-
A recombinant fungal photolyase autonomously enters human cell nuclei to fix UV-induced DNA lesions.Biotechnology Letters Jun 2024Solar ultraviolet radiations induced DNA damages in human skin cells with cyclobutane pyrimidine dimers (CPD) and (6-4) photoproducts (6-4PPs) as the most frequent...
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.
Topics: Humans; Deoxyribodipyrimidine Photo-Lyase; Ultraviolet Rays; Cell Nucleus; DNA Damage; DNA Repair; Recombinant Proteins; Pyrimidine Dimers; Fungal Proteins
PubMed: 38523200
DOI: 10.1007/s10529-024-03474-3 -
Mutation Research 2024Human xeroderma pigmentosum variant (XP-V) patients are mutated in the POLH gene, responsible for encoding the translesion synthesis (TLS) DNA polymerase eta (Pol eta).... (Review)
Review
Human xeroderma pigmentosum variant (XP-V) patients are mutated in the POLH gene, responsible for encoding the translesion synthesis (TLS) DNA polymerase eta (Pol eta). These patients suffer from a high frequency of skin tumors. Despite several decades of research, studies on Pol eta still offer an intriguing paradox: How does this error-prone polymerase suppress mutations? This review examines recent evidence suggesting that cyclobutane pyrimidine dimers (CPDs) are instructional for Pol eta. Consequently, it can accurately replicate these lesions, and the mutagenic effects induced by UV radiation stem from the deamination of C-containing CPDs. In this model, the deamination of C (forming a U) within CPDs leads to the correct insertion of an A opposite to the deaminated C (or U)-containing dimers. This intricate process results in C>T transitions, which represent the most prevalent mutations detected in skin cancers. Finally, the delayed replication in XP-V cells amplifies the process of C-deamination in CPDs and increases the burden of C>T mutations prevalent in XP-V tumors through the activity of backup TLS polymerases.
Topics: DNA-Directed DNA Polymerase; Humans; Pyrimidine Dimers; Ultraviolet Rays; Mutagenesis; Skin Neoplasms; Xeroderma Pigmentosum; DNA Repair; DNA Replication; Mutation; DNA Damage
PubMed: 37984186
DOI: 10.1016/j.mrfmmm.2023.111840 -
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 -
Physiologia Plantarum 2023The single-stranded DNA/RNA binding protein WHIRLY1 is a major chloroplast nucleoid-associated protein required for the compactness of nucleoids. Most nucleoids in...
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.
Topics: Pyrimidine Dimers; Plant Proteins; Chloroplasts; Ultraviolet Rays; DNA
PubMed: 37882276
DOI: 10.1111/ppl.14049 -
Genes, Chromosomes & Cancer Jan 2024Polyketide synthase (pks) island harboring Escherichia coli are, under the right circumstances, able to produce the genotoxin colibactin. Colibactin is a risk factor for...
Polyketide synthase (pks) island harboring Escherichia coli are, under the right circumstances, able to produce the genotoxin colibactin. Colibactin is a risk factor for the development of colorectal cancer and associated with mutational signatures SBS88 and ID18. This study explores colibactin-associated mutational signatures in biallelic NTHL1 and MUTYH patients. Targeted Next Generation Sequencing (NGS) was performed on colorectal adenomas and carcinomas of one biallelic NTHL and 12 biallelic MUTYH patients. Additional fecal metagenomics and genome sequencing followed by mutational signature analysis was conducted for the NTHL1 patient. Targeted NGS of the NTHL1 patient showed somatic APC variants fitting SBS88 which was confirmed using WGS. Furthermore, fecal metagenomics revealed pks genes. Also, in 1 out of 11 MUTYH patient a somatic variant was detected fitting SBS88. This report shows that colibactin may influence development of colorectal neoplasms in predisposed patients.
Topics: Humans; Adenomatous Polyposis Coli; Mutation; Colorectal Neoplasms; Deoxyribonuclease (Pyrimidine Dimer)
PubMed: 37795928
DOI: 10.1002/gcc.23208 -
Photochemistry and Photobiology 2024Prolonged ultraviolet exposure results in the formation of cyclobutane pyrimidine dimers (CPDs) in RNA. Consequently, prebiotic photolesion repair mechanisms should have...
Prolonged ultraviolet exposure results in the formation of cyclobutane pyrimidine dimers (CPDs) in RNA. Consequently, prebiotic photolesion repair mechanisms should have played an important role in the maintenance of the structural integrity of primitive nucleic acids. 2,6-Diaminopurine is a prebiotic nucleobase that repairs CPDs with high efficiency when incorporated into polymers. We investigate the electronic deactivation pathways of 2,6-diaminopurine-2'-deoxyribose and 9-methyl-2,6-diaminopurine in acetonitrile and aqueous solution to shed light on the photophysical and excited state properties of the 2,6-diaminopurine chromophore. Evidence is presented that both are photostable compounds exhibiting similar deactivation mechanisms upon the population of the S (ππ* L ) state at 290 nm. The mechanism involves deactivation through the C2- and C6-reaction coordinates and >99% of the excited state population decays through nonradiative pathways involving two conical intersections with the ground state. The radiative and nonradiative lifetimes are longer in aqueous solution compared to acetonitrile. While τ is similar in both derivatives, τ is ca. 1.5-fold longer in 2,6-diaminopurine-2'-deoxyribose due to a more efficient trapping in the S (ππ* L ) minimum. Therefore, 2,6-diaminopurine could have accumulated in significant quantities during prebiotic times to be incorporated into non-canonical RNA and play a significant role in its photoprotection.
Topics: Deoxyribose; 2-Aminopurine; Water; RNA; Acetonitriles
PubMed: 38018292
DOI: 10.1111/php.13887 -
Biochemistry Aug 2023G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging...
Post- and Pre-Radiolabeling Assays for Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures.
G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via CPD formation without perturbing their dynamics, after which the CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting , T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the , T=(T) CPD-containing DNA to the tetranucleotide pT=(pT) corresponding to CPD formation between the underlined T's of two separate dinucleotides while degrading the adjacent TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric , T=(T) CPD.
Topics: Humans; G-Quadruplexes; DNA; Pyrimidine Dimers; Ultraviolet Rays; DNA Damage
PubMed: 37459251
DOI: 10.1021/acs.biochem.3c00155 -
Carcinogenesis Aug 2023Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells...
Xeroderma pigmentosum variant (XP-V) is an autosomal recessive disease with an increased risk of developing cutaneous neoplasms in sunlight-exposed regions. These cells are deficient in the translesion synthesis (TLS) DNA polymerase eta, responsible for bypassing different types of DNA lesions. From the exome sequencing of 11 skin tumors of a genetic XP-V patients' cluster, classical mutational signatures related to sunlight exposure, such as C>T transitions targeted to pyrimidine dimers, were identified. However, basal cell carcinomas also showed distinct C>A mutation spectra reflecting a mutational signature possibly related to sunlight-induced oxidative stress. Moreover, four samples carry different mutational signatures, with C>A mutations associated with tobacco chewing or smoking usage. Thus, XP-V patients should be warned of the risk of these habits. Surprisingly, higher levels of retrotransposon somatic insertions were also detected when the tumors were compared with non-XP skin tumors, revealing other possible causes for XP-V tumors and novel functions for the TLS polymerase eta in suppressing retrotransposition. Finally, the expected high mutation burden found in most of these tumors renders these XP patients good candidates for checkpoint blockade immunotherapy.
Topics: Humans; Xeroderma Pigmentosum; Retroelements; Mutation; DNA Repair; Skin Neoplasms; Ultraviolet Rays
PubMed: 37195263
DOI: 10.1093/carcin/bgad030 -
Journal of Photochemistry and... Aug 2023The application of a far-ultraviolet C (UVC) light emitting diode (LED) of 233 nm showed significant bactericidal efficacy at an applied dose between 20 and 80 mJ cm...
The application of a far-ultraviolet C (UVC) light emitting diode (LED) of 233 nm showed significant bactericidal efficacy at an applied dose between 20 and 80 mJ cm as reported recently. In addition, only minor epidermal DNA lesions were observed in ex vivo human skin and in vitro epidermal models <10% of the minimal erythema dose of UVB radiation. To broaden the potential range of applications of such systems, e.g. to include postoperative application on wounds for the purpose of decontamination, we assessed how a disruption of normal anatomic skin structure and function influences the skin damage induced by light from 233 nm far-UVC LEDs. Thus, we induced superficial skin wounds by mechanical detachment of the stratum corneum in ex vivo human skin. Barrier-disruption of the skin could be successfully determined by measuring an increase in the transepidermal water loss (TEWL) and the stratum corneum loss could be determined morphologically by 2-photon microscopy (2-PM). After far-UVC irradiation of the skin, we screened the tissue for the development of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). The abundance of DNA lesions was elevated in wound skin in comparison to intact skin after irradiation with far-UVC. However, no increase in DNA lesions was detected when artificial wound exudate consisting of cell culture medium and serum was applied to the disrupted skin surface prior to irradiation. This effect agrees with the results of ray tracing simulations of the absorption of far-UVC light incident on a superficial skin wound. Interestingly, no significant deviations in radical formation between intact skin and superficially wounded skin were detected after far-UVC irradiation as analyzed by electron paramagnetic resonance (EPR) spectroscopy. In conclusion, 233 nm LED light at a dose of 60 mJ/cm could be applied safely on superficial wounds for the purpose of skin antisepsis as long as the wounds are covered with wound fluid.
Topics: Humans; Skin; Pyrimidine Dimers; Epidermis; DNA; Ultraviolet Rays; DNA Damage
PubMed: 37481791
DOI: 10.1016/j.jphotobiol.2023.112757 -
The Journal of Physical Chemistry... Feb 2024Despite the widely recognized importance of noncovalent interactions involving aromatic rings in many fields, our understanding of the underlying forces and structural...
Despite the widely recognized importance of noncovalent interactions involving aromatic rings in many fields, our understanding of the underlying forces and structural patterns, especially the impact of heteroaromaticity, is still incomplete. Here, we investigate the relaxation processes that follow inner-valence ionization in a range of molecular dimers involving various combinations of benzene, pyridine, and pyrimidine, which initiate an ultrafast intermolecular Coulombic decay process. Multiparticle coincidence momentum spectroscopy, combined with calculations, enables us to explore the principal orientations of these fundamental dimers and, thus, to elucidate the influence of N heteroatoms on the relative preference of the aromatic π-stacking, H-bonding, and CH-π interactions and their dependence on the number of nitrogen atoms in the rings. Our studies reveal a sensitive tool for the structural imaging of molecular complexes and provide a more complete understanding of the effects of N heteroatoms on the noncovalent aromatic interactions at the molecular level.
PubMed: 38299504
DOI: 10.1021/acs.jpclett.3c02979