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Photochemical & Photobiological... Aug 2013The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences...
The kinetics of thymine-thymine cyclobutane pyrimidine dimer (TT-CPD) formation was studied at 23 thymine-thymine base steps in two 247-base pair DNA sequences irradiated at 254 nm. Damage was assayed site-specifically and simultaneously on both the forward and reverse strands by detecting emission from distinguishable fluorescent labels at the 5'-termini of fragments cleaved at CPD sites by T4 pyrimidine dimer glycosylase and separated by gel electrophoresis. The total DNA strand length of nearly 1000 bases made it possible to monitor damage at all 9 tetrads of the type XTTY, where X and Y are non-thymine bases. TT-CPD yields for different tetrads were found to vary by as much as an order of magnitude, but similar yields were observed at all instances of a given tetrad. Kinetic analysis of CPD formation at 23 distinct sites reveals that both the formation and reversal photoreactions depend sensitively on the identity of the nearest-neighbour bases on the 5' and the 3' side of a photoreactive TT base step. The lowest formation and reversal rates occur when two purine bases flank a TT step, while the highest formation and reversal rates are observed for tetrads with at least one flanking C. Overall, the results show that the probabilities of CPD formation and photoreversal depend principally on interactions with nearest-neighbour bases.
Topics: Base Sequence; DNA; Kinetics; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 23727985
DOI: 10.1039/c3pp50078k -
The Journal of Biological Chemistry Nov 2017Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During... (Comparative Study)
Comparative Study
Ribonucleotides are the natural analogs of deoxyribonucleotides, which can be misinserted by DNA polymerases, leading to the most abundant DNA lesions in genomes. During replication, DNA polymerases tolerate patches of ribonucleotides on the parental strands to different extents. The majority of human DNA polymerases have been reported to misinsert ribonucleotides into genomes. However, only PrimPol, DNA polymerase α, telomerase, and the mitochondrial human DNA polymerase (hpol) γ have been shown to tolerate an entire RNA strand. Y-family hpol η is known for translesion synthesis opposite the UV-induced DNA lesion cyclobutane pyrimidine dimer and was recently found to incorporate ribonucleotides into DNA. Here, we report that hpol η is able to bind DNA/DNA, RNA/DNA, and DNA/RNA duplexes with similar affinities. In addition, hpol η, as well as another Y-family DNA polymerase, hpol κ, accommodates RNA as one of the two strands during primer extension, mainly by inserting dNMPs opposite unmodified templates or DNA lesions, such as 8-oxo-2'-deoxyguanosine or cyclobutane pyrimidine dimer, even in the presence of an equal amount of the DNA/DNA substrate. The discovery of this RNA-accommodating ability of hpol η redefines the traditional concept of human DNA polymerases and indicates potential new functions of hpol η .
Topics: 8-Hydroxy-2'-Deoxyguanosine; Base Pair Mismatch; DNA Primers; DNA Replication; DNA-Directed DNA Polymerase; Deoxyguanosine; Electrophoretic Mobility Shift Assay; Humans; Kinetics; Nucleic Acid Heteroduplexes; Nucleic Acid Hybridization; Oligodeoxyribonucleotides; Oligoribonucleotides; Pyrimidine Dimers; RNA; Recombinant Proteins; Reverse Transcription; Substrate Specificity; Transcription Elongation, Genetic
PubMed: 28972162
DOI: 10.1074/jbc.M117.809723 -
The Journal of Biological Chemistry May 2023The 3D organization of the eukaryotic genome is crucial for various cellular processes such as gene expression and epigenetic regulation, as well as for maintaining...
The 3D organization of the eukaryotic genome is crucial for various cellular processes such as gene expression and epigenetic regulation, as well as for maintaining genome integrity. However, the interplay between UV-induced DNA damage and repair with the 3D structure of the genome is not well understood. Here, we used state-of-the-art Hi-C, Damage-seq, and XR-seq datasets and in silico simulations to investigate the synergistic effects of UV damage and 3D genome organization. Our findings demonstrate that the peripheral 3D organization of the genome shields the central regions of genomic DNA from UV-induced damage. Additionally, we observed that potential damage sites of pyrimidine-pyrimidone (6-4) photoproducts are more prevalent in the nucleus center, possibly indicating an evolutionary pressure against those sites at the periphery. Interestingly, we found no correlation between repair efficiency and 3D structure after 12 min of irradiation, suggesting that UV radiation alters the genome's 3D organization in a short period of time. Interestingly, however, 2 h after UV induction, we observed more efficient repair levels in the center of the nucleus relative to the periphery. These results have implications for understanding the etiology of cancer and other diseases, as the interplay between UV radiation and the 3D genome may play a role in the development of genetic mutations and genomic instability.
Topics: DNA Damage; DNA Repair; Epigenesis, Genetic; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 37028766
DOI: 10.1016/j.jbc.2023.104679 -
The Journal of Biological Chemistry Sep 1987Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the...
Escherichia coli DNA photolyase binds to DNA containing pyrimidine dimers with high affinity and then breaks the cyclobutane ring joining the two pyrimidines of the dimer in a light- (300-500 nm) dependent reaction. In order to determine the structural features important for this level of specificity, we have constructed a 43 base pair (bp) long DNA substrate that contains a thymine dimer at a unique location and studied its interaction with photolyase. We find that the enzyme protects a 12-16-bp region around the dimer from DNase I digestion and only a 6-bp region from methidium propyl-EDTA-Fe (II) digestion. Chemical footprinting experiments reveal that photolyase contacts the phosphodiester bond immediately 5' and the 3 phosphodiester bonds immediately 3' to the dimer but not the phosphodiester bond between the two thymines that make up the dimer. Methylation protection and interference experiments indicate that the enzyme makes major groove contacts with the first base 5' and the second base 3' to the dimer. These data are consistent with photolyase binding in the major groove over a 4-6-bp region. However, major groove contacts cannot be of major significance in substrate recognition as the enzyme binds equally well to a thymine dimer in a 44-base long single strand DNA and protects a 10-nucleotide long region around the dimer from DNase I digestion. It is therefore concluded that the unique configuration of the phosphodiester backbone in the strand containing the pyrimidine dimer, as well as the cyclobutane ring of the dimer itself are the important structural determinants of the substrate for recognition by photolyase.
Topics: Base Sequence; Computer Graphics; DNA Damage; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Lyases; Models, Molecular; Nucleic Acid Conformation; Pyrimidine Dimers; Substrate Specificity
PubMed: 3308872
DOI: No ID Found -
The Journal of Biological Chemistry Oct 2015Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer...
Sunlight-induced C to T mutation hot spots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. The C and 5-methyl-C in CPDs are not stable and deaminate to U and T, respectively, which leads to the insertion of A by the DNA damage bypass polymerase η, thereby defining a probable mechanism for the origin of UV-induced C to T mutations. Deamination rates for T(m)CG CPDs have been found to vary 12-fold with rotational position in a nucleosome in vitro. To determine the influence of nucleosome structure on deamination rates in vivo, we determined the deamination rates of CPDs at TCG sites in a stably positioned nucleosome within the FOS promoter in HeLa cells. A procedure for in vivo hydroxyl radical footprinting with Fe-EDTA was developed, and, together with results from a cytosine methylation protection assay, we determined the translational and rotational positions of the TCG sites. Consistent with the in vitro observations, deamination was slower for one CPD located at an intermediate rotational position compared with two other sites located at outside positions, and all were much faster than for CPDs at non-TCG sites. Photoproduct formation was also highly suppressed at one site, possibly due to its interaction with a histone tail. Thus, it was shown that CPDs of TCG sites deaminate the fastest in vivo and that nucleosomes can modulate both their formation and deamination, which could contribute to the UV mutation hot spots and cold spots.
Topics: Bacterial Proteins; Base Sequence; Chromatin Assembly and Disassembly; DNA Methylation; DNA-Directed DNA Polymerase; Deamination; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Gene Expression; HeLa Cells; Histones; Humans; Hydroxyl Radical; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation; Nucleosomes; Promoter Regions, Genetic; Pyrimidine Dimers; Recombinant Fusion Proteins; Ultraviolet Rays
PubMed: 26354431
DOI: 10.1074/jbc.M115.673301 -
The Journal of Biological Chemistry May 2015Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class...
Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.
Topics: Agrobacterium tumefaciens; Binding Sites; Crystallography, X-Ray; Cytochromes; DNA Damage; Deoxyribodipyrimidine Photo-Lyase; Enzyme Stability; Evolution, Molecular; Flavin-Adenine Dinucleotide; Models, Molecular; Nucleic Acid Conformation; Oxidation-Reduction; Protein Structure, Tertiary; Pyrimidine Dimers; Tetrahydrofolates; Ultraviolet Rays
PubMed: 25784552
DOI: 10.1074/jbc.M115.637868 -
International Journal of Molecular... Feb 2021The high incidence of skin cancers in the Caucasian population is primarily due to the accumulation of DNA damage in epidermal cells induced by chronic ultraviolet B...
The high incidence of skin cancers in the Caucasian population is primarily due to the accumulation of DNA damage in epidermal cells induced by chronic ultraviolet B (UVB) exposure. UVB-induced DNA photolesions, including cyclobutane-pyrimidine dimers (CPDs), promote mutations in skin cancer driver genes. In humans, CPDs are repaired by nucleotide excision repair (NER). Several commonly used and investigational medications negatively influence NER in experimental systems. Despite these molecules' ability to decrease NER activity in vitro, the role of these drugs in enhancing skin cancer risk is unclear. In this study, we investigated four molecules (veliparib, resveratrol, spironolactone, and arsenic trioxide) with well-known NER-inhibitory potential in vitro, using UVB-irradiated CHO epithelial and HaCaT immortalized keratinocyte cell lines. Relative CPD levels, hypoxanthine phosphoribosyltransferase gene mutation frequency, cell viability, cell cycle progression, and protein expression were assessed. All four molecules significantly elevated CPD levels in the genome 24 h after UVB irradiation. However, veliparib, spironolactone, and arsenic trioxide reduced the mutagenic potential of UVB, while resveratrol did not alter UVB-induced mutation formation. UVB-induced apoptosis was enhanced by spironolactone and arsenic-trioxide treatment, while veliparib caused significantly prolonged cell cycle arrest and increased autophagy. Spironolactone also enhanced the phosphorylation level of mammalian target of rapamycin (mTOR), while arsenic trioxide modified UVB-driven mitochondrial fission. Resveratrol induced only mild changes in the cellular UVB response. Our results show that chemically inhibited NER does not result in increased mutagenic effects. Furthermore, the UVB-induced mutagenic potential can be paradoxically mitigated by NER-inhibitor molecules. We identified molecular changes in the cellular UVB response after NER-inhibitor treatment, which may compensate for the mitigated DNA repair. Our findings show that metabolic cellular response pathways are essential to consider in evaluating the skin cancer risk-modifying effects of pharmacological compounds.
Topics: Animals; Arsenic Trioxide; Autophagy; Benzimidazoles; CHO Cells; Cell Cycle Checkpoints; Cell Line, Transformed; Cell Survival; Cricetulus; DNA Damage; DNA Repair; HaCaT Cells; Humans; Hypoxanthine Phosphoribosyltransferase; Melanoma; Mutation Rate; Pyrimidine Dimers; Resveratrol; Skin; Skin Neoplasms; Spironolactone; Ultraviolet Rays
PubMed: 33562002
DOI: 10.3390/ijms22041638 -
Nucleic Acids Research Dec 2023Ultraviolet light generates cyclobutane pyrimidine dimer (CPD) and pyrimidine 6-4 pyrimidone (6-4PP) photoproducts that cause skin malignancies if not repaired by...
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.
Topics: Humans; DNA; DNA Damage; DNA Helicases; DNA Repair; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 37933861
DOI: 10.1093/nar/gkad974 -
The Journal of Biological Chemistry Mar 2012C-to-T mutations are a hallmark of UV light and, in humans, occur preferentially at methylated Py(m)CG sites, which are also sites of preferential cyclobutane pyrimidine...
C-to-T mutations are a hallmark of UV light and, in humans, occur preferentially at methylated Py(m)CG sites, which are also sites of preferential cyclobutane pyrimidine dimer (CPD) formation. In response, cells have evolved DNA damage bypass polymerases, of which polymerase η (pol η) appears to be specifically adapted to synthesize past cis-syn CPDs. Although T=T CPDs are stable, CPDs containing C or 5-methylcytosine ((m)C) are not and spontaneously deaminate to U or T at pH 7 and 37 °C over a period of hours or days, making their preparation and study difficult. Furthermore, there is evidence to suggest that, depending on solvent polarity, a C or an (m)C in a CPD can adopt three tautomeric forms, one of which could code as T. Although many in vitro studies have established that synthesis past T or U in a CPD by pol η occurs in a highly error-free manner, the only in vitro evidence that synthesis past C or (m)C in a CPD also occurs in an error-free manner is for an (m)C in the 5'-position of an (m)C=T CPD. Herein, we describe the preparation and characterization of an oligodeoxynucleotide containing a CPD of a T(m)CG site, one of the major sites of C methylation and C-to-T mutations found in the p53 gene of basal and squamous cell cancers. We also demonstrate that both yeast and human pol η synthesize past the 3'-(m)C CPD in a >99% error-free manner, consistent with the highly water-exposed nature of the active site.
Topics: Catalytic Domain; CpG Islands; DNA; DNA Damage; DNA Methylation; DNA-Directed DNA Polymerase; Humans; Pyrimidine Dimers; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 22262850
DOI: 10.1074/jbc.M111.333591 -
Toxicologic Pathology Jun 2016Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts usually created picoseconds after an ultraviolet (UV) photon is...
Mutations in sunlight-induced melanoma arise from cyclobutane pyrimidine dimers (CPDs), DNA photoproducts usually created picoseconds after an ultraviolet (UV) photon is absorbed at thymine or cytosine. Surprisingly, we found that, in melanocytes, CPDs were generated for hours after UVA or UVB exposure. These "dark CPDs" constituted the majority of CPDs in cultured human and murine melanocytes and in mouse skin, and they were most prominent in skin containing pheomelanin, the melanin responsible for blonde and red hair. The mechanism was also a surprise. Dark cyclobutane pyrimidine dimers (CPDs) arise when ultraviolet (UV)-induced superoxide and nitric oxide combine to form peroxynitrite, one of the few biological molecules capable of exciting an electron. This process, termed "chemiexcitation," is the source of bioluminescence in lower organisms. Excitation occurred in fragments of melanin, creating a quantum triplet state that had the energy of a UV photon but which induced CPDs by radiationless energy transfer to DNA. UVA and peroxynitrite also solubilized melanin and permeabilized the nuclear membrane, allowing melanin to enter. Melanin is evidently carcinogenic as well as protective. Chemiexcitation may also trigger pathogenesis in internal tissues because the same chemistry should arise wherever superoxide and nitric oxide arise near cells that contain melanin.
Topics: Animals; DNA Damage; Humans; Melanins; Melanocytes; Melanoma; Mice; Pyrimidine Dimers; Skin Neoplasms; Ultraviolet Rays
PubMed: 26951162
DOI: 10.1177/0192623316632072