Photochemical DNA Research Articles

Spore Photoproduct Lyase Catalyzes Specific Repair of the 5R but Not the 5S Spore Photoproduct

Bacterial spores are remarkable in their resistance to chemical and physical stresses, including exposure to UV radiation. The unusual UV resistance of bacterial spores is a result of the unique photochemistry of spore DNA, which results in accumulation of 5-thyminyl-5,6-dihydrothymine (spore photoproduct, or SP), coupled with the efficient repair of accumulated damage by the enzyme spore photoproduct lyase (SPL). SPL is a member of the radical AdoMet superfamily of enzymes, and utilizes an iron-sulfur cluster and S-adenosylmethionine to repair SP by a direct reversal mechanism initiated by H atom abstraction from C-6 of the thymine dimer. While two distinct diastereomers of SP (5R or 5S) could in principle be formed upon UV irradiation of bacterial spores, only the 5R configuration is possible for SP formed from adjacent thymines in double helical DNA, due to the constraints imposed by the DNA structure; the 5S configuration is possible in less well-defined DNA structures or as an interstrand cross-link. We report here results from HPLC and MS analysis of in vitro enzymatic assays on stereochemically defined SP substrates demonstrating that SPL specifically repairs only the 5R isomer of SP. The observation that 5R-SP, but not 5S-SP, is a substrate for SPL is consistent with the expectation that 5R is the SP isomer produced in vivo upon UV irradiation of bacterial spore DNA.

Molecular Basis of DNA Photodimerization: Intrinsic Production of Cyclobutane Cytosine Dimers

Based on CASPT2 results, the present contribution establishes for the first time that cytosine photodimer formation (C< >C) is mediated along the triplet and singlet manifold by a singlet-triplet crossing, (T1/S0)X, and by a conical intersection, (S1/S0)CI, respectively. The former can be accessed in a barrierless way from a great variety of photochemical avenues and exhibits a covalent single bond between the ethene C6-C6' carbon atoms of each monomer. The efficiency of the stepwise triplet mechanism, however, would be modulated by the effectiveness of the intersystem crossing mechanism. The results provide the grounds for the understanding of the potential photogenotoxicity of endogenous and exogenous compounds via triplet-triplet sensitization, with a lower bound for cytosine oligonucleotides predicted to be 2.70 eV, and give support to the traditional view of the primary role of triplet excited states in the photochemistry of DNA, a well-known source of photoproducts in solution under triplet photosensitization conditions. The function played by singlet excimers (excited dimers) to explain both the red-shifted fluorescence and photoreaction is highlighted. A rationale on the pronounced wavelength dependence of the observed fluorescence is offered. Geometrical arrangements at the time of light irradiation close to, but energetically above, (S1/S0)CI are suggested as reactive orientations that become prone to produce C< >C directly, with no energy barrier. Because of the outstanding intrinsic ability of cytosine to form stable relaxed excimers, the system located near the bound relaxed excimer has to accumulate enough vibrational energy to surmount a small barrier of 0.2 eV to reach (S1/S0)CI, making the overall process to proceed at a slower relative rate as compared to other compounds such as thymine, which is not susceptible of forming so stable excimers.

Autonomous DNA Computing Machine Based on Photochemical Gate Transition

We report the construction of a one-pot autonomous DNA computing machine based on photochemical gate transition (photocleavage, hybridization, and photoligation), and we performed binary digit additions using this machine. In our method, both photochemical DNA manipulations previously reported, photoligation via 5-carboxyvinyldeoxyuridene (cvU) containing ODN and photocleavage via carbazole-modified ODN, were employed. The binary digit additions were autonomously carried out by one-time irradiation at 366 nm in the single test tube. The fluorescence readout by the DNA chip was in good agreement with the correct answer of binary digit additions. We believe that this system is easily applicable to correlation analysis between SNPs as well as other binary digit processing, such as subtraction.

Photochemical DNA cleavage by novel water-soluble sulfonated dihydroxy phosphorus(V) tetrabenzotriazacorrole

The photosensitizing properties of a novel water-soluble phthalocyanine-like photosensitizer, sulfonated dihydroxy phosphorus(V) tetrabenzotriazacorrole {P(OH)(2)TBCS(n)}, have been reported. It is relevant for the use of this dye as photodynamic sensitizer. The compound exhibited significant photocleavage of supercoiled (SC) pUC19 DNA. The photooxygenation and photocleavage to DNA showed high efficiency in generating singlet oxygen.

Photochemical DNA Manipulation and DNA Analysis by Photoresponsive Artificial DNA

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

Parallel processable light-driven DNA logic gate

Here, we report the construction of a photochemical DNA logic circuit made entirely of DNA and implemented with sequence-specific photo-cleavage (SSPC) of photocrosslinking sites enclosed in the gate strand. As example, we demonstrate the performance of logic operations including NOT, AND, OR, and full-adder using a SSPC reaction.

N-Hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione as a Photochemical Hydroxyl-radical Source: Photochemistry and Oxidative Damage of DNA (Strand Breaks) and 2′-Deoxyguanosine (8-oxodG Formation)¶

N-Hydroxy-4-(4-chlorophenyl)thiazole-2(3H)-thione as a Photochemical Hydroxyl-radical Source: Photochemistry and Oxidative Damage of DNA (Strand Breaks) and 2′-Deoxyguanosine (8-oxodG Formation)¶

Low ionic strength reduces cytosine photoreactivity in UVC-irradiated isolated DNA

Exposure of DNA to far-UV radiation leads to the formation of several types of dimeric lesions, including cyclobutane dimers and (6-4) photoproducts. In order to gain insights into the main parameters driving DNA photochemistry, the effect of ionic strength on the yield of formation of these photoproducts was investigated in UVC-irradiated samples of isolated genomic DNA. The main consequence of lowering the ionic strength was a decrease in the UVC-induced formation of thymine-cytosine and cytosine-cytosine photoproducts. The reactivity of thymine-thymine and cytosine-thymine doublets was hardly affected. Evidence was obtained against a major role played by duplex denaturation in these observations. A more likely explanation is a change in the DNA structure as the result of a larger extent of protonation at low counter-ions concentration.

Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals

A number of mechanisms are responsible for the resistance of spores of Bacillus species to heat, radiation and chemicals and for spore killing by these agents. Spore resistance to wet heat is determined largely by the water content of spore core, which is much lower than that in the growing cell protoplast. A lower core water content generally gives more wet heat-resistant spores. The level and type of spore core mineral ions and the intrinsic stability of total spore proteins also play a role in spore wet heat resistance, and the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP) protects DNA against wet heat damage. However, how wet heat kills spores is not clear, although it is not through DNA damage. The alpha/beta-type SASP are also important in spore resistance to dry heat, as is DNA repair in spore outgrowth, as Bacillus subtilis spores are killed by dry heat via DNA damage. Both UV and gamma-radiation also kill spores via DNA damage. The mechanism of spore resistance to gamma-radiation is not well understood, although the alpha/beta-type SASP are not involved. In contrast, spore UV resistance is due largely to an alteration in spore DNA photochemistry caused by the binding of alpha/beta-type SASP to the DNA, and to a lesser extent to the photosensitizing action of the spore core's large pool of dipicolinic acid. UV irradiation of spores at 254 nm does not generate the cyclobutane dimers (CPDs) and (6-4)-photoproducts (64PPs) formed between adjacent pyrimidines in growing cells, but rather a thymidyl-thymidine adduct termed spore photoproduct (SP). While SP is formed in spores with approximately the same quantum efficiency as that for generation of CPDs and 64PPs in growing cells, SP is repaired rapidly and efficiently in spore outgrowth by a number of repair systems, at least one of which is specific for SP. Some chemicals (e.g. nitrous acid, formaldehyde) again kill spores by DNA damage, while others, in particular oxidizing agents, appear to damage the spore's inner membrane so that this membrane ruptures upon spore germination and outgrowth. There are also other agents such as glutaraldehyde for which the mechanism of spore killing is unclear. Factors important in spore chemical resistance vary with the chemical, but include: (i) the spore coat proteins that likely react with and detoxify chemical agents; (ii) the relative impermeability of the spore's inner membrane that restricts access of exogenous chemicals to the spore core; (iii) the protection of spore DNA by its saturation with alpha/beta-type SASP; and (iv) DNA repair for agents that kill spores via DNA damage. Given the importance of the killing of spores of Bacillus species in the food and medical products industry, a deeper understanding of the mechanisms of spore resistance and killing may lead to improved methods for spore destruction.

2P490 Photochemical DNA Manipulation via artifitial photoresponsive base(51. New methods and tools (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P490 Photochemical DNA Manipulation via artifitial photoresponsive base(51. New methods and tools (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Effect of denaturation on the photochemistry of pyrimidine bases in isolated DNA

The influence of denaturation on DNA photochemistry was studied by quantifying the yield of formation of all possible bipyrimidine photolesions within isolated genomic DNA samples exposed to UVC radiation. Effects of DNA melting was studied either by carrying out irradiation over a wide range of temperature (0–90 °C) or by decreasing the ionic strength of the solution at 30 °C. A first observation was a much larger decrease in the photoreactivity upon increasing the temperature in single-stranded than in double-stranded DNA. Secondly, formation of trans, syn cyclobutane dimers and, to a lesser extent, modification in the ratio between the yields of cyclobutane dimers and (6-4) photoproducts, were found to be other main features associated with denaturation. These results emphasize the modulating role of structure in the yield and nature of UV-induced DNA damage.

Molecular Design, Chemical Synthesis and Biological Activity of Artificial and DNA Photocleaving Carbohydrate‐Intercalator Hybrids

AbstractFor Abstract see ChemInform Abstract in Full Text.

Photosensitization of DNA by dipicolinic acid, a major component of spores of Bacillus species

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.

Computational chemistry applied to studies of organic contaminants in the environment: Examples based on benzo[a]pyrene

The topics of DNA adduct structure, biodegradation mechanisms, photo-chemistry, and adsorption of polycyclic aromatic hydrocarbons (PAHs) are discussed using benzo[a]pyrene and its partial oxidation products as an example. The use of classical mechanical, semi-empirical, and ab initio computational techniques are discussed in terms of their ability to answer important questions regarding the environmental fate of this important carcinogenic compound. The role of H-bonding variation with computational technique was analyzed and significant errors are likely when this parameter is predicted by classical force field simulations or semi-empirical calculations. The stability of the conformations of benzo[a]pyrene also change with computational method although the density functional theory (DFT) and second-order Moller-Plessett (MP2) methods used here converge to similar relative energies. Analysis of the relationship among HOMO-LUMO gaps, triplet and singlet excitation energies, and phototoxicity suggests that the HOMO-LUMO gaps are not the true parameter related to phototoxicity but a correlation between HOMO-LUMO gaps and excitation energies gives the appearance of a cause-and-effect relationship. Methods for predicting the water-soot partition coefficient of PAHs and the complexation of partial oxidation products with cations and metals in the environment are also discussed.

Use of DNA Repair Enzymes in Electrochemical Detection of Damage to DNA Bases in Vitro and in Cells

Electrochemical measurements at mercury or solid amalgam electrodes offer a highly sensitive detection of DNA strand breaks. On the other hand, electrochemical detection of damage to DNA bases at any electrode is usually much less sensitive. In this paper, we propose a new voltammetric method for the detection of the DNA base damage based on enzymatic conversion of the damaged DNA bases to single-strand breaks (ssb), single-stranded (ss) DNA regions, or both. Supercoiled DNA exposed to UV light was specifically cleaved by T4 endonuclease V, an enzyme recognizing pyrimidine dimers, the major products of photochemical DNA damage. Apurinic sites (formed in dimethyl sulfate-modified DNA) were determined after treating the DNA with E. coli exonuclease III, an enzyme introducing ssb at the abasic sites and degrading one of the DNA strands. The ssb or ssDNA regions, or both, were detected by adsorptive transfer stripping alternating current voltammetry at the mercury electrode. This technique offers much better sensitivity and selectivity of DNA base damage detection than any other electrochemical method. It is not limited to DNA damage in vitro, but it can detect also DNA base damage induced in living bacterial cells.

Solution of a SAT Problem on a Photochemical DNA Computer

Abstract The photochemical DNA computing via 5-carboxyvinyl-deoxyuridine (cvU) in anchor oligodeoxynucleotides (ODNs) in order to tether the multiple “DNA words” was demonstrated. A new MARK and UNMARK operation based on the cvU mediated reversible DNA photoligation has been developed for multiple-words DNA computing. The utility of this operation for DNA computing was demonstrated by solving a satisfiability problem (SAT problem) in which information was encoded in three tandem words.

Effects of the Binding of α/β-type Small, Acid-soluble Spore Proteins on the Photochemistry of DNA in Spores of Bacillus subtilis and In Vitro¶

The main lesion produced in DNA by UV-C irradiation of spores of Bacillus subtilis is 5-thyminyl-5,6-dihydrothymine (spore photoproduct [SP]). In contrast, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP) are the main photolesions in other cell types. The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with alpha/beta-type small, acid-soluble spore proteins (SASP). Using high-performance liquid chromatography-mass spectrometry analysis of the photoproducts, we showed that in wild-type B. subtilis spores (1) UV-C irradiation generates almost exclusively SP with little if any CPD and 6-4PP; (2) the SP generated is approximately 99% of the intrastrand derivative, but approximately 1% is in the interstrand form; and (3) there is no detectable formation of the SP analog between adjacent C and T residues. UV-C irradiation of spores lacking the majority of their alpha/beta-type SASP gave less SP than with wild-type spores and significant levels of CPD and 6-4PP. The binding of an alpha/beta-type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPD and 6-4PP, and a concomitant increase in the yield of SP, although levels of interstrand photoproducts were extremely low.

Effects of the Binding of α/β‐type Small, Acid‐soluble Spore Proteins on the Photochemistry of DNA in Spores of Bacillus subtilis and In Vitro¶

ABSTRACTThe main lesion produced in DNA by UV‐C irradiation of spores of Bacillus subtilis is 5‐thyminyl‐5,6‐dihydrothymine (spore photoproduct [SP]). In contrast, cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6–4PP) are the main photolesions in other cell types. The novel photochemistry of spore DNA is accounted for in part by its reduced hydration, but largely by the saturation of spore DNA with α/β‐type small, acid‐soluble spore proteins (SASP). Using high‐performance liquid chromatography‐mass spectrometry analysis of the photoproducts, we showed that in wild‐type B. subtilis spores (1) UV‐C irradiation generates almost exclusively SP with little if any CPD and 6–4PP; (2) the SP generated is ∼99% of the intrastrand derivative, but ∼1% is in the interstrand form; and (3) there is no detectable formation of the SP analog between adjacent C and T residues. UV‐C irradiation of spores lacking the majority of their α/β‐type SASP gave less SP than with wild‐type spores and significant levels of CPD and 6–4PP. The binding of an α/β‐type SASP to isolated DNA either in dry films or in aqueous solution led to a large decrease in the yield of CPD and 6–4PP, and a concomitant increase in the yield of SP, although levels of interstrand photoproducts were extremely low.

糖‐インターカレーター複合型人工ＤＮＡ光切断分子の分子デザインと機能発現制御

Studies of the interaction between the small molecules and DNA, especially the effects of the structural characteristics of the small molecules on the DNA interaction, are very important for the design of DNA targeting new drugs. In this context, the development of photochemical DNA cleaving agents, which selectively cleave DNA by irradiation with light of a specific wavelength under mild conditions and without any additives such as metals and reducing agents, is very interesting from chemical and biological standpoints and offers considerable potential in medicine especially in the post-genome era. In this article, the molecular design, chemical synthesis, DNA photocleaving property and photo-selective cytotoxicity of such novel and artificial light activatable DNA cleaving agents, which consist of DNA intercalator and carbohydrate, are described

Hydroxyl Radical is the Active Species in Photochemical DNA Strand Scission by Bis(peroxo)vanadium(V) Phenanthroline

Bis(peroxo)vanadium(V) complexes are widely investigated as anticancer agents. They exert their antitumor and cyctotoxic effects through inhibition of tyrosine phosphatases and DNA cleavage, respectively. The latter process remains poorly understood. The mechanism of DNA cleavage by NH(4)[(phen)V(O)(eta(2)-O(2))(2)] (phen = 1,10-phenanthroline) was investigated. Kinetic studies on DNA cleavage revealed that the complex is a single-strand nicking agent with no specificity. EPR experiments using 2,2,6,6-tetramethyl-4-piperidone (TMP) and 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) as spin-traps for singlet oxygen and hydroxyl radical, respectively, implicated hydroxyl radical production upon photodecomposition of bis(peroxo)vanadium(V). This was corroborated by benzoate inhibition of DNA strand scission and stoichiometric oxidation of 2-propanol to acetone upon irradiation of bis(peroxo)vanadium(V) phenanthroline. High-resolution polyacrylamide gel analysis of the vanadium cleavage reaction and [Fe(II)EDTA](2)(-)/H(2)O(2) resulted in comigration of "ladder" pattern bands, which superimposed when both reactions were run on the same lane. These findings identify hydroxyl radical produced from the photooxidation of the peroxo ligand on vanadium as the active species in DNA cleavage.