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Proceedings of the National Academy of... Jan 2024Noncoding mutation hotspots have been identified in melanoma and many of them occur at the binding sites of E26 transformation-specific (ETS) proteins; however, their...
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 and , 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.
Topics: Humans; Melanoma; Cytosine; Deamination; Skin Neoplasms; Mutation; Pyrimidine Dimers; Binding Sites; Ultraviolet Rays; DNA Damage; DNA Repair
PubMed: 38241433
DOI: 10.1073/pnas.2310854121 -
Journal of the American Chemical Society Jan 2024The dynamics of excited electronic states in self-assembled structures formed between silver(I) ions and cytosine-containing DNA strands or monomeric cytosine...
The dynamics of excited electronic states in self-assembled structures formed between silver(I) ions and cytosine-containing DNA strands or monomeric cytosine derivatives were investigated by time-resolved infrared (TRIR) spectroscopy and quantum mechanical calculations. The steady-state and time-resolved spectra depend sensitively on the underlying structures, which change with pH and the nucleobase and silver ion concentrations. At pH ∼ 4 and low dC strand concentration, an intramolecularly folded i-motif is observed, in which protons, and not silver ions, mediate C-C base pairing. However, at the higher strand concentrations used in the TRIR measurements, dC strands associate pairwise to yield duplex structures containing C-Ag-C base pairs with a high degree of propeller twisting. UV excitation of the silver ion-mediated duplex produces a long-lived excited state, which we assign to a triplet excimer state localized on a pair of stacked cytosines. The computational results indicate that the propeller-twisted motifs induced by metal-ion binding are responsible for the enhanced intersystem crossing that populates the triplet state and not a generic heavy atom effect. Although triplet excimer states have been discussed frequently as intermediates in the formation of cyclobutane pyrimidine dimers, we find neither computational nor experimental evidence for cytosine-cytosine photoproduct formation in the systems studied. These findings provide a rare demonstration of a long-lived triplet excited state that is formed in a significant yield in a DNA duplex, demonstrating that supramolecular structural changes induced by metal ion binding profoundly affect DNA photophysics.
Topics: Base Pairing; Silver; DNA; Cytosine; Protons
PubMed: 38215466
DOI: 10.1021/jacs.3c08793 -
Scientific Reports Jan 2024DNA polymerase eta (Polη) is the only translesion synthesis polymerase capable of error-free bypass of UV-induced cyclobutane pyrimidine dimers. A deficiency in Polη...
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.
Topics: Humans; Middle Aged; DNA Damage; Mutation, Missense; Proline; Pyrimidine Dimers; Ultraviolet Rays; Xeroderma Pigmentosum; Female
PubMed: 38212351
DOI: 10.1038/s41598-023-51120-1 -
International Journal of Molecular... Dec 2023Chronic exposure to ultraviolet (UV) radiation is known to induce the formation of DNA photo-adducts, including cyclobutane pyrimidine dimers (CPDs) and Dewar valence...
Chronic exposure to ultraviolet (UV) radiation is known to induce the formation of DNA photo-adducts, including cyclobutane pyrimidine dimers (CPDs) and Dewar valence derivatives (DVs). While CPDs usually occur at higher frequency than DVs, recent studies have shown that the latter display superior selectivity and significant stability in interaction with the human DNA/topoisomerase 1 complex (TOP1). With the aim to deeply investigate the mechanism of interaction of DVs with TOP1, we report here four all-atom molecular dynamic simulations spanning one microsecond. These simulations are focused on the stability and conformational changes of two DNA/TOP1-DV complexes in solution, the data being compared with the biomimetic thymine dimer counterparts. Results from root-mean-square deviation (RMSD) and root-mean-square fluctuation (RMSF) analyses unequivocally confirmed increased stability of the DNA/TOP1-DV complexes throughout the simulation duration. Detailed interaction analyses, uncovering the presence of salt bridges, hydrogen bonds, water-mediated interactions, and hydrophobic interactions, as well as pinpointing the non-covalent interactions within the complexes, enabled the identification of specific TOP1 residues involved in the interactions over time and suggested a potential TOP1 inhibition mechanism in action.
Topics: Humans; Molecular Dynamics Simulation; DNA Topoisomerases, Type I; Biomimetics; DNA Adducts; Data Interpretation, Statistical; Pyrimidine Dimers
PubMed: 38203410
DOI: 10.3390/ijms25010234 -
Journal of Photochemistry and... Feb 2024The most prevalent solar ultraviolet radiation is ultraviolet-A (UVA) radiation. It is the inducer of reactive oxygen species (ROS), a potent mediator of inflammation...
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-1β (IL-1β), 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.
Topics: Humans; Ultraviolet Rays; Reactive Oxygen Species; DNA Damage; Pyrimidine Dimers; Inflammation
PubMed: 38194816
DOI: 10.1016/j.jphotobiol.2024.112841 -
Photochemistry and Photobiology Jan 2024Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of...
Ultraviolet B (UVB) radiation represents a major carcinogen for the development of all skin cancer types. Mechanistically, UVB induces damage to DNA in the form of lesions, including cyclobutane pyrimidine dimers (CPDs). Disruption of the functional repair processes, such as nucleotide excision repair (NER), allows persistence of DNA damage and contributes to skin carcinogenesis. Recent work has implicated m A RNA methylation and its regulatory proteins as having critical roles in facilitating UVB-induced DNA damage repair. However, the biological functions of the m A reader YTHDC2 are unknown in this context. Here, we show that YTHDC2 inhibition enhances the repair of UVB-induced DNA damage. We discovered that YTHDC2 inhibition increased the expression of PTEN while it decreased the expression of the PRC2 component SUZ12 and the levels of the histone modification H3K27me3. However, none of these functions were causally linked to the improvements in DNA repair, suggesting that the mechanism utilized by YTHDC2 may be unconventional. Moreover, inhibition of the m A writer METTL14 reversed the effect of YTHDC2 inhibition on DNA repair while inhibition of the m A eraser FTO mimicked the effect of YTHDC2 inhibition, indicating that YTHDC2 may regulate DNA repair through the m A pathway. Finally, compared to normal human skin, YTHDC2 expression was upregulated in human cutaneous squamous cell carcinomas (cSCC), suggesting that it may function as a tumor-promoting factor in skin cancer. Taken together, our findings demonstrate that the m A reader YTHDC2 plays a role in regulating UVB-induced DNA damage repair and may serve as a potential biomarker in cSCC.
PubMed: 38190286
DOI: 10.1111/php.13904 -
Biomolecules Dec 2023The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by...
The activity of β-ureidopropionase, which catalyses the last step in the degradation of uracil, thymine, and analogous antimetabolites, is cooperatively regulated by the substrate and product of the reaction. This involves shifts in the equilibrium of the oligomeric states of the enzyme, but how these are achieved and result in changes in enzyme catalytic competence has yet to be determined. Here, the regulation of human β-ureidopropionase was further explored via site-directed mutagenesis, inhibition studies, and cryo-electron microscopy. The active-site residue E207, as well as H173 and H307 located at the dimer-dimer interface, are shown to play crucial roles in enzyme activation. Dimer association to larger assemblies requires closure of active-site loops, which positions the catalytically crucial E207 stably in the active site. H173 and H307 likely respond to ligand-induced changes in their environment with changes in their protonation states, which fine-tunes the active-site loop stability and the strength of dimer-dimer interfaces and explains the previously observed pH influence on the oligomer equilibrium. The correlation between substrate analogue structure and effect on enzyme assembly suggests that the ability to favourably interact with F205 may distinguish activators from inhibitors. The cryo-EM structure of human β-ureidopropionase assembly obtained at low pH provides first insights into the architecture of its activated state. and validates our current model of the allosteric regulation mechanism. Closed entrance loop conformations and dimer-dimer interfaces are highly conserved between human and fruit fly enzymes.
Topics: Humans; Catalytic Domain; Allosteric Regulation; Cryoelectron Microscopy; Mutagenesis, Site-Directed
PubMed: 38136634
DOI: 10.3390/biom13121763 -
Biochimie May 2024Many prokaryotic Argonaute (pAgo) proteins act as programmable nucleases that use small guide DNAs for recognition and cleavage of complementary target DNA. Recent...
Many prokaryotic Argonaute (pAgo) proteins act as programmable nucleases that use small guide DNAs for recognition and cleavage of complementary target DNA. Recent studies suggested that pAgos participate in cell defense against invader DNA and may also be involved in other genetic processes, including DNA replication and repair. The ability of pAgos to recognize specific targets potentially make them an invaluable tool for DNA manipulations. Here, we demonstrate that DNA-guided DNA-targeting pAgo nucleases from three bacterial species, DloAgo from Dorea longicatena, CbAgo from Clostridium butyricum and KmAgo from Kurthia massiliensis, can sense site-specific modifications in the target DNA, including 8-oxoguanine, thymine glycol, ethenoadenine and pyrimidine dimers. The effects of DNA modifications on the activity of pAgos strongly depend on their positions relative to the site of cleavage and are comparable to or exceed the effects of guide-target mismatches at corresponding positions. For all tested pAgos, the strongest effects are observed when DNA lesions are located at the cleavage position. The results demonstrate that DNA cleavage by pAgos is strongly affected by DNA modifications, thus making possible their use as sensors of DNA damage.
Topics: Bacterial Proteins; Argonaute Proteins; DNA; DNA Damage; Guanine; Clostridium butyricum; Thymine
PubMed: 38128776
DOI: 10.1016/j.biochi.2023.12.006 -
Nucleic Acids Research Feb 2024Nucleotide excision repair (NER) removes helix-distorting DNA lesions and is therefore critical for genome stability. During NER, DNA is unwound on either side of the...
Nucleotide excision repair (NER) removes helix-distorting DNA lesions and is therefore critical for genome stability. During NER, DNA is unwound on either side of the lesion and excised, but the rules governing incision site selection, particularly in eukaryotic cells, are unclear. Excision repair-sequencing (XR-seq) sequences excised NER fragments, but analysis has been limited because the lesion location is unknown. Here, we exploit accelerated cytosine deamination rates in UV-induced CPD (cyclobutane pyrimidine dimer) lesions to precisely map their locations at C to T mismatches in XR-seq reads, revealing general and species-specific patterns of incision site selection during NER. Our data indicate that the 5' incision site occurs preferentially in HYV (i.e. not G; C/T; not T) sequence motifs, a pattern that can be explained by sequence preferences of the XPF-ERCC1 endonuclease. In contrast, the 3' incision site does not show strong sequence preferences, once truncated reads arising from mispriming events are excluded. Instead, the 3' incision is partially determined by the 5' incision site distance, indicating that the two incision events are coupled. Finally, our data reveal unique and coupled NER incision patterns at nucleosome boundaries. These findings reveal key principles governing NER incision site selection in eukaryotic cells.
Topics: Cytosine; Deamination; DNA Damage; Excision Repair; Eukaryotic Cells
PubMed: 38109317
DOI: 10.1093/nar/gkad1195 -
Chemico-biological Interactions Jan 2024Cyclobutane 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...
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 Hg. 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.
Topics: Animals; Humans; Zebrafish; Excision Repair; Thallium; DNA Repair; DNA Damage; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 38104746
DOI: 10.1016/j.cbi.2023.110837