Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) II. Evidence for heterogeneous light-dependent repair activities

Abstract. Two biological effects of UV radiation upon Smittia eggs are observed, both of which seem to be associated with the formation of pyrimidine dimers in the RNA (largely ribosomal) of the eggs. While irradiation of the anterior pole region causes the formation of an aberrant segment pattern (double abdomen induction), irradiation of entire eggs leads to an arrest of their development (inactiva-tion). Both UV effects are photoreversible with different action spectra of the photoreactivating light. A dose rate dependence of the photoreactivation can be observed after both UV effects. The saturating dose rate is about 6 W/m² (at 440 nm) after UV induction of double abdomens. Upon UV inactivation, the saturating dose rate level for the photoreactivating light is much higher, and a single light flash causes both a considerable biological reactivation and the disappearance of about 7 × 10⁹ pyrimidine dimers from the total RNA per egg. The results indicate the presence of heterogeneous light-dependent repair activities acting upon UV induced pyrimidine dimers in the RNA of the eggs.     

The α‐L‐Threofuranosyl‐(3′→2′)‐oligonucleotide System (‘TNA'): Synthesis and Pairing Properties

Our studies of α‐L ‐Threofuranosyl‐(3′→2′)‐oligonucleotides (‘TNA') are part of a systematic experimental inquiry into the base‐pairing properties of potentially natural nucleic acid alternatives taken from RNA's close structural neighborhood. TNA is an efficient Watson‐Crick base‐pairing system and has the capability of informational cross‐pairing with both RNA and DNA. This property, together with the system's constitutional and (presumed) generational simplicity, warrants special scrutiny of TNA in the context of the search for chemical clues to RNA's origin.     

Photorepair of pyrimidine dimers in the epidermis of the marsupial Monodelphis domestica

Abstract Induction and fate of ultraviolet radiation-induced pyrimidine dimers in DNA have been measured in the epidermis of the marsupial, Monodelphis domestica, using damage-specific endonucleases from Micrococcus luteus. Approximately 90% of the dimers are lost when irradiated animals are subjected to photoreactivating light for 180 min. No loss of dimers was detected when the animals were held for a similar period of time in the dark. The capacity of these epithelial cells to photorepair pyrimidine dimers may provide a useful whole animal system in which to determine the role of pyrimidine dimers in photobiological responses of the skin.     

Photoreactivating Enzyme for (6–4) Photoproducts in Cultured Goldfish Cells

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6–4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6–4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6–4) photoproducts and its dissociation from (6–4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of pro-tein(s) to (6–4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA pho-toreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6–4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6–4) photolyase in cultured goldfish cells as in Dro-sophila, Xenopus and Crotalus.     

Monodelphis domestica: a new animal model for studies in photodermatology

The ability to photoreverse pyrimidine dimers in DNA of the South American opossum Monodelphis domestica provides a powerful tool with which to probe the role of pyrimidine dimers in ultraviolet radiation (UVR)-induced histopathologic changes of the skin of this mammal. We have observed that post-UVR exposure to photoreactivation light not only reversed pyrimidine dimers in epidermal DNA, but also suppressed the capacity of UVR to induce macroscopic and microscopic changes in the skin of M. domestica.     

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING I: REACTIONS INVOLVING PYRIMIDINE RADICAL CATION INTERMEDIATES

The light-induced splitting of pyrimidine dimers was studied using the electron acceptor anthraquinone-2-sulfonate (AQS) as a photosensitizer. To this end, photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed on a series of pyrimidine monomers and dimers. The CIDNP spectra demonstrate the existence of both the dimer radical cation, which is formed by electron transfer from the dimer to the photoexcited sensitizer AQS*, and its dissociation product, the monomer radical cation. In spectra of 1,1′-trimethylene bridged cis,syn pyrimidine dimers, polarization is observed that originates from a spin-sorting process in the dimer radical pair. This points to a relatively long lifetime of the dimer radical cation involved, which is presumably due to stabilization by the trimethylene bridge. Polarization originating from a dimer radical pair is detected in the spectrum of trans,anti (1,3-dimethyluracil) dimer as well. The spectra of the bridged pyrimidines also demonstrate the reversibility of the dissociation of dimer radical cation into monomer radical cation, which is concluded from the observation of polarization in the dimer as a result of spin sorting in the monomer radical pair.     

PHOTO-CIDNP STUDY OF PYRIMIDINE DIMER SPLITTING II: REACTIONS INVOLVING PYRIMIDINE RADICAL ANION INTERMEDIATES

A series of photo-CIDNP (chemically induced dynamic nuclear polarization) experiments were performed on pyrimidine monomers and dimers, using the electron-donor Nα-acetyltryptophan (AcTrp) as a photosensitizer. The CIDNP spectra give evidence for the existence of both the dimer radical anion, which is formed by electron transfer from the excited AcTrp* to the dimer, and its dissociation product, the monomer radical anion. The AcTrp spectra are completely different from those obtained with an oxidizing sensitizer like anthraquinone-2-sulfonate, because of different unpaired electron spin density distributions in pyrimidine radical anion and cation. In the spectra of the anti (1,3-dimethyluracil) dimers, polarization is detected that originates from a spin-sorting process in the dimer radical pair, pointing to a relatively long lifetime of the dimer radical anions involved. Although the dimer radical anions of the 1,1′-trimethylene-bridged pyrimidines may have a relatively long lifetime as well, their protons have only very weak hyperfine interaction, which explains why no polarization originating from the dimer radical pair is detected. In the spectra of the bridged pyrimidines, polarized dimer protons are observed as a result of spin sorting in the monomer radical pair, from which it follows that the dissociation of dimer radical anion into monomer radical anion is reversible. A study of CIDNP intensities as a function of pH shows that a pH between 3 and 4 is optimal for observing monomer polarization that originates from spin-sorting in the monomer radical pair. At higher pH the geminate recombination polarization is partly cancelled by escape polarization arising in the same product.     

FORMATION OF PYRIMIDINE DIMERS IN SIMIAN VIRUS 40 CHROMOSOMES AND DNA in vitro: EFFECTS OF SALT

Abstract— Simian virus 40 chromosomes were used to determine whether packaging of DNA into chromatin affected the yield of cyclobutane pyrimidine dimers introduced by ultraviolet light (254 nm). SV40 chromatin and purified SV40 DNA (radioactively labeled with different isotopes) were mixed and irradiated in vitro . The proteins were extracted and pyrimidine dimers detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. When irradiation was carried out in the presence of at least 0.05 M NaCl the same number of dimers were formed in chromatin as in free DNA. Irradiation in the absence of NaCl, however, reduced the relative yield of dimers in chromatin to 89% of that in free DNA. Different methods of chromatin preparation did not influence these results.     

THE FORMATION OF PHOTOREVERSIBLE CYCLOBUTANE-TYPE PYRIMIDINE DIMERS IN ULTRAVIOLET-IRRADIATED POTATO VIRUS X*

Abstract— Photoreversible cyclobutane-type pyrimidine dimers were found in ³²P-labeled RN A isolated from intact potato virus X irradiated with 254 nm light. The number of dimers was correlated with the biological infectivity of the ultraviolet-treated virus. Assuming that incorporated ³²P is homogeneously distributed in the viral RNA chain, it can be calculated that about 5·7±1·7 dimers are present in each strand of virus per lethal biological hit.     

Photoreactivation of RNA in UV-irradiated insect eggs (Smittia sp., Chironomidae, Diptera) I. Photosensitized production and light-dependent disappearance of pyrimidine dimers

Abstract. Irradiation of Smittia eggs with UV during intravitelline cleavage causes the formation of pyrimidine dimers in the (largely ribosomal) RNA of the eggs. The yield of dimers is wavelength-dependent in a way that strongly suggests the involvement of photosensitizing egg components. Illumination of UV-irradiated eggs with light (380 or 400 nm) causes both photoreactivation of the eggs and mono-merization of the pyrimidine dimers in their RNA. The photoreactivable sector of the biological damage is correlated with the amount of pyrimidine dimers present in the RNA after inactivation of the eggs with UV of different wavelengths. The data are regarded as the first direct evidence that the photoreactivation of a eukaryotic organism is correlated with the light-dependent (and apparently enzymatic) monomerization of pyrimidine dimers in RNA.     

Action of photoreactivating light on pyrimidine heteroadduct in bacteria

Abstract— In stationary-phase Escherichia coli B/r, photoreactivation (PR) at 313 nm of ultraviolet (u.v.) killing is inefficient compared with PR at 405 nm, and can be explained solely by photoenzymatic reversal of pyrimidine dimers. In Staphylococcus epidermidis, PR shows a maximum at 313 nm, suggesting that this organism shows the Type III PR proposed by Jagger et al.[5] for Streptomyces strains. Reversal of pyrimidine dimers is not sufficient to explain this PR. The mechanism of Type III PR remains unknown. With both S. epidermidis and E. coli B/r, the amount of uracil–thymine heteroadduct in DNA hydrolysates decreases if the cells are given a post-u.v. treatment at 313 nm, but no decrease is observed if the post-u.v. treatment is at 405 nm. The biological significance of this adduct and of its removal is not clear. It may play a role in Type III PR.     

Incandescent lamps can produce pyrimidine dimers in DNA

DNA molecules that have been exposed to light from a 150 W incandescent spot lamp are nicked by the Micrococcus luteus endonuclease specific for cyclobutyl-type pyrimidine dimers. The production of these enzyme-sensitive sites increases with increasing spot lamp exposure. These sites have been confirmed to be pyrimidine dimers by their property of being photoreversed by an E. coli photoreactivating enzyme. The emission spectrum of the lamp shows detectable output at wavelengths less than 320 nm. These results indicate that the sensitivity of the techniques utilized in this work can be used to detect low levels of contaminating UV radiation.     

Regioselectivity and Competition of the Paternò–Büchi Reaction and Triplet–Triplet Energy Transfer between Triplet Benzophenones and Pyrimidines: Control by Triplet Energy Levels

The photochemical reaction of a pyrimidine and a ketone occurs either as a Paternò–Büchi (PB) reaction or as energy transfer (ET) from the triplet ketone to the pyrimidine. It is rare for the two types of reactions to occur concurrently, and their competitive mechanism remains unknown. In this work, two classes of products, regioisomeric oxetane(s) ( 2 , 3 ) from a PB reaction and three isomeric dimers of 5‐fluoro‐1,3‐dimethyl uracil (FDMU) ( 4 – 6 ) from a photosensitized dimerization of FDMU, are obtained through the UV irradiation of FDMU with various benzophenones (BPs). The ratio of the two products (oxetanes to dimers) reveals that the two competitive reactions depend strongly on the triplet energy levels (E_T) of the BPs. The BPs with higher E_T values lead to higher proportions of dimers, whereas those with lower E_T values give higher proportions of oxetane(s), with the generation of just two regioisomeric oxetanes for the BP with the lowest E_T of the eight BPs investigated. The ratio of the two oxetanes ( 2 : 3 ) decreases with the BP E_T value. The competitive mechanism for the two types of photochemical reactions is demonstrated through quenching experiments and investigation of temperature effects. Kinetic analysis shows that the rate constants of the two [2+2] photocycloadditions are comparable. Furthermore, in combination with the results of previous studies, we have gained insight into the dependence of the photochemical type and the regioselectivity in the PB reaction on the triplet energy gaps (ΔE) between the pyrimidines and ketones. For ketones with higher E_T values than the pyrimidines, the photochemical reaction is a photosensitized dimerization of the pyrimidine. In the opposite case, a PB reaction occurs, and the lower the E_T of the ketones, the lower the ratio of oxetanes ( 2 : 3 ). When the E_T of values of the ketones are close to those of the pyrimidines, the two reactions occur concurrently, and the higher the E_T of the ketones, the higher the proportion of the dimers. The ratio of oxetanes ( 2 : 3 ) decreases with the E_T value of the BPs.     

PHOTOREVERSIBLE UV-INACTIVATION OF MESSENGER RNA IN AN INSECT EMBRYO (SMITTIA SPEC., CHIRONOMIDAE, DIPTERA)

Smittia embryos were UV-irradiated during intravitelline cleavage. At this stage, nuclei are heavily shielded by yolk-rich cytoplasm, and do not synthesize detectable amounts of RNA. Irradiation at 265, 285 and 295 nm wavelength caused biological inactivation, and pyrimidine dimer formation in maternal RNA as described earlier (Kalthoff, 1976; Jäckie and Kalthoff, 1978). In addition, we observed marked effects on protein synthesis: (1) The overall rate of [³⁵S]-methionine incorporation in vivo was reduced to less than half of the normal rate. (2) Two-dimensional gel electrophoresis revealed quantitative variations in the synthetic rate of some polypeptides, and the appearance of new ones in UV-irradiated embryos. (3) Translation of polyadenylated RNA from Smittia embryos in a cell-free system was inhibited by UV irradiation in vivo. (4) The apparent degradation, during early embryogenesis, of maternal polyadenylated RNA was retarded in UV-irradiated embryos. Exposure to light (400 nm) after UV caused partial photoreversal of all UV effects observed. Both the photoreactivable sector of UV-inactivation, and the photoreactivated portion of UV inhibition of protein synthesis, were correlated with the amounts of pyrimidine dimers generated in maternal RNA by UV irradiation at the three wavelengths used. These correlated effects were produced most efficiently by 295 nm radiation, indicating the involvement of photosensitizing components in the embryos. Our data show, for the first time to our knowledge, that animal mRNA, after UV irradiation, can be photoreactivated in vivo. Moreover, our results strongly suggest that the photorepairable lesions consist of pyrimidine dimers generated in a photosensitized reaction.     

PHOTOCHEMISTRY OF KETONES IN SOLUTION-49* A STUDY OF PHOTOSENSITIZED SPLITTING OF DIMETHYLTHYMINE DIMERS

Abstract— Several high energy ketone triplet sensitizers, e.g. carvone, camphor, 3-methylcyclohexanone, benzoin and 3-methylindanone, were studied as photosensitizers for the splitting of dimethylthymine dimers. The absence of splitting in all cases and the lack of quenching of benzoin and 3-methylindanone triplets by the trans-anti dimer of dimethylthymine strongly suggests that cleavage of dimethylthymine dimers cannot be achieved by a triplet mechanism on irradiation at Λ> 300 nm. The absence of optical rotation in the recovered chiral cis-anti dimethylthymine dimer after sensitized photolysis (12% splitting) in the presence of (—)-tryptophan suggests that. in highly polar solvents, such as methanol, where reaction probably takes place according to an electron transfer mechanism involving ion-pair intermediates, close approach of the sensitizer and substrate does not occur. To the extent that these results can be extrapolated to sensitized cleavage of cis-syn pyrimidine dimers in DNA brought about by action of photoreactivating enzyme or conventional photosensitizers, a mechanism involving dimer triplet states appears highly unlikely.     

INTRODUCTION OF PYRIMIDINE DIMERS INTO DIFFERENT INTRACELLULAR FORMS OF SIMIAN VIRUS 40

We examined the production of pyrimidine dimers by UV radiation in different intracellular forms of simian virus 40 DNA. Virus and chromatin or previrions were selectively labeled with [^l4C]-thymidine and [³H]-thymidine, respectively, in the same monolayer of infected cells. Viral DNA was extracted immediately after irradiation, and pyrimidine dimers were detected as sites sensitive to the UV-endonuclease encoded by bacteriophage T4. No difference in the number of dimers introduced into chromatin, previrions. or virions was detected.     

On the Formation of Thymine Photodimers in Thymine Single Strands and Calf Thymus DNA

Solar light leads to thymine dimers that are mutagenic and primary cause of skin cancer. Here, we report absorption and synchrotron radiation circular dichroism (CD) spectra of T_n single strands with different number n of bases (n = 2–7, 10, 11) recorded after various 254 nm irradiation times. From a principal component analysis of the CD spectra, we extract fingerprint spectra of both the cyclobutane pyrimidine dimer (CPD) and the pyrimidine (6‐4) pyrimidone photoadduct (64PP). Extending the CD measurements to the vacuum ultraviolet region in combination with systematic examinations of size effects is a new approach to gain insight on the dimeric photoproducts. We find a simple linear correlation between n and average number of dimers formed after 1 h of irradiation. The probability for a thymine to engage in a dimer increases from 32% for n = 2 to 41% for n = 11, which implies limited effects of terminal thymines, i.e., the reaction does not occur preferentially at the extremities of the single strands as previously stated. It is even possible to form two dimers with only two bridging thymines. Finally, experiments conducted on calf thymus DNA provided a similar signature of the photodimer, but differences are also evident.     

PHOTOREACTIVATION and EXCISION REPAIR OF UV INDUCED PYRIMIDINE DIMERS IN THE UNICELLULAR CYANOBACTERIUM GLOEOCAPSA ALPICOLA (SYNECHOCYSTIS PCC 6308)

Abstract— The survival curve obtained after UV irradiation of the unicellular cyanobacterium Synecho-cystis is typical of a DNA repair competent organism. Inhibition of DNA replication, by incubating cells in the dark, increased resistance to the lethal effects of UV at higher fluences. Exposure of irradiated cells to near ultraviolet light(350–500 nm) restored viability to pre-irradiation levels. In order to measure DNA repair activity, techniques have been developed for the chromatographic analysis of pyrimidine dimers in Synechocystis. The specificity of this method was established using a haploid strain of Sacchar-omyces cerevisiae. In accordance with the physiological responses of irradiated cells to photoreactivating light, pyrimidine dimers were not detected after photoreactivation treatment. Incubation of irradiated cells under non-photoreactivating growth conditions for 15 h resulted in complete removal of pyrimidine dimers. It is concluded that Synechocystis contains photoreactivation and excision repair systems for the removal of pyrimidine dimers.     

PHOTOREVERSIBLE PROTON DISSOCIATION AND ASSOCIATION IN PEA PHYTOCHROME AND ITS CHROMOPEPTIDES

Abstract— Effect of red-light irradiation on the medium pH at 10d?C was measured and compared among unbuffered solutions of the 121-kDa native pea (Pisum sativum cv. Alaska) phytochrome and its 114- and 62-kDa fragments in a red-light-absorbing form (P_r), all of which converted to far-red-light-absorbing form (Pf_r) on red-light irradiation. Red-light irradiation induced alkalinization in the solutions of the phytochrome and the fragments in the pH range 6.6-7.2 and 6.2-7.8, respectively. The amount of protons taken up by the 121-kDa phytochrome was less than one half of that of the 114-kDa fragment. Red-light irradiation induced acidification in the solutions of the 114- and the 62-kDa fragments above pH 7.8. In the solutions of the 121-kDa phytochrome, however, the irradiation induced no pH change at pH 7.2-8.2, and only a slight acidification at pH 8.2-8.7, which may be ascribed to a small amount of contamination from the 114-kDa fragment. All these red-light-induced pH changes were reversible following exposure to far-red light. The 7-kDa polypeptide(s) of the native 121-kDa phytochrome, which is lacking in the 114-kDa fragment, thus, prohibited proton transfer between phytochrome and the medium. A red-light-induced pH change was also measured in unbuffered solutions of the 39-kDa fragment of the phytochrome and of the 114-kDa fragment in the presence of 0.8 mM soyasaponin I. The 39-kDa fragment showed partially photoreversible conversion between a spectral form having an absorption maximum at 659 nm (P₆₅₉) and a bleached form, P_***. The 114-kDa fragment in the presence of the saponin showed a photoreversible conversion between P65V and Pb,. Exposure of P₆₅₉ from the 39-kDa fragment and from the 114-kDa fragment in the presence of the saponin to red light, caused acidification of the medium in the pH range 6.8-8.8 and 7.2-9.0, respectively, but no change at pH 6.2-6.8 and 6.4-7.2, respectively. The acidification of the latter was reversible following a far-red-light irradiation, but that of the former was only partially photoreversible. Proton uptake of phytochrome was inhibited by tryptic degradation to the 39-kDa fragment and also by the presence of the saponin. Only proton release was observed during the photoconversion from P₆₅₉ and P_***hl. It is suggested that a phytochrome molecule has possible site(s) for both proton release and for uptake and that the proton release reaction may be correlated to the photoconversion process(es) prior to the bleached intermediate (I_***) of phytochrome.     

FORMATION OF 5-HYDROXYMETHYLCYTOSINE-CONTAINING PYRIMIDINE DIMERS IN UV-IRRADIATED BACTERIOPHAGE T4 DNA

Cyclobutyl pyrimidine dimers composed of 5-hydroxymethylcytosine and thymine (5HMC>T dimer for a mutant of T4 ( denV ) that is unable to excise pyrimidine dimers from its DNA. The ability of 5HMC to form dimers suggests that other modified pyrimidines such as 5-methylcytosine can participate in dimer formation, particularly at the UV wavelengths in sunlight likely to be responsible for the induction of skin cancer.