Influence of Phage Proteins on Formation of Specific UV DMA Photoproducts in Phage T7

Phage T7 can be used as a biological UV dosimeter. Its reading is proportional to the inactivation rate expressed in HT7 units. To understand the influence of phage proteins on the formation of DNA UV photoproducts, cyclobutane pyrimidine dimers (CPD) and (6-4)photoproducts ((6-4)PD) were determined in T7 DNA exposed to UV radiation under different conditions: intraphage T7 DNA, isolated T7 DNA and heated phage. To investigate the effects of various wavelengths, seven different UV sources have been used. The CPD and (6-4)PD were determined by lesion-specific antibodies in an immunodot-blot assay. Both photoproducts were HT7 dose-dependently produced in all three objects by every irradiation source in the biologically relevant UV dose range (1-10 HT7). The CPD to (6-4)PD ratios increased with the increasing effective wavelength of the irradiation source and were similar in intraphage T7 DNA, isolated DNA and heated phage with all irradiation sources. However, a significant decrease in the yield of both photoproducts was detected in isolated T7 DNA and in heated phage compared to intraphage DNA, the decrease was dependent on the irradiation source. Both photoproducts were affected the same way in isolated T7 DNA and heated phage, respectively. The yield of CPD and (6-4)PD was similar in B, C-like and A conformational states of isolated T7 DNA, indicating that the conformational switch in the DNA is not the decisive factor in photoproduct formation. The most likely explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in an increased rate of dimerization and (6-4)PD production of adjacent based in intraphage T7 DNA.     

Photoreactivation of killing in Streptomyces. II. Kinetics of formation and photolysis of pyrimidine dimers and adducts in S. coelicolor and S. griseus PHR-1

Abstract— A pyrimidine adduct, 6-4‘-[pyrimidine-2’-one] thymine (PO-T)?, observed in DNA hydrolysates of 254-nm ultraviolet (u.v.) irradiated conidia of Streptomyces coelicolor, increases linearly with u.v. dose up to 2 × 10⁵ ergs/mm². Yields of thymine dimer (T○) and uracil-thymine dimer (U○) level off at much lower doses. Initial relative rates of formation of these u.v. photoproducts are: 1:1.3:4.8 for PO-T, T○ and U○, respectively. Similar results were obtained with a Streptomyces griseus mutant, PHR-1. An equation is derived to estimate the ratio of the amount of PO-T to the total amount of thymine-derived photoproducts at low (biological) u.v. doses. The observed PO-T fractions compare well with the calculated values. Rapid photolysis of the precursor of PO-T was observed by post-u. v. treatment at 313 nm of conidia of S. coelicolor and of S. griseus PHR-1. The photolysis was much slower at 365 nm and did not occur at all at 405 nm. Pyrimidine dimers were not appreciably affected by post-u. v. treatment at the above wavelengths in these Streptomyces strains. Both of these strains are phenotypically photoreactivation-deficient, and the present results indicate that they do not possess active photoreactivating enzyme. In earlier papers[3,4,5], the pyrimidine adduct found in acid hydrolysates of DNA was loosely referred to as “uracil-thymine adduct (U-T adduct)”. Such terminology is not strictly correct. The pyrimidine adduct in acid hydrolysates is PO-T (sometimes called P₂B), which could theoretically result from removal of ammonia from a C-T adduct or removal of water from a U-T adduct (see [6]).     

Excision of cytosine-thymine adduct from the DNA of ultraviolet-irradiated Micrococcus radiodurans

Abstract— 6-4‘-[pyrimidin-2’-one]-thymine (PO-T), a deamination product of a cytosine–thymine adduct accounted for about 17 per cent of the total detectable thymine-derived photoproducts in u.v.-irradiated Micrococcus radiodurans. On incubation of the irradiated cells, oligonucleotides containing the photoproducts were released into the medium. After various periods of incubation the different photoproducts were isolated both from the cells and from the medium. Analysis of these different photoproducts showed that during the excision process the ratio of PO–T to total thymine-derived products remained constant both in the cells and in the medium. This shows that the precursor to PO–T is excised at the same rate as the other thymine-derived photoproducts by the dark repair mechanisms exhibited by this radiation-resistant organism.     

Validation of phage T7 biological dosimeter by quantitative polymerase chain reaction using short and long segments of phage T7 DNA

Phage T7 can be used as a biological dosimeter; its reading, the biologically effective dose (BED), is proportional to the inactivation rate |ln (n/n0)|. For the measurement of DNA damage in phage T7 dosimeter, a quantitative polymerase chain reaction (QPCR) methodology has been developed using 555 and 3826 bp fragments of phage T7 DNA. Both optimized reactions are so robust that an equally good amplification was obtained when intact phage T7 was used in the reaction mixture. In the biologically relevant dose range a good correlation was obtained between the BED of the phage T7 dosimeter and the amount of ultraviolet (UV) photoproducts determined by QPCR with both fragments under the effect of five various UV sources. A significant decrease in the yield of photoproducts was detected by QPCR in isolated T7 DNA and in heated phage compared with intraphage DNA with all irradiation sources. Because the yield of photoproducts was the same in B, C and A conformational states of T7 DNA, a possible explanation for modulation of photoproduct frequency in intraphage T7 DNA is that the presence of bound phage proteins induces an alteration in DNA structure that can result in increased induction of photoproducts.     

Are isolated nucleic acid bases really planar? A Car-Parrinello molecular dynamics study

Car-Parrinello molecular dynamics simulations of the flexibility of isolated DNA bases have been carried out. The comparison of lowest ring out-of-plane vibrations calculated by using MP2/cc-pvdz and BLYP/PW methods reveals that the DFT method with the plane wave basis set reasonably reproduces out-of-plane deformability of the pyrimidine ring in nucleic acid bases and could be used for reliable modeling of conformational flexibility of nucleobases. The conformational phase space of pyrimidine rings in thymine, cytosine, guanine, and adenine has been investigated by using the ab initio Car-Parrinello molecular dynamics method. It is demonstrated that all nucleic acid bases are highly flexible molecules and possess a nonplanar effective conformation of the pyrimidine ring despite the fact that the planar geometry corresponds to a minimum on the potential energy surface. The population of the planar geometry of the pyrimidine ring does not exceed 30%. Among the nonplanar conformations of the pyrimidine rings, the boat-like and half-chair conformations are the most populated.     

Conformational properties of a phototautomerizable nucleoside biomarker for phenolic carcinogen exposure

We have characterized the conformational properties of the C8-deoxyguanosine (C8-dG) nucleoside adduct, 8-(2"-hydroxyphenyl)-2'-dG (1), which is a potential biomarker for exposure to phenolic carcinogens. Adduct 1 possesses the unique ability to phototautomerize, through an excited-state intramolecular proton transfer (ESIPT) process, to generate its keto form. This tautomerization depends on the presence of an intramolecular hydrogen (H)-bond between the phenolic OH and the imine nitrogen (N7) and has permitted insight into the equilibrium ground states of adduct 1. The results of our studies demonstrate that adduct 1 undergoes an ESIPT despite preferring a nonplanar "twisted" conformation that is imposed by the deoxyribose (dR) sugar moiety. Interestingly, a planar conformation of adduct 1 is also formed in certain aprotic solvents due to the anchoring effect of the intramolecular H-bond. Our results provide a basis for future studies aimed at determining the conformations of adduct 1 within DNA that will aid in our understanding of phenol-mediated carcinogenesis.     

A proposed bioactive conformation of Peptide T

The conformational profiles of Peptide T, (5–8)Peptide T, [Abu⁵](4–8)Peptide T and (4–8)Peptide T were computed independently to assess the geometrical characteristics of the bioactive conformation of Peptide T. The conformational profiles of the peptides were computed within the molecular mechanics framework using an effective dielectric constant of 80. The conformational space was thoroughly sampled using an iterative simulated annealing protocol. The bioactive conformation was assessed by pairwise cross comparisons of each of the unique low energy conformations found for each of the different analogs studied. After a putative bioactive conformation was selected, in order to further validate our hypothesis the conformational profile of the potent compound cyclo(Thr-Thr-Asn-Tyr-Thr-Asp) was computed and the putative bioactive conformation was found. The conformation exhibits a pseudo -turn involving the side chain of Thr⁵ and the carbonyl oxygen of Tyr⁷ forming a C12 ring.     

PREFERENTIAL INHIBITION OF NUCLEOSOME ASSEMBLY BY ULTRAVIOLET-INDUCED (6-4)PHOTOPRODUCTS

We reconstituted nucleosomes in vitro using two kinds of damaged pBR322 plasmid DNA carrying cyclobutane pyrimidine dimers (CPD) or (6-4)photoproducts. The results indicate that nucleosome assembly is inhibited preferentially by (6-4)photoproducts compared with CPD, suggesting that the regions carrying (6-4)photoproducts retain their nucleosome-free form, i.e. linker-like conformation until completion of the repair processes.     

UV INDUCED (6-4) PHOTOPRODUCTS ARE DISTRIBUTED DIFFERENTLY THAN CYCLOBUTANE DIMERS IN NUCLEOSOMES

We have compared the distributions of two stable UV photoproducts in nucleosome core DNA at the single-nucleotide level using a T4 polymerase-exonuclease mapping procedure. The distribution of pyrimidine-pyrimidone (6-4) dimers was uncovered by reversing the major UV photo-product, cis-syn cyclobutane pyrimidine dimer, with E. coli DNA photolyase and photoreactivating light. Whereas the distribution of total UV photoproducts in nucleosome core DNA forms a striking 10.3 base periodic pattern, the distribution of (6-4) dimers is much more random throughout the nucleosome core domain. Therefore, histone-DNA interactions in nucleosomes strongly modulate formation of the major class of UV-induced photoproducts, while having either a constant effect or no effect on (6-4) dimer formation.     

Photoinduced Damage to Cellular DNA: Direct and Photosensitized Reactions(†)

The survey focuses on recent aspects of photochemical reactions to cellular DNA that are implicated through the predominant formation of mostly bipyrimidine photoproducts in deleterious effects of human exposure to sunlight. Recent developments in analytical methods have allowed accurate and quantitative measurements of the main DNA photoproducts in cells and human skin. Highly mutagenic CC and CT bipyrimidine photoproducts, including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) are generated in low yields with respect to TT and TC photoproducts. Another striking finding deals with the formation of Dewar valence isomers, the third class of bipyrimidine photoproducts that is accounted for by UVA-mediated isomerization of initially UVB generated 6-4PPs. Cyclobutadithymine (T<>T) has been unambiguously shown to be involved in the genotoxicity of UVA radiation. Thus, T<>T is formed in UVA-irradiated cellular DNA according to a direct excitation mechanism with a higher efficiency than oxidatively generated DNA damage that arises mostly through the Type II photosensitization mechanism. C<>C and C<>T are repaired at rates intermediate between those of T<>T and 6-4TT. Evidence has been also provided for the occurrence of photosensitized reactions mediated by exogenous agents that act either in an independent way or through photodynamic effects.     

Effects of the binding of alpha/beta-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.     

Adjacent and Nonadjacent Dipyrimidine Photoproducts as Intrinsic Probes of DNA Secondary and Tertiary Structure†

While the photochemistry of duplex DNA has been extensively studied, the photochemistry of nonduplex DNA structures is largely unexplored. Because the structure and stereochemistry of DNA photoproducts depend on the secondary structure and conformation of the DNA precursor, they can serve as intrinsic probes of DNA structure. This review focuses on the structures and stereoisomers of pyrimidine dimer photoproducts arising from adjacent and nonadjacent pyrimidines in A, B and denatured DNA, bulge loops, G-quadruplexes and reverse Hoogsteen hairpins and methods for their detection.     

DNA-mediated charge transport requires conformational motion of the DNA bases: elimination of charge transport in rigid glasses at 77 K

We have proposed that DNA-mediated charge transport (CT) is gated by base motions, with only certain base conformations being CT-active; a CT-active conformation can be described as a domain, a transiently extended pi-orbital defined dynamically by DNA sequence. Here, to explore these CT-active conformations, we examine the yield of base-base CT between photoexcited 2-aminopurine (Ap*) and guanine in DNA in rigid LiCl glasses at 77 K, where conformational rearrangement is effectively eliminated. Duplex DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)nG (n = 0-4). The yield of CT was monitored through fluorescence quenching of Ap* by G. We find, first, that the emission intensity of Ap* in all DNA duplexes increases dramatically upon cooling and becomes comparable to free Ap*. This indicates that all quenching of Ap* in duplex DNA is a dynamic process that requires conformational motion of the DNA bases. Second, DNA-mediated CT between Ap* and G is not observed at 77 K; rather than hindering the ability of DNA to transport charge, conformational motion is required. Moreover, the lack of DNA-mediated CT at 77 K, even through the shortest bridge, suggests that the static structures adopted upon cooling do not represent optimum CT-active conformations. These observations are consistent with our model of conformationally gated CT. Through conformational motion of the DNA bases, CT-active domains form and break-up transiently, both facilitating and limiting CT.     

PHOTO-OXIDATION OF THYMINE SENSITIZED BY 2-METHYL-1,4-NAPHTHOQUINONE: ANALYSIS OF PRODUCTS INCLUDING THREE NOVEL PHOTO-DIMERS

Abstract —The near-UV photosensitization of thymine by 2-methyl-1,4-naphthoquinone in oxygenated aqueous solution leads to the formation of nine major thymine-derived products. Six of the photoproducts, representing 70% of the initial thymine loss, are also known to be formed by γ-ray or X-ray irradiation of oxygenated water solutions of thymine. They include the cis and trans isomers of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine, the cis and trans thymine glycols, 5-hydroxy-5-methylhydantoin, and N-formyl-N'-pyruvylurea. The three remaining major products, accounting for a further 25% of the initial thymine loss, are thymine di-adducts of a type not previously reported. Preliminary characterization of these novel photoproducts, based on their UV absorption, mass spectra, and proton and carbon-13 nuclear magnetic resonance spectra, suggests that in each of the three, the thymine moieties are linked by an N(1)-C(5) or N(1)-C(6) bond. A thymine cation radical is implicated as the common precursor of all 9 products. Accordingly. pyrimidine photosensitization by 2-methyl-1,4-naphthoquinone serves as a model system for studying the chemical consequences of radiation-induced ionization of the DNA bases.     

Electrospray-mass spectrometry characterization and measurement of far-UV-induced thymine photoproducts

Far-UV-induced formation of dimeric pyrimidine photoproducts within DNA is a major cause of the carcinogenic effects of solar light. The chemical structure of this class of lesion has been mostly determined by studies on model compounds. The present work is aimed at providing mass spectrometry data on the thymine-thymine photoproducts, including the diastereoisomers of the cyclobutane dimer, the (6-4) adduct, the related Dewar valence isomer and the spore photoproduct. Fragmentation mass spectra of the modified bases, nucleosides, dinucleoside monophosphates and dinucleotides were recorded following electrospray ionization with either triple-quadrupolar or ion-trap detection. The results showed differences in fragmentation pattern between the different types of photoproducts. In addition, a drastic effect of the diastereoisometry was observed for the cyclobutane dimers. A sensitive detection technique has been developed for the analysis of dinucleoside monophosphate photoproducts by high-performance liquid chromatography associated with mass spectrometry in the negative mode with multiple reaction-monitoring detection.     

Biological responses to the simulated Martian UV radiation of bacteriophages and isolated DNA

Mars is considered as a main target for astrobiologically relevant exploration programmes. In this work the effect of simulated Martian solar UV radiation was examined on bacteriophage T7 and on isolated T7 DNA. A decrease of the biological activity of phages, characteristic changes in the absorption spectrum and in the electrophoretic pattern of isolated DNA/phage and the decrease of the amount of PCR products were detected indicating damage of isolated and intraphage T7 DNA by UV radiation. Further mechanistic insights into the UV-induced formation of intraphage/isolated T7 DNA photoproducts were gained from the application of appropriate enzymatic digestion and neutral/alkaline agarose gel electrophoresis. Our results showed that intraphage DNA was about ten times more sensitive to simulated Martian UV radiation than isolated T7 DNA indicating the role of phage proteins in the DNA damage. Compared to solar UV radiation the total amount of DNA damage determined by QPCR was about ten times larger in isolated DNA and phage T7 as well, and the types of the DNA photoproducts were different, besides cyclobutane pyrimidine dimers (CPD), double-strand breaks (dsb), and single-strand breaks (ssb), DNA-protein cross-links were produced as well. Surprisingly, energy deposition as low as 4-6eV corresponding to 200-400nm range could induce significant amount of ssb and dsb in phage/isolated DNA (in phage the ratio of ssb/dsb was approximately 23%/12% and approximately 32%/19% in isolated DNA). 5-8% of the CPD, 3-5% of the AP (apurinic/apyrimidinic) sites were located in clusters in DNA/phage, suggesting that clustering of damage occur in the form of multiple damaged sites and these can have a high probability to produce strand breaks. The amount of total DNA damage in samples which were irradiated in Tris buffer was reduced by a factor approximately 2, compared to samples in phosphate buffer, suggesting that some of the photoproducts were produced via radicals.     

Far-UV photochemistry and photosensitization of 2'-deoxycytidylyl-(3'-5')-thymidine: isolation and characterization of the main photoproducts.

Far-UV irradiation of 2'-deoxycytidylyl-(3'-5')-thymidine (dCpT) gave rise to the pyrimidine (6-4) pyrimidone adduct and its Dewar valence isomer as the main photoproducts. The absolute configuration of the former adduct was determined and its photoisomerization studied. A comparison of the alkali lability of both compounds showed that hydrolysis of the phosphodiester bond occurs for the Dewar valence isomer but not for its (6-4) precursor. In addition, the trans-syn and cis-syn cyclobutane dimers of dCpT were obtained by acetophenone photosensitization and characterized. Finally, the deamination rate constants for this series of compounds were shown to be dramatically influenced by the nature and the configuration of the photoproducts.     

Crystal Structure of the T(6‐4)C Lesion in Complex with a (6‐4) DNA Photolyase and Repair of UV‐Induced (6‐4) and Dewar Photolesions

UV‐light irradiation induces the formation of highly mutagenic lesions in DNA, such as cis‐syn cyclobutane pyrimidine dimers (CPD photoproducts), pyrimidine(6‐4)pyrimidone photoproducts ((6‐4) photoproducts) and their Dewar valence isomers ((Dew) photoproducts). Here we describe the synthesis of defined DNA strands containing these lesions by direct irradiation. We show that all lesions are efficiently repaired except for the T(Dew)T lesion, which cannot be cleaved by the repair enzyme under our conditions. A crystal structure of a T(6‐4)C lesion containing DNA duplex in complex with the (6‐4) photolyase from Drosophila melanogaster provides insight into the molecular recognition event of a cytosine derived photolesion for the first time. In light of the previously postulated repair mechanism, which involves rearrangement of the (6‐4) lesions into strained four‐membered ring repair intermediates, it is surprising that the not rearranged T(6‐4)C lesion is observed in the active site. The structure, therefore, provides additional support for the newly postulated repair mechanism that avoids this rearrangement step and argues for a direct electron injection into the lesion as the first step of the repair reaction performed by (6‐4) DNA photolyases.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

UNIQUE DNA REPAIR PROPERTY OF AN ULTRAVIOLET-SENSITIVE (radC MUTANT OF Dictyostelium discoideum

Abstract— Dictyostelium discoideum is an organism that shows higher UV resistance than other organisms, such as Escherichia coli and human cultured cells. We examined the removal of cyclobutane pyrimidine dimers (CPD) and 6–4 photoproducts from DNA in the radC mutant and the wild-type strain using an enzyme-linked immunosorbent assay with monoclonal antibodies. Wild-type cells excised more than 90% of both CPD and 6–4 photoproducts within 4 h. Dictyostelium discoideum appeared to have a special repair system, because 6–4 photoproducts were repaired faster than CPD in E. coli and human cultured cells. In radC mutant cells, although only 50% of CPD were excised from DNA within 8 h, effective removal of 6–4 photoproducts (80% in 8 h) was observed. Excision repair-deficient mutants generally cannot remove both CPD and 6–4 photoproducts. Though the radC mutant shows deficient excision repair, it can remove 6–4 photoproducts to a moderate degree. These results suggest that D. discoideum has two kinds of repair systems, one mainly for CPD and the other for 6–4 photoproducts, and that the radC mutant has a defect mainly in the repair enzyme for CPD.