with/without UVA, and collagen I was measured with a GlasBoxPlus device. II) A lotion, with/without AAComplex (1%) was applied topically to skin explants, following UVA irradiation, and quantified for health-related biomarkers (TNFalpha, histamine, and MMP-1). III) A broad spectrum sunscreen with SPF 46 and a skincare serum containing AAComplex (2%) were assessed using epidermal equivalents, in the presence of UV irradiation, for effects on IL-1alpha, thymine dimers, Ki-67, filaggrin and Nrf2. RESULTS: I) Collagen I synthesis in dermal fibroblasts was significantly decreased after UVA compared to without UV. The presence of AAComplex prevented this decrease. II) UVA irradiation of skin explants increased histamine, TNFα, and MMP-1. Hydrocortisone aceponate cream significantly decreases all 3 biomarkers. AAComplex contained lotion also significantly decreased all 3 biomarkers, the no AAComplex control lotion only reduced histamine. III) With the regimen of sunscreen + AAComplex contained skincare serum, the significant reduction in IL-1alpha was observed along with a complete recovery of Ki-67 and stimulation of filaggrin and Nrf2T. No thymine dimer positive cell was observed indicating the most positive skin impact from the regiment.  Conclusion: This research using different human skin models demonstrated that AAComplex can provide protection and damage repair caused by UV, at the ingredient level also when formulated in a serum or lotion formula. Skin may be best protected from UV damage when the regimen is used.   J Drugs Dermatol. 2024;23(5):366-375. doi:10.36849/JDD.7916.

Urinary thymidine dimer excretion reflects personal ultraviolet radiation exposure levels.

Exposure to ultraviolet radiation (UVR) leads to skin DNA damage, specifically in the form of cyclobutane pyrimidine dimers, with thymidine dimers being the most common. Quantifying these dimers can indicate the extent of DNA damage resulting from UVR exposure. Here, a new liquid chromatography-mass spectrometry (LC-MS) method was used to quantify thymidine dimers in the urine after a temporary increase in real-life UVR exposure. Healthy Danish volunteers (n = 27) experienced increased UVR exposure during a winter vacation. Individual exposure, assessed via personally worn electronic UVR dosimeters, revealed a mean exposure level of 32.9 standard erythema doses (SEDs) during the last week of vacation. Morning urine thymidine dimer concentrations were markedly elevated both 1 and 2 days post-vacation, and individual thymidine dimer levels correlated with UVR exposure during the last week of the vacation. The strongest correlation with erythema-weighted personal UVR exposure (Power model, r2 = 0.64, p < 0.001) was observed when both morning urine samples were combined to measure 48-h thymidine dimer excretion, whereas 24-h excretion based on a single sample provided a weaker correlation (Power model, r2 = 0.55, p < 0.001). Sex, age, and skin phototype had no significant effect on these correlations. For the first time, urinary thymidine dimer excretion was quantified by LC-MS to evaluate the effect of a temporary increase in personal UVR exposure in a real-life setting. The high sensitivity to elevated UVR exposure and correlation between urinary excretion and measured SED suggest that this approach may be used to quantify DNA damage and repair and to evaluate photoprevention strategies.

Evidence for persistent UV-induced DNA damage and altered DNA damage response in xeroderma pigmentosa patient corneas.

Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by injury to the ocular surface due to exposure to ultraviolet (UV) radiation. UV-induced damage in the cells leads to the formation of cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts that are repaired by the NER (Nucleotide Excision Repair) pathway. Mutations in the genes coding for NER proteins, as reported in XP patients, would lead to sub-optimal damage repair resulting in clinical signs varying from photo-keratitis to cancerous lesions on the ocular surface. Here, we aimed to provide evidence for the accumulation of DNA damage and activation of DNA repair pathway proteins in the corneal cells of patients with XP. Corneal buttons of patients who underwent penetrating keratoplasty were stained to quantify DNA damage and the presence of activated DNA damage response proteins (DDR) using specific antibodies. Positive staining for pH2A.X and thymidine dimers confirmed the presence of DNA damage in the corneal cells. Positive cells were found in both control corneas and XP samples however, unlike normal tissues, positive cells were found in all cell layers of XP samples indicating that these cells were sensitive to very low levels of UV. pH2A.X-positive cells were significantly more in XP corneas (p < 0.05) indicating the presence of double strand breaks in these tissues. A positive expression of phosphorylated-forms of DDR proteins was noted in XP corneas (unlike controls) such as ataxia telangiectasia mutated/Rad-3 related proteins (ATM/ATR), breast cancer-1 and checkpoint kinases-1 and -2. Nuclear localization of XPA was noted in XP samples which co-localized (calculated using Pearson's correlation) with pATM (0.9 ± 0.007) and pATR (0.6 ± 0.053). The increased presence of these in the nucleus confirms that unresolved DNA damage was accumulating in these cells thereby leading to prolonged activation of the damage response proteins. An increase in pp53 and TUNEL positive cells in the XP corneas indicated cell death likely driven by the p53 pathway. For comparison, cultured normal corneal epithelial cells were exposed to UV-radiation and stained for DDR proteins at 3, 6 and 24 h after irradiation to quantify the time taken by cells with intact DDR pathway to repair damage. These cells, when exposed to UV showed nuclear translocation of DDR proteins at 3 and 6 h which reduced significantly by 24 h confirming that the damaged DNA was being actively repaired leading to cell survival. The persistent presence of the DDR proteins in XP corneas indicates that damage is being actively recognized and DNA replication is stalled, thereby causing accumulation of damaged DNA leading to cell death, which would explain the cancer incidence and cell loss reported in these patients.

A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions.

Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent lesions. CPDs are repaired much slower than 6-4PPs by the nucleotide excision repair pathway, which are thus the major lesions that interfere with key cellular processes and give rise to gene mutations, possibly resulting in skin cancer. In prokaryotes and multicellular eukaryotes other than placental mammals, CPDs can be rapidly repaired by CPD photolyases in one simple enzymatic reaction using the energy of blue light. In this study, we aim to construct recombinant CPD photolyases that can autonomously enter human cell nuclei to fix UV-induced CPDs. A fly cell penetration peptide and a viral nucleus localization signal peptide were recombined with a fungal CPD photolyase to construct a recombinant protein. This engineered CPD photolyase autonomously crosses cytoplasm and nuclear membrane of human cell nuclei, which then efficiently photo-repairs UV-induced CPD lesions in the genomic DNA. This further protects the cells by increasing SOD activity, and decreasing cellular ROSs, malondialdehyde and apoptosis.

Poaceae plants transfer cyclobutane pyrimidine dimer photolyase to chloroplasts for ultraviolet-B resistance.

Photoreactivation enzyme that repairs cyclobutane pyrimidine dimer (CPD) induced by ultraviolet-B radiation, commonly called CPD photolyase (PHR) is essential for plants living under sunlight. Rice (Oryza sativa) PHR (OsPHR) is a unique triple-targeting protein. The signal sequences required for its translocation to the nucleus or mitochondria are located in the C-terminal region but have yet to be identified for chloroplasts. Here, we identified sequences located in the N-terminal region, including the serine-phosphorylation site at position 7 of OsPHR, and found that OsPHR is transported/localized to chloroplasts via a vesicle transport system under the control of serine-phosphorylation. However, the sequence identified in this study is only conserved in some Poaceae species, and in many other plants, PHR is not localized to the chloroplasts. Therefore, we reasoned that Poaceae species need the ability to repair CPD in the chloroplast genome to survive under sunlight and have uniquely acquired this mechanism for PHR chloroplast translocation.

UV-C LED-induced cyclobutane pyrimidine dimer formation, lesion repair and mutagenesis in the biofilm-forming diatom, Navicula incerta.

The use of ultraviolet-C (UV-C) irradiation in marine biofouling control is a relatively new and potentially disruptive technology. This study examined effects of UV-C exposure on the biofilm-forming diatom, Navicula incerta. UV-C-induced mutations were identified via Illumina HiSeq. A de novo genome was assembled from control sequences and reads from UV-C-exposed treatments were mapped to this genome, with a quantitative estimate of mutagenesis then derived from the frequency of single nucleotide polymorphisms. UV-C exposure increased cyclobutane pyrimidine dimer (CPD) abundance with a direct correlation between lesion formation and fluency. Cellular repair mechanisms gradually reduced CPDs over time, with the highest UV-C fluence treatments having the fastest repair rates. Mutation abundances were, however, negatively correlated with CPD abundance suggesting that UV-C exposure may influence lesion repair. The threshold fluence for CPD formation exceeding CPD repair was >1.27 J cm-2. Fluences >2.54 J cm-2 were predicted to inhibit repair mechanisms. While UV-C holds considerable promise for marine antifouling, diatoms are just one, albeit an important, component of marine biofouling communities. Determining fluence thresholds for other representative taxa, highlighting the most resistant, would allow UV-C treatments to be specifically tuned to target biofouling organisms, whilst limiting environmental effects and the power requirement.

Global repair is the primary nucleotide excision repair subpathway for the removal of pyrimidine-pyrimidone (6-4) damage from the Arabidopsis genome.

Ultraviolet (UV) component of solar radiation impairs genome stability by inducing the formation of pyrimidine-pyrimidone (6-4) photoproducts [(6-4)PPs] in plant genomes. (6-4)PPs disrupt growth and development by interfering with transcription and DNA replication. To resist UV stress, plants employ both photoreactivation and nucleotide excision repair that excises oligonucleotide containing (6-4)PPs through two subpathways: global and transcription-coupled excision repair (TCR). Here, we analyzed the genome-wide excision repair-mediated repair of (6-4)PPs in Arabidopsis thaliana and found that (6-4)PPs can be repaired by TCR; however, the main subpathway to remove (6-4)PPs from the genome is global repair. Our analysis showed that open chromatin genome regions are more rapidly repaired than heterochromatin regions, and the repair level peaks at the promoter, transcription start site and transcription end site of genes. Our study revealed that the repair of (6-4)PP in plants showed a distinct genome-wide repair profile compared to the repair of other major UV-induced DNA lesion called cyclobutane pyrimidine dimers (CPDs).

Homozygous substitution of threonine 191 by proline in polymerase η causes Xeroderma pigmentosum variant.

DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη function is associated with the human disease Xeroderma pigmentosum variant (XPV). We hereby report the case of a 60-year-old woman known for XPV and carrying a Polη Thr191Pro variant in homozygosity. We further characterize the variant in vitro and in vivo, providing molecular evidence that the substitution abrogates polymerase activity and results in UV sensitivity through deficient damage bypass. This is the first functional molecular characterization of a missense variant of Polη, whose reported pathogenic variants have thus far been loss of function truncation or frameshift mutations. Our work allows the upgrading of Polη Thr191Pro from 'variant of uncertain significance' to 'likely pathogenic mutant', bearing direct impact on molecular diagnosis and genetic counseling. Furthermore, we have established a robust experimental approach that will allow a precise molecular analysis of further missense mutations possibly linked to XPV. Finally, it provides insight into critical Polη residues that may be targeted to develop small molecule inhibitors for cancer therapeutics.

High UV damage and low repair, but not cytosine deamination, stimulate mutation hotspots at ETS binding sites in melanoma.

Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their formation mechanism and functional impacts are not fully understood. Here, we used UV (Ultraviolet) damage sequencing data and analyzed cyclobutane pyrimidine dimer (CPD) formation, DNA repair, and CPD deamination in human cells at single-nucleotide resolution. Our data show prominent CPD hotspots immediately after UV irradiation at ETS binding sites, particularly at sites with a conserved TTCCGG motif, which correlate with mutation hotspots identified in cutaneous melanoma. Additionally, CPDs are repaired slower at ETS binding sites than in flanking DNA. Cytosine deamination in CPDs to uracil is suggested as an important step for UV mutagenesis. However, we found that CPD deamination is significantly suppressed at ETS binding sites, particularly for the CPD hotspot on the 5' side of the ETS motif, arguing against a role for CPD deamination in promoting ETS-associated UV mutations. Finally, we analyzed a subset of frequently mutated promoters, including the ribosomal protein genes RPL13A and RPS20, and found that mutations in the ETS motif can significantly reduce the promoter activity. Thus, our data identify high UV damage and low repair, but not CPD deamination, as the main mechanism for ETS-associated mutations in melanoma and uncover important roles of often-overlooked mutation hotspots in perturbing gene transcription.

In vitro photoprotective potential of aryl-sandwiched (thio)semicarbazones against UVA mediated cellular and DNA damage.

The most prevalent solar ultraviolet radiation is ultraviolet-A (UVA) radiation. It is the inducer of reactive oxygen species (ROS), a potent mediator of inflammation and photocarcinogenesis. Regular application of sunscreens containing UVA filters is an effective preventive measure in mitigating the risk associated with the formation of dermal carcinoma. Therefore, the development of new photoprotective agents is of great need. The current work examined the in vitro photoprotection of the aryl-linked (thio)semicarbazone derivatives against UVA-mediated DNA damage, inflammation, reactive nitrogen species (RNS), and ROS. Except for the inflammatory cytokine assay, which was carried out on the human monocytic leukemia (THP-1) cell line, all tests were conducted on the human dermal fibroblast (BJ) cell line. In comparison to benzophenone (reference compound), the compound (2Z, 2'Z)-2,2'-(1,3-Phenylenebis (methanylylidene)) bis (hydrazine-1-carbothioamide) (DD-21) demonstrated considerable protection against UVA-induced damage. Compared to the UVA-irradiated control, DD-21 significantly decreased the levels of nitric oxide (NO) and ROS (p < 0.001). In the presence of DD-21, the release of UVA-induced pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß), was also significantly reduced (p < 0.05). Moreover, it was observed that DD-21 protected the cells from UVA-mediated DNA strand breaks and also inhibited the formation of cyclobutane pyrimidine dimers (CPDs) upon comparison to the UVA-exposed control cells (p < 0.001). In conclusion, the findings of this study revealed that DD-21 exhibits remarkable photoprotective properties, thus demonstrating its potential as a candidate UVA filter.

Genome-wide impact of cytosine methylation and DNA sequence context on UV-induced CPD formation.

Exposure to ultraviolet (UV) light is the primary etiological agent for skin cancers because UV damages cellular DNA. The most frequent form of UV damage is the cyclobutane pyrimidine dimer (CPD), which consists of covalent linkages between neighboring pyrimidine bases in DNA. In human cells, the 5' position of cytosine bases in CG dinucleotides is frequently methylated, and methylated cytosines in the TP53 tumor suppressor are often sites of mutation hotspots in skin cancers. It has been argued that this is because cytosine methylation promotes UV-induced CPD formation; however, the effects of cytosine methylation on CPD formation are controversial, with conflicting results from previous studies. Here, we use a genome-wide method known as CPD-seq to map UVB- and UVC-induced CPDs across the yeast genome in the presence or absence in vitro methylation by the CpG methyltransferase M.SssI. Our data indicate that cytosine methylation increases UVB-induced CPD formation nearly 2-fold relative to unmethylated DNA, but the magnitude of induction depends on the flanking sequence context. Sequence contexts with a 5' guanine base (e.g., GCCG and GTCG) show the strongest induction due to cytosine methylation, potentially because these sequence contexts are less efficient at forming CPD lesions in the absence of methylation. We show that cytosine methylation also modulates UVC-induced CPD formation, albeit to a lesser extent than UVB. These findings can potentially reconcile previous studies, and define the impact of cytosine methylation on UV damage across a eukaryotic genome.

Thallium(I) induces a prolonged inhibition of (6-4)photoproduct binding and UV damage excision repair activities in zebrafish (Danio rerio) embryos via protein inactivation.

Cyclobutane pyrimidine dimer (CPD) and (6-4)photoproduct (6-4 PP) are two major types of UV-induced DNA lesion and 6-4 PP is more mutagenic than CPD. Activated by lesion detection, nucleotide excision repair (NER) eliminates CPDs and 6-4 PPs. Thallium (Tl) is a toxic metal existing primarily as Tl+ in the aquatic environment. Ingestion of Tl+-contaminated foods and water is a major route of human poisoning. As Tl+ may inhibit enzyme activities via binding to sulfhydryl groups, this study explored if Tl+ could intensify UV mutagenicity by inactivating NER-linked damage recognition factors using zebrafish (Danio rerio) embryo as a model system. Incubation of Tl+ (as thallium nitrate) at 0.1-0.4 µg/mL with zebrafish extracts for 20 min caused a concentration-dependent inhibition of 6-4 PP binding activities as shown by a photolesion-specific band shift assay, while CPD binding activities were insensitive to Tl+. The ability of Tl+ to suppress 6-4 PP detection was stronger than that of Hg2+. Exposure of zebrafish embryos at 1 h post fertilization (hpf) to Tl+ at 0.4-1 µg/mL for 9 or 71 h also specifically inhibited 6-4 PP detection, indicating that Tl+ induced a prolonged inhibition of 6-4 PP sensing ability primarily via its direct interaction with damage recognition molecules. Tl+-mediated inhibition of 6-4 PP binding in embryos at distinct stages resulted in a suppression of NER capacity monitored by a transcription-based DNA repair assay. Our results revealed the potential of Tl+ to enhance UV mutagenicity by disturbing the removal of 6-4 PP through repressing the lesion detection step of NER.

Differing structures and dynamics of two photolesions portray verification differences by the human XPD helicase.

Ultraviolet light generates cyclobutane pyrimidine dimer (CPD) and pyrimidine 6-4 pyrimidone (6-4PP) photoproducts that cause skin malignancies if not repaired by nucleotide excision repair (NER). While the faster repair of the more distorting 6-4PPs is attributed mainly to more efficient recognition by XPC, the XPD lesion verification helicase may play a role, as it directly scans the damaged DNA strand. With extensive molecular dynamics simulations of XPD-bound single-strand DNA containing each lesion outside the entry pore of XPD, we elucidate strikingly different verification processes for these two lesions that have very different topologies. The open book-like CPD thymines are sterically blocked from pore entry and preferably entrapped by sensors that are outside the pore; however, the near-perpendicular 6-4PP thymines can enter, accompanied by a displacement of the Arch domain toward the lesion, which is thereby tightly accommodated within the pore. This trapped 6-4PP may inhibit XPD helicase activity to foster lesion verification by locking the Arch to other domains. Furthermore, the movement of the Arch domain, only in the case of 6-4PP, may trigger signaling to the XPG nuclease for subsequent lesion incision by fostering direct contact between the Arch domain and XPG, and thereby facilitating repair of 6-4PP.

Visualizing the DNA repair process by a photolyase at atomic resolution.

Photolyases, a ubiquitous class of flavoproteins, use blue light to repair DNA photolesions. In this work, we determined the structural mechanism of the photolyase-catalyzed repair of a cyclobutane pyrimidine dimer (CPD) lesion using time-resolved serial femtosecond crystallography (TR-SFX). We obtained 18 snapshots that show time-dependent changes in four reaction loci. We used these results to create a movie that depicts the repair of CPD lesions in the picosecond-to-nanosecond range, followed by the recovery of the enzymatic moieties involved in catalysis, completing the formation of the fully reduced enzyme-product complex at 500 nanoseconds. Finally, back-flip intermediates of the thymine bases to reanneal the DNA were captured at 25 to 200 microseconds. Our data cover the complete molecular mechanism of a photolyase and, importantly, its chemistry and enzymatic catalysis at work across a wide timescale and at atomic resolution.

Filming DNA repair at the atomic level.

Dissection of multistep catalysis by a photoenzyme could inspire green chemistry applications.

WHIRLY1-deficient chloroplasts display enhanced formation of cyclobutane pyrimidine dimers during exposure to UV-B radiation.

The single-stranded DNA/RNA binding protein WHIRLY1 is a major chloroplast nucleoid-associated protein required for the compactness of nucleoids. Most nucleoids in chloroplasts of WHIRLY1-knockdown barley plants are less compact compared to nucleoids in wild-type plants. The reduced compaction leads to an enhanced optical cross-section, which may cause the plastid DNA to be a better target for damaging UV-B radiation. To investigate this hypothesis, primary foliage leaves, chloroplasts, and nuclei from wild-type and WHIRLY1-knockdown plants were exposed to experimental UV-B radiation. Thereafter, total, genomic and plastid DNA were isolated, respectively, and analyzed for the occurrence of cyclobutane pyrimidine dimers (CPDs), which is a parameter for genome stability. The results of this study revealed that WHIRLY1-deficient chloroplasts had strongly enhanced DNA damages, whereas isolated nuclei from the same plant line were not more sensitive than nuclei from the wild-type, indicating that WHIRLY1 has different functions in chloroplasts and nucleus. This supports the hypothesis that the compaction of nucleoids may provide protection against UV-B radiation.

UV-driven self-repair of cyclobutane pyrimidine dimers in RNA.

Nucleic acids can be damaged by ultraviolet (UV) irradiation, forming structural photolesions such as cyclobutane-pyrimidine-dimers (CPD). In modern organisms, sophisticated enzymes repair CPD lesions in DNA, but to our knowledge, no RNA-specific enzymes exist for CPD repair. Here, we show for the first time that RNA can protect itself from photolesions by an intrinsic UV-induced self-repair mechanism. This mechanism, prior to this study, has exclusively been observed in DNA and is based on charge transfer from CPD-adjacent bases. In a comparative study, we determined the quantum yields of the self-repair of the CPD-containing RNA sequence, GAU = U to GAUU (0.23%), and DNA sequence, d(GAT = T) to d(GATT) (0.44%), upon 285 nm irradiation via UV/Vis spectroscopy and HPLC analysis. After several hours of irradiation, a maximum conversion yield of ∼16% for GAU = U and ∼33% for d(GAT = T) was reached. We examined the dynamics of the intermediate charge transfer (CT) state responsible for the self-repair with ultrafast UV pump - IR probe spectroscopy. In the dinucleotides GA and d(GA), we found comparable quantum yields of the CT state of ∼50% and lifetimes on the order of several hundred picoseconds. Charge transfer in RNA strands might lead to reactions currently not considered in RNA photochemistry and may help understanding RNA damage formation and repair in modern organisms and viruses. On the UV-rich surface of the early Earth, these self-stabilizing mechanisms likely affected the selection of the earliest nucleotide sequences from which the first organisms may have developed.

APE1-dependent base excision repair of DNA photodimers in human cells.

UV irradiation induces "bulky" DNA photodimers such as (6-4)-photoproducts and cyclobutane pyrimidine dimers that are removed by nucleotide excision repair, a complex process defective in the sunlight-sensitive and cancer-prone disease xeroderma pigmentosum. Some bacteria and lower eukaryotes can also repair photodimers by enzymatically simpler mechanisms, but such pathways have not been reported in normal human cells. Here, we have identified such a mechanism. We show that normal human cells can employ a DNA base excision repair process involving NTH1, APE1, PARP1, XRCC1, and FEN1 to rapidly remove a subset of photodimers at early times following UVC irradiation. Loss of these proteins slows the early rate of repair of photodimers in normal cells, ablates their residual repair in xeroderma pigmentosum cells, and increases UVC sensitivity ∼2-fold. These data reveal that human cells can excise photodimers using a long-patch base excision repair process that functions additively but independently of nucleotide excision repair.

Mapping the recognition pathway of cyclobutane pyrimidine dimer in DNA by Rad4/XPC.

UV radiation-induced DNA damages have adverse effects on genome integrity and cellular function. The most prevalent UV-induced DNA lesion is the cyclobutane pyrimidine dimer (CPD), which can cause skin disorders and cancers in humans. Rad4/XPC is a damage sensing protein that recognizes and repairs CPD lesions with high fidelity. However, the molecular mechanism of how Rad4/XPC interrogates CPD lesions remains elusive. Emerging viewpoints indicate that the association of Rad4/XPC with DNA, the insertion of a lesion-sensing ß-hairpin of Rad4/XPC into the lesion site and the flipping of CPD's partner bases (5'-dA and 3'-dA) are essential for damage recognition. Characterizing these slow events is challenging due to their infrequent occurrence on molecular time scales. Herein, we have used enhanced sampling and molecular dynamics simulations to investigate the mechanism and energetics of lesion recognition by Rad4/XPC, considering multiple plausible pathways between the crystal structure of the Rad4-DNA complex and nine intermediate states. Our results shed light on the most likely sequence of events, their potential coupling and energetics. Upon association, Rad4 and DNA form an encounter complex in which CPD and its partner bases remain in the duplex and the BHD3 ß-hairpin is yet to be inserted into the lesion site. Subsequently, sequential base flipping occurs, with the flipping of the 5'-dA base preceding that of the 3'-dA base, followed by the insertion of the BHD3 ß-hairpin into the lesion site. The results presented here have significant implications for understanding the molecular basis of UV-related skin disorders and cancers and for paving the way for novel therapeutic strategies.

Far-UVC- and UVB-induced DNA damage depending on skin type.

Far-UVC radiation sources of wavelengths 222 nm and 233 nm represent an interesting potential alternative for the antiseptic treatment of the skin due to their high skin compatibility. Nevertheless, no studies on far-UVC-induced DNA damage in different skin types have been published to date, which this study aims for. After irradiating the skin with far-UVC of the wavelengths 222 and 233 nm as well as broadband UVB, the tissue was screened for cyclobutane pyrimidine dimer-positive (CPD+ ) cells using immunohistochemistry. The epidermal DNA damage was lower in dark skin types than in fair skin types after irradiation at 233 nm. Contrary to this, irradiation at 222 nm caused no skin type-dependent differences, which can be attributed to the decreased penetration depth of radiation. UVB showed the relatively strongest differences between light and dark skin types when using a suberythemal dose of 3 mJ/cm2 . As melanin is known for its photoprotective effect, we evaluated the ratio of melanin content in the stratum basale and stratum granulosum in samples of different skin types using two-photon excited fluorescence lifetime imaging (TPE-FLIM) finding a higher ratio up to skin type IV-V. As far-UVC is known to penetrate only into the upper layers of the viable skin, the aforementioned melanin ratio could explain the less pronounced differences between skin types after irradiation with far-UVC compared to UVB.