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Frontiers in Microbiology 2022Photosynthetic organisms are continuously exposed to solar ultraviolet radiation-B (UV-B) because of their autotrophic lifestyle. UV-B provokes DNA damage, such as...
Photosynthetic organisms are continuously exposed to solar ultraviolet radiation-B (UV-B) because of their autotrophic lifestyle. UV-B provokes DNA damage, such as cyclobutane pyrimidine dimers (CPD) or pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs). The cryptochrome/photolyase family (CPF) comprises flavoproteins that can bind damaged or undamaged DNA. Photolyases (PHRs) are enzymes that repair either CPDs or 6-4 PPs. A natural bifunctional CPD/(6-4)- PHR (PhrSph98) was recently isolated from the UV-resistant bacteria sp. UV9. In this work, phylogenetic studies of bifunctional CPD/(6-4)- photolyases and their evolutionary relationship with other CPF members were performed. Amino acids involved in electron transfer and binding to FAD cofactor and DNA lesions were conserved in proteins from proteobacteria, planctomycete, bacteroidete, acidobacteria and cyanobacteria clades. Genome analysis revealed that the cyanobacteria sp. PCC 7335 encodes a two-gene assembly operon coding for a and a . Operon structure was validated by RT-qPCR analysis and the polycistronic transcript accumulated after 15 min of UV-B irradiation. Conservation of structure and evolution is discussed. This study provides evidence for a UV-B inducible PHR operon that encodes a CPD/(6-4)- photolyase homolog with a putative bifunctional role in the repair of CPDs and 6-4 PPs damages in oxygenic photosynthetic organisms.
PubMed: 36386616
DOI: 10.3389/fmicb.2022.981788 -
Biomedicines Oct 2022Despite significant developments in renal cell carcinoma (RCC) detection and molecular pathology, mortality has been steadily rising. Advanced RCC remains an incurable...
Despite significant developments in renal cell carcinoma (RCC) detection and molecular pathology, mortality has been steadily rising. Advanced RCC remains an incurable disease. Better clinical management tools, i.e., RCC biomarkers, have yet to emerge. Thymine-dimers (TDs) were traditionally considered photo-dependent pre-mutagenic lesions, occurring exclusively during ultra-violet light exposure. Non-oxidative, direct, and preferential byproducts of DNA photochemical reactions, TDs, have recently shown evidence regarding UVR-independent formation. In this study, we investigate, for the first time, TD expression within RCC tumor tissue and tumor-adjacent healthy renal parenchyma using a TD-targeted IHC monoclonal antibody, clone KTM53. Remarkably, out of the 54 RCCs evaluated, 77.8% showed nuclear TD-expression in RCC tumor tissue and 37% in the tumor-adjacent healthy renal parenchyma. A comprehensive report regarding quantitative/qualitative TD-targeted immunostaining was elaborated. Two main distribution models for TD expression within RCC tumor tissue were identified. Statistical analysis showed significant yet moderate correlations regarding TD-positivity in RCC tissue/tumor-adjacent healthy renal parenchyma and TNM stage at diagnosis/lymphatic dissemination, respectively, indicating possible prognostic relevance. We review possible explanations for UVR-independent TD formation and molecular implications regarding RCC carcinogenesis. Further rigorous molecular analysis is required in order to fully comprehend/validate the biological significance of this newly documented TD expression in RCC.
PubMed: 36359193
DOI: 10.3390/biomedicines10112673 -
Biochimie Mar 2023Nei Like DNA Glycosylase 1 (NEIL1) is a DNA glycosylase, which specifically processes oxidative DNA damage by initiating base excision repair. NEIL1 recognizes and...
Nei Like DNA Glycosylase 1 (NEIL1) is a DNA glycosylase, which specifically processes oxidative DNA damage by initiating base excision repair. NEIL1 recognizes and removes bases, primarily oxidized pyrimidines, which have been damaged by endogenous oxidation or exogenous mutagenic agents. NEIL1 functions through a combined glycosylase/AP (apurinic/apyrimidinic)-lyase activity, whereby it cleaves the N-glycosylic bond between the DNA backbone and the damaged base via its glycosylase activity and hydrolysis of the DNA backbone through beta-delta elimination due to its AP-lyase activity. In our study we investigated our hypothesis proposing that the cancer resistance of the bowhead whale can be associated with a better DNA repair with NEIL1 being upregulated or more active. Here, we report the molecular cloning and characterization of three transcript variants of bowhead whale NEIL1 of which two were homologous to human transcripts. In addition, a novel NEIL1 transcript variant was found. A differential expression of NEIL mRNA was detected in bowhead eye, liver, kidney, and muscle. The A-to-I editing of NEIL1 mRNA was shown to be conserved in the bowhead and two adenosines in the 242Lys codon were subjected to editing. A mass spectroscopy analysis of liver and eye tissue failed to demonstrate the existence of a NEIL1 isoform originating from RNA editing. Recombinant bowhead and human NEIL1 were expressed in E. coli and assayed for enzymatic activity. Both bowhead and human recombinant NEIL1 catalyzed, with similar efficiency, the removal of a 5-hydroxyuracil lesion in a DNA bubble structure. Hence, these results do not support our hypothesis but do not refute the hypothesis either.
Topics: Animals; Humans; Bowhead Whale; Escherichia coli; DNA Repair; DNA Glycosylases; Cloning, Molecular; DNA; RNA, Messenger; Lyases; Escherichia coli Proteins; Deoxyribonuclease (Pyrimidine Dimer)
PubMed: 36334646
DOI: 10.1016/j.biochi.2022.10.014 -
RSC Advances Oct 2022The "RAS-RAF-MEK-ERK" pathway is an important signaling pathway in melanoma. BRAF (70-90%) is the most common mutation in this pathway. BRAF inhibitors have four types...
The "RAS-RAF-MEK-ERK" pathway is an important signaling pathway in melanoma. BRAF (70-90%) is the most common mutation in this pathway. BRAF inhibitors have four types of conformers: type I (αC-IN/DFG-IN), type II (αC-IN/DFG-OUT), type I (αC-OUT/DFG-IN), and type I/II (αC-OUT/DFG-OUT). First- and second-generation BRAF inhibitors show resistance to BRAF and are ineffective against malignancies induced by dimer BRAF mutants causing 'paradoxical' activation. In the present study, we performed molecular modeling of pyrimidine-sulfonamide hybrids inhibitors using 3D-QSAR, molecular docking, and molecular dynamics simulations. Previous reports reveal the importance of pyrimidine and sulfonamide moieties in the development of BRAF inhibitors. Analysis of 3D-QSAR models provided novel pyrimidine sulfonamide hybrid BRAF inhibitors. The designed compounds share similarities with several structural moieties present in first- and second-generation BRAF inhibitors. A total library of 88 designed compounds was generated and molecular docking studies were performed with them. Four molecules (T109, T183, T160, and T126) were identified as hits and selected for detailed studies. Molecular dynamics simulations were performed at 900 ns and binding was calculated. Based on molecular docking and simulation studies, it was found that the designed compounds have better interactions with the core active site [the nucleotide (ADP or ATP) binding site, DFG motif, and the phospho-acceptor site (activation segment) of BRAF protein than previous inhibitors. Similar to the FDA-approved BRAF inhibitors the developed compounds have [αC-OUT/DFG-IN] conformation. Compounds T126, T160 and T183 interacted with DIF (Leu505), making them potentially useful against BRAF resistance and malignancies induced by dimer BRAF mutants. The synthesis and biological evaluation of the designed molecules is in progress, which may lead to some potent BRAF selective inhibitors.
PubMed: 36329938
DOI: 10.1039/d2ra05751d -
Cells Oct 2022Proteins that recognize specific DNA sequences or structural elements often find their cognate DNA lesions in a processive mode, in which an enzyme binds DNA...
Proteins that recognize specific DNA sequences or structural elements often find their cognate DNA lesions in a processive mode, in which an enzyme binds DNA non-specifically and then slides along the DNA contour by one-dimensional diffusion. Opposite to the processive mechanism is distributive search, when an enzyme binds, samples and releases DNA without significant lateral movement. Many DNA glycosylases, the repair enzymes that excise damaged bases from DNA, use processive search to find their cognate lesions. Here, using a method based on correlated cleavage of multiply damaged oligonucleotide substrates we investigate the mechanism of lesion search by three structurally related DNA glycosylases-bacterial endonuclease VIII (Nei) and its mammalian homologs NEIL1 and NEIL2. Similarly to another homologous enzyme, bacterial formamidopyrimidine-DNA glycosylase, NEIL1 seems to use a processive mode to locate its targets. However, the processivity of Nei was notably lower, and NEIL2 exhibited almost fully distributive action on all types of substrates. Although one-dimensional diffusion is often regarded as a universal search mechanism, our results indicate that even proteins sharing a common fold may be quite different in the ways they locate their targets in DNA.
Topics: Animals; Deoxyribonuclease (Pyrimidine Dimer); DNA-Formamidopyrimidine Glycosylase; DNA Repair; DNA Glycosylases; DNA; Oligonucleotides; Mammals
PubMed: 36291061
DOI: 10.3390/cells11203192 -
Molecules (Basel, Switzerland) Oct 2022Cannabidiol (CBD) has emerged as a phytocannabinoid with various beneficial effects for the skin, including anti-photoaging effects, but its mechanisms of action are not...
Cannabidiol (CBD) has emerged as a phytocannabinoid with various beneficial effects for the skin, including anti-photoaging effects, but its mechanisms of action are not fully elucidated. The study assessed CBD's photoprotective effects against acute ultraviolet B (UVB)-induced damage in HaCaT human keratinocyte cells and murine skin tissue. CBD (8 μM) alleviated UVB-induced cytotoxicity, apoptosis, and G2/M cell cycle arrest in HaCaT cells. The contents of γH2AX and cyclobutane pyrimidine dimers were decreased after CBD treatment. CBD reduced the production of reactive oxygen species and modulated the expression of antioxidant-related proteins such as nuclear factor erythroid 2-related factor 2 in UVB-stimulated HaCaT cells. Furthermore, CBD mitigated the UVB-induced cytotoxicity by activating autophagy. In addition, a cream containing 5% CBD showed effectiveness against UVB-induced photodamage in a murine model. The CBD cream improved the skin's condition by lowering the photodamage scores, reducing abnormal skin proliferation, and decreasing expression of the inflammation-related protein cyclooxygenase-2 in UVB-irradiated skin tissue. These findings indicate that CBD might be beneficial in alleviating UVB-induced skin damage in humans. The photoprotective effects of CBD might be attributed to its modulatory effects on redox homeostasis and autophagy.
Topics: Animals; Antioxidants; Autophagy; Cannabidiol; Cyclooxygenase 2; DNA Damage; Humans; Keratinocytes; Mice; Pyrimidine Dimers; Reactive Oxygen Species; Ultraviolet Rays
PubMed: 36235276
DOI: 10.3390/molecules27196740 -
International Journal of Molecular... Oct 2022Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome...
Oxidative DNA base lesions in DNA are repaired through the base excision repair (BER) pathway, which consequently plays a vital role in the maintenance of genome integrity and in suppressing mutagenesis. 8-oxoguanine DNA glycosylase (OGG1), endonuclease III-like protein 1 (NTH1), and the endonuclease VIII-like proteins 1-3 (NEIL1-3) are the key enzymes that initiate repair through the excision of the oxidized base. We have previously identified that the E3 ubiquitin ligase tripartite motif 26 (TRIM26) controls the cellular response to oxidative stress through regulating both NEIL1 and NTH1, although its potential, broader role in BER is unclear. We now show that TRIM26 is a central player in determining the response to different forms of oxidative stress. Using siRNA-mediated knockdowns, we demonstrate that the resistance of cells to X-ray radiation and hydrogen peroxide generated as a consequence of depletion can be reversed through suppression of selective DNA glycosylases. In particular, a knockdown of or can enhance sensitivity and DNA repair rates in response to X-rays, whereas a knockdown of or can produce the same effect in response to hydrogen peroxide. Our study, therefore, highlights the importance of TRIM26 in balancing cellular DNA glycosylase levels required for an efficient BER response.
Topics: Cell Survival; DNA; DNA Damage; DNA Glycosylases; DNA Repair; Deoxyribonuclease (Pyrimidine Dimer); Endonucleases; Hydrogen Peroxide; Oxidative Stress; RNA, Small Interfering; Ubiquitin-Protein Ligases
PubMed: 36232914
DOI: 10.3390/ijms231911613 -
Microbiology Spectrum Dec 2022Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the...
Light quality is a significant factor for living organisms that have photosensory systems, such as rhodopsin, a seven alpha-helical transmembrane protein with the retinal chromophore. Here, we report, for the first time, the function of new rhodopsin, which is an inverted 7-transmembrane protein, isolated from Trichococcus flocculiformis heliorhodopsin (TfHeR) works as a regulatory helper rhodopsin that binds with class 2 cyclobutane pyrimidine dimer (CPDII) photolyase to broaden the spectrum and upregulate DNA repair activity. We have confirmed their interaction through isothermal titration calorimetry (dissociation constant of 21.7 μM) and identified the charged residues for the interaction. Based on and experiments, we showed that the binding of heliorhodopsin with photolyase improved photolyase activity by about 3-fold to repair UV-caused DNA damage. Also, the DNA repair activity of TfHeR/ photolyase (TfPHR) was observed in the presence of green light. Our results suggested that heliorhodopsin directly controls the activity of photolyase and coevolves to broaden the activity spectrum by protein-protein interaction. This study reports a function for Heliorhodopsin working as a regulatory helper rhodopsin that with CPDII photolyase to broaden the spectrum and upregulating the DNA repair activity. Our results suggested that heliorhodopsin directly controls photolyase activity and coevolves to broaden the DNA repair capacity by protein-protein interaction.
Topics: Deoxyribodipyrimidine Photo-Lyase; Rhodopsin; Pyrimidine Dimers; DNA Repair
PubMed: 36219103
DOI: 10.1128/spectrum.02215-22 -
Frontiers in Molecular Biosciences 2022Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA... (Review)
Review
Faithful DNA replication is essential for all life. A multi-protein complex called the replisome contains all the enzymatic activities required to facilitate DNA replication, including unwinding parental DNA and synthesizing two identical daughter molecules. Faithful DNA replication can be challenged by both intrinsic and extrinsic factors, which can result in roadblocks to replication, causing incomplete replication, genomic instability, and an increased mutational load. This increased mutational load can ultimately lead to a number of diseases, a notable example being cancer. A key example of a roadblock to replication is chemical modifications in the DNA caused by exposure to ultraviolet light. Protein dynamics are thought to play a crucial role to the molecular pathways that occur in the presence of such DNA lesions, including potential damage bypass. Therefore, many assays have been developed to study these dynamics. In this review, we discuss three methods that can be used to study protein dynamics during replisome-lesion encounters in replication reactions reconstituted from purified proteins. Specifically, we focus on ensemble biochemical assays, single-molecule fluorescence, and cryo-electron microscopy. We discuss two key model DNA replication systems, derived from and . The main methods of choice to study replication over the last decades have involved biochemical assays that rely on ensemble averaging. While these assays do not provide a direct readout of protein dynamics, they can often be inferred. More recently, single-molecule techniques including single-molecule fluorescence microscopy have been used to visualize replisomes encountering lesions in real time. In these experiments, individual proteins can be fluorescently labeled in order to observe the dynamics of specific proteins during DNA replication. Finally, cryo-electron microscopy can provide detailed structures of individual replisome components, which allows functional data to be interpreted in a structural context. While classic cryo-electron microscopy approaches provide static information, recent developments such as time-resolved cryo-electron microscopy help to bridge the gap between static structures and dynamic single-molecule techniques by visualizing sequential steps in biochemical pathways. In combination, these techniques will be capable of visualizing DNA replication and lesion encounter dynamics in real time, whilst observing the structural changes that facilitate these dynamics.
PubMed: 36213113
DOI: 10.3389/fmolb.2022.968424 -
Computational and Structural... 2022Exposure to ultraviolet (UV) light causes the formation of mutagenic cyclobutane pyrimidine dimers (CPDs) in cellular DNA. Previous studies have revealed that CPD...
Exposure to ultraviolet (UV) light causes the formation of mutagenic cyclobutane pyrimidine dimers (CPDs) in cellular DNA. Previous studies have revealed that CPD formation in nucleosomes, the building blocks of chromatin, shows a striking ∼10 base pair (bp) periodic pattern. CPD formation is suppressed at positions where the DNA minor groove faces toward the histone octamer (minor-in) and elevated CPD formation at positions where the minor groove faces away from the histone octamer (minor-out). However, the molecular mechanism underlying this nucleosome photofootprint is unclear. Here, we analyzed ∼180 high-resolution nucleosome structures to characterize whether differences in DNA mobility or conformation are responsible for the CPD modulation in nucleosomes. Our results indicate that differences in DNA mobility cannot explain CPD modulation in nucleosome. Instead, we find that the sharp DNA bending around the histone octamer results in DNA conformations with structural parameters more susceptible to UV damage formation at minor-out positions and more resistant to CPD formation at minor-in positions. This analysis reveals the molecular mechanism responsible for periodic modulation of CPD formation and UV mutagenesis in nucleosomal DNA.
PubMed: 36212527
DOI: 10.1016/j.csbj.2022.08.071