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Journal of Bacteriology Jan 1979An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads...
An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.
Topics: DNA Polymerase I; DNA Repair; DNA, Bacterial; DNA-Directed DNA Polymerase; Escherichia coli; Exonucleases; Kinetics; Mutation; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 368015
DOI: 10.1128/jb.137.1.234-242.1979 -
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 -
Photochemistry and Photobiology Jan 2019While is it well known that human telomeric DNA sequences can adopt G-quadruplex structures, some promoters sequences have also been found to form G-quadruplexes, and...
While is it well known that human telomeric DNA sequences can adopt G-quadruplex structures, some promoters sequences have also been found to form G-quadruplexes, and over 40% of promoters contain putative G-quadruplex-forming sequences. Because UV light has been shown to crosslink human telomeric G-quadruplexes by cyclobutane pyrimidine dimer (CPD) formation between T's on adjacent loops, UV light might also be able to photocrosslink G-quadruplexes in promoters. To investigate this possibility, 15 potentially UV-crosslinkable G-quadruplex-forming sequences found in a search of human DNA promoters were UVB irradiated in vitro, and three were confirmed to have formed nonadjacent CPDs by mass spectrometry. In addition to nonadjacent T=T CPDs found in human telomeric DNA, a nonadjacent T=U CPD was discovered that presumably arose from deamination of a nonadjacent T=C CPD. Analysis of the three sequences by circular dichroism, melting temperature analysis and chemical footprinting confirmed the presence of G-quadruplexes that could explain the formation of the nonadjacent CPDs. The formation of nonadjacent CPDs from the sequences in vitro suggests that they might be useful probes for the presence of non-B DNA structures, such as G-quadruplexes, in vivo, and if they were to form in vivo, might also have significant biological consequences.
Topics: G-Quadruplexes; Humans; Mass Spectrometry; Photochemical Processes; Promoter Regions, Genetic; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 30084501
DOI: 10.1111/php.12991 -
The Journal of Biological Chemistry Aug 2017Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The replisome transiently stalls at leading-strand... (Comparative Study)
Comparative Study
Genome integrity relies on the ability of the replisome to navigate ubiquitous DNA damage during DNA replication. The replisome transiently stalls at leading-strand template lesions and can either reinitiate replication downstream of the lesion or recruit specialized DNA polymerases that can bypass the lesion via translesion synthesis. Previous results had suggested that the replicase might play a role in lesion bypass, but this possibility has not been tested in reconstituted DNA replication systems. We report here that the DNA polymerase III holoenzyme in a stalled replisome can directly bypass a single cyclobutane pyrimidine dimer or abasic site by translesion synthesis in the absence of specialized translesion synthesis polymerases. Bypass efficiency was proportional to deoxynucleotide concentrations equivalent to those found and was dependent on the frequency of primer synthesis downstream of the lesion. Translesion synthesis came at the expense of lesion-skipping replication restart. Replication of a cyclobutane pyrimidine dimer was accurate, whereas replication of an abasic site resulted in mainly -1 frameshifts. Lesion bypass was accompanied by an increase in base substitution frequency for the base preceding the lesion. These findings suggest that DNA damage at the replication fork can be replicated directly by the replisome without the need to activate error-prone pathways.
Topics: DNA Breaks, Single-Stranded; DNA Polymerase III; DNA Replication; DNA, Bacterial; DNA-Directed DNA Polymerase; Escherichia coli; Escherichia coli Proteins; Frameshift Mutation; Furans; Holoenzymes; Multienzyme Complexes; Protein Multimerization; Pyrimidine Dimers; Replication Origin
PubMed: 28642369
DOI: 10.1074/jbc.M117.800441 -
Biochimica Et Biophysica Acta Feb 2005More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon... (Review)
Review
More than 50 years ago, initial experiments on enzymatic photorepair of ultraviolet (UV)-damaged DNA were reported [Proc. Natl. Acad. Sci. U. S. A. 35 (1949) 73]. Soon after this discovery, it was recognized that one enzyme, photolyase, is able to repair UV-induced DNA lesions by effectively reversing their formation using blue light. The enzymatic process named DNA photoreactivation depends on a non-covalently bound cofactor, flavin adenine dinucleotide (FAD). Flavins are ubiquitous redox-active catalysts in one- and two-electron transfer reactions of numerous biological processes. However, in the case of photolyase, not only the ground-state redox properties of the FAD cofactor are exploited but also, and perhaps more importantly, its excited-state properties. In the catalytically active, fully reduced redox form, the FAD absorbs in the blue and near-UV ranges of visible light. Although there is no direct experimental evidence, it appears generally accepted that starting from the excited singlet state, the chromophore initiates a reductive cleavage of the two major DNA photodamages, cyclobutane pyrimidine dimers and (6-4) photoproducts, by short-distance electron transfer to the DNA lesion. Back electron transfer from the repaired DNA segment is believed to eventually restore the initial redox states of the cofactor and the DNA nucleobases, resulting in an overall reaction with net-zero exchanged electrons. Thus, the entire process represents a true catalytic cycle. Many biochemical and biophysical studies have been carried out to unravel the fundamentals of this unique mode of action. The work has culminated in the elucidation of the three-dimensional structure of the enzyme in 1995 that revealed remarkable details, such as the FAD-cofactor arrangement in an unusual U-shaped configuration. With the crystal structure of the enzyme at hand, research on photolyases did not come to an end but, for good reason, intensified: the geometrical structure of the enzyme alone is not sufficient to fully understand the enzyme's action on UV-damaged DNA. Much effort has therefore been invested to learn more about, for example, the geometry of the enzyme-substrate complex, and the mechanism and pathways of intra-enzyme and enzyme <-->DNA electron transfer. Many of the key results from biochemical and molecular biology characterizations of the enzyme or the enzyme-substrate complex have been summarized in a number of reviews. Complementary to these articles, this review focuses on recent biophysical studies of photoreactivation comprising work performed from the early 1990s until the present.
Topics: Biophysics; Catalysis; DNA Damage; DNA Repair; Deoxyribodipyrimidine Photo-Lyase; Flavin-Adenine Dinucleotide; Light; Models, Chemical; Photochemistry; Protein Binding; Protein Conformation; Pyrimidine Dimers; Ultraviolet Rays
PubMed: 15721603
DOI: 10.1016/j.bbabio.2004.02.010 -
PLoS Genetics Apr 2022Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure....
Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert, but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y', and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA.
Topics: Chromatin; DNA; DNA Damage; DNA Repair; Heterochromatin; Pyrimidine Dimers; RNA, Untranslated; Saccharomyces cerevisiae; Telomere; Transcription, Genetic; Ultraviolet Rays
PubMed: 35486666
DOI: 10.1371/journal.pgen.1010167 -
The Journal of Investigative Dermatology Jun 1991We compared the susceptibilities of cultured melanocytes and keratinocytes to dimer induction in DNA by monochromatic ultraviolet (UV) radiation. Keratinocytes as well... (Comparative Study)
Comparative Study
We compared the susceptibilities of cultured melanocytes and keratinocytes to dimer induction in DNA by monochromatic ultraviolet (UV) radiation. Keratinocytes as well as melanocytes were derived from human foreskin, grown as a monolayer in petri dishes, covered with phosphate-buffered saline containing 0.1% glucose, and irradiated. UV irradiation was carried out at 254, 297, and 302 nm as well as with a light source emitting predominantly 312 nm. The induction of pyrmidine dimers was assessed by determination of the number of T4 endonuclease V-sensitive sites (ESS). We found a slightly higher response for dimer induction in melanocytes at 254, 297, and 302 nm; this difference was only significant at the 297-nm wavelength. Action spectra for pyrimidine dimer induction were derived from the exposure-response data obtained. The action spectra mimic to a large degree the action spectra for dimer induction in other cultured mammalian cells. The repair rate during a post-irradiation period lasting up to 24 h was substantially the same for the two cell types. The percentage of T4 endonuclease V-sensitive sites (ESS) remaining 9 and 24 h after irradiation was 45% and 30%, respectively.
Topics: Cells, Cultured; DNA; DNA Repair; Deoxyribonuclease (Pyrimidine Dimer); Endodeoxyribonucleases; Humans; Keratinocytes; Melanocytes; Pyrimidine Dimers; Ultraviolet Rays; Viral Proteins
PubMed: 2045680
DOI: 10.1111/1523-1747.ep12475443 -
Photochemistry and Photobiology Sep 2022Ultraviolet B-light (UVB) has been often used as a "physiological" UV in photobiology studies. How representative and equivalent these studies are compared to the effect...
Ultraviolet B-light (UVB) has been often used as a "physiological" UV in photobiology studies. How representative and equivalent these studies are compared to the effect of the sunlight is always of great interest. We now characterized the spectrum and intensity of two commonly used UV sources, a UVB lamp and a UVA-340 lamp which simulate the solar spectrum in the UVB/UVA range in the presence or absence of a UVB band pass filter that reduces >80% UVA from the UVA-340 lamp. The spectrum of each lamp was used in computational modeling for skin penetration. The effects of the lamps on endoplasmic reticulum (ER)-stress response and DNA damage in cultured keratinocytes HaCaT cells were analyzed. Our data show that the UVB lamp is a better inducer for both eIF2α phosphorylation and PERK modification, as well as a better reducer of ATF6 expression. The UVB lamp is also the best inducer of gamma-H2AX expression and cyclobutane pyrimidine dimers formation. However, the UVA-340 lamp is a better inducer for ATF4 expression. Our results indicate that different spectral characteristics of UV lamps can produce different results for the activation of the ER-stress responses and the differences do not always follow a defined pattern.
Topics: DNA Damage; Pyrimidine Dimers; Skin; Sunlight; Ultraviolet Rays
PubMed: 34932214
DOI: 10.1111/php.13585 -
Biochimica Et Biophysica Acta.... Nov 2022Ultraviolet (UV) light irradiation generates pyrimidine dimers on DNA, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Such dimers distort the...
Ultraviolet (UV) light irradiation generates pyrimidine dimers on DNA, such as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Such dimers distort the high-order DNA structure and prevent transcription and replication. The nucleotide excision repair (NER) system contributes to resolving this type of DNA lesion. There are two pathways that recognize pyrimidine dimers. One acts on transcribed strands of DNA (transcription-coupled NER), and the other acts on the whole genome (global genome-NER; GG-NER). In the latter case, DNA damage-binding protein 2 (DDB2) senses pyrimidine dimers with several histone modification enzymes. We previously reported that histone acetyltransferase binding to ORC1 (HBO1) interacts with DDB2 and facilitates recruitment of the imitation switch chromatin remodeler at UV-irradiated sites via an unknown methyltransferase. Here, we found that the phosphorylated histone methyltransferase mixed lineage leukemia 1 (MLL1) was maintained at UV-irradiated sites in an HBO1-dependent manner. Furthermore, MLL1 catalyzed histone H3K4 methylation and recruited the chromatin remodeler bromodomain adjacent to zinc finger domain 1A (BAZ1A)/ATP-utilizing chromatin assembly and remodeling factor 1 (ACF1). Depletion of MLL1 suppressed BAZ1A accumulation at UV-irradiated sites and inhibited the removal of CPDs. These data indicate that the DDB2-HBO1-MLL1 axis is essential for the recruitment of BAZ1A to facilitate GG-NER.
Topics: Chromatin; Chromosomal Proteins, Non-Histone; DNA Damage; DNA Repair; Humans; Leukemia; Pyrimidine Dimers
PubMed: 35940372
DOI: 10.1016/j.bbamcr.2022.119332 -
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