Transient reduction in the tRNA 4-thiouridine content induced by ultraviolet A during post-irradiation growth in Enterobacter cloacae

The influence of previous exposure to ultraviolet-A radiation (UVA) was studied on the susceptibility of Enterobacter cloacae to undergo the growth delay effect. Comparison of growth curves corresponding to irradiated and control cells showed that a previous treatment with UVA almost abolished the growth delay effect. UV absorption spectra of tRNA, and reverse phase HPLC analysis of hydrolysed tRNA, demonstrated a low content of 4-thiouridine in E. cloacae cells grown after UVA exposure at low doses. Since 4-thiouridine is the UVA target responsible for initiation of growth delay, this observation explained the influence of previous exposure to UVA on the susceptibility of this organism to undergo growth delay. A similar but weaker alteration was found when Escherichia coli was assayed. The results suggest that, in addition to cross-linking with cytidine residues, the content of 4-thiouridine in tRNA may be modified by UVA by an unknown mechanism.     

ROLES OF THE RELA+ GENE AND OF 4-THIOURIDINE IN NEAR-ULTRAVIOLET (334 nm) RADIATION INHIBITION OF INDUCED SYNTHESIS OF TRYPTOPHANASE IN ESCHERICHIA COLI B/r

Abstract— Near-ultraviolet radiation (near UV; 300–380 nm) is known to inhibit the induced synthesis of tryptophanase by tryptophan in Escherichia coli , showing an action spectrum similar to that for near-UV-induced growth delay. The present work shows that a rel A mutant of E. coli B/r exhibits 50% as much monochromatic near-UV (334 nm) inhibition of tryptophanase induction as the wild type, and that a mutant lacking 4-thiouridine, an unusual nucleoside in tRNA, exhibits < 10% as much inhibition of tryptophanase induction. These findings indicate that 4-thiouridine is almost the sole chromophore for this effect in E. coli B/r, but that only 50% of the effect operates by a mechanism utilizing the rel A ⁺ gene product; growth delay appears not to be primarily involved.     

BASE-SPECIFIC DAMAGE INDUCED BY 4-THIOURIDINE PHOTOSENSITIZATION WITH 334-nm RADIATION IN M13 PHAGE DNA

Abstract— Site-specific DNA damage caused by 334-nm radiation in the presence of the rare Escherichia coli base 4-thiouridine was investigated in vitro by detecting the sites of the termination of DNA synthesis with irradiated M13 phage DNA used as a template. Single-strand breakage was also examined. The results indicate that 334-nm radiation at very low fluences in the presence of 4-thiouridine induces termination of strand synthesis at thymine base sites and at the base immediately prior to thymine. Termination at these sites was diminished by treatment with hot piperidine. Strand cleavage by piperidine treatments was observed preferentially at the guanine site, but only after irradiation at much larger fluences. It is hypothesized that at low fluences 4-thiouridine forms photoadducts with thymine that block DNA synthesis, while at high fluences the guanine site is damaged via oxygen species.     

DNA BREAKAGE CAUSED BY 334-nm ULTRAVIOLET LIGHT IS ENHANCED BY NATURALLY OCCURRING NUCLEIC ACID COMPONENTS AND NUCLEOTIDE COENZYMES

Abstract— The induction of breaks in DNA in vitro caused by 334-nm UV radíation is enhanced by the following compounds (fluence enhancement factors and concentrations used in parentheses): 4-thiouridine (6.9, 1 m M ), 5-methylamino-2-thiouridine (7.5, 1 m M ), 2-thiouracil (41.0, 1 m M ), riboflavin (14.4.0.1 m M ), and the oxidized (6.8, 1 m M ) and reduced (3.4, 1 m M ) forms of nicotinamide adenine dinucleotide. Anoxia and diazobicyclo(2.2.2)octane reduce the number of DNA breaks caused by 334-nm radiation plus 4-thiouridine by 70 and 76%, respectively.     

PHOTOCHEMISTRY OF 4-THIOURIDINE AND THYMINE

Abstract— When thymine is irradiated in aqueous solution with monochromatic 334-nm UV radiation in the presence of 4-thiouridine a photoproduct of thymine is formed, as shown by thin-layer chromatography and autoradiography. The quantum yield for the formation of thymine photoproduct (θ=0.017) is greater than that for cytosine photoproduct formation (θ= 0.0015). The identity of the photoproduct is not known: one possibility is the formation of an adduct between the sensitizer and the base yielding a pyrimidine-pyrimidone type of photoproduct.     

Generation of thiyl radicals by the photolysis of 5-iodo-4-thiouridine

The photochemistry of 2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine (3) in deoxygenated 1:1 CH(3)CN-H(2)O pH 5.8 (phosphate buffer) solution has been studied by means of steady-state and nanosecond laser flash photolysis methods. Under steady-state irradiation (lambda > or = 334 nm), the stable photoproducts were iodide ion, 2',3',5'-tri-O-acetyl-4-thiouridine (4), and two disulfides. The disulfides were the symmetrical bis-(2',3',5'-tri-O-acetyl-5-iodo-4-thiouridine) (5) and unsymmetrical 6, which contains both 4-thiouridine and 5-iodo-4-thiouridine residues. The formation of the dehalogenated photoproduct suggests that C(5)-I bond cleavage is a primary photochemical step. Attempts to scavenge the resulting C(5)-centered radical by suitable addends, bis-(N-alpha-acetyl)cystine-bis-N-ethylamide or benzene, were unsuccessful. Analysis of the photoproducts formed under these conditions showed that the S-atom is the reactive center. The photoproduct 4, obtained by irradiation of 3 in CD(3)CN-H(2)O, followed by reversed-phase HPLC isolation using nonlabeled eluents, did not contain deuterium. An analogous experiment performed in CH(3)CN-D(2)O gave deuterated product 4-d with 88% of the deuterium incorporated at C(5). Transient absorption observed upon laser excitation (lambda= 308 nm) of 3 was assigned to the 4-uridinylthiyl radical on the basis of the similarity of this spectrum with that obtained upon laser photolysis of the disulfide: bis-(2',3',5'-tri-O-acetyl-4-thiouridine) 14. On the basis of the results of steady-state and laser photolysis studies, a mechanism of the photochemical reaction of 3 is proposed. The key mechanistic step is a transformation of the C(5)-centered radical formed initially by C(5)-I bond cleavage into a long-lived S-centered radical via a 1,3-hydrogen shift. Theoretical calculations confirmed that the long-lived S-centered radical is the most stable radical derived from the 4-thiouracil residue.     

A5–4 PYRIMIDINE-PYRIMIDONE PHOTOPRODUCT PRODUCED FROM MIXTURES OF THYMINE AND 4-THIOURIDINE IRRADIATED WITH 334 nm LIGHT

The nucleoside 4-thiouridine, present in some bacterial tRNA species, is known to be a chromophore and a target for near-UV light-induced growth delay and also mediates both photoprotection and near-UV cell killing in various bacterial strains. To investigate the photoreaction of 4-thiouridine with DNA or its precursors, we irradiated aqueous mixtures of thymine and 4-thiouridine with 334 nm light and then separated photoproducts using two or more stages of reversed-phase high performance liquid chromatography. The two equally abundant major photoproducts were analyzed by UV absorbance spectrophotometry, fast-atom bombardment and electron-impact mass spectrometry, and ¹H- and ¹³C-NMR spectroscopy, and have been identified as two diastereomers of 6-hydroxy-5-[1-(β-D-erythro-pentofuranosyl)-4′-pyrimidin-2′-one]dihydrothymine (o⁶hThy[5-4]Pdo), of molecular weight = 370.32. These two diastereomers, although stable at room temperature or below, are interconvertible by heating (90d?C for 5 min) in aqueous solution. The possible biological significance of this photoproduct is discussed, and an application as a crosslinker for oligonucleotides to selectively block replication is suggested.     

PHOTOHEMOLYSIS SENSITIZED BY PSORALEN: RECIPROCITY LAW IS NOT FULFILLED

The effect of the intensity of ultraviolet-A (UV-A) radiation (366 nm) on delayed photohemolysis sensitized by psoralen (PUV-A hemolysis) was studied. It was shown that PUV-A hemolysis induced by UV-A radiation at low fluence rate (20 W m-2) develops according to the well-known colloid-osmotic mechanism: there was no threshold dose of PUV-A treatment. After irradiation all the cells were hemolysed. The rate of PUV-A hemolysis was proportional to the square of the fluence. Hemolysis was delayed in the presence of sucrose. When the fluence rate of UV-A radiation was increased to 150 W m-2, the character of PUV-A hemolysis changed drastically. A threshold fluence appeared, below which PUV-A hemolysis was not induced. At fluences slightly exceeding the threshold, only part of the cells in the suspension were lysed. The dependence of the portion of hemolysing cells on fluence was S-shaped. Increasing the fluence resulted in complete (100%) hemolysis. The rate of complete hemolysis decreased at higher fluences, but was many-fold higher than the rate of low-intensity PUV-A hemolysis at equal fluences. The main features of high intensity PUV-A hemolysis (dependences on fluence and temperature, effect of sucrose) were the same for the hemolysis induced by the addition of previously photooxidized psoralen. We suggest that high intensity PUV-A hemolysis is induced with participation of photooxidized psoralen as an intermediate.     

Adaptation to UVA radiation of E. coli growing in continuous culture

Adaptive responses of bacteria to physical or chemical stresses in the laboratory or in the environment are of great interest. Here we investigated the ability of Escherichia coli growing in continuous culture to adapt to UVA radiation. It was shown that E. coli indeed expressed an adaptive response to UVA irradiation at an intensity of 50W/m(2). Cells grown in continuous culture with complex medium (diluted Luria Bertani broth) at dilution rates of 0.7h(-1), 0.5h(-1) and 0.3h(-1) were able to maintain growth under UVA irradiation after a transient reduction of specific growth rate and recovery. In contrast, slow-growing cells (D=0.05h(-1)) were unable to induce enough protection capacity to maintain growth under UVA irradiation. We propose that faster growing E. coli cells have a higher adaptive flexibility to UVA light-stress than slow-growing cells. Furthermore it was shown with flow cytometry and viability stains that at a dilution rate of 0.3h(-1) only a small fraction (1%) of the initial cell population survived UVA light-stress. Adapted cells were significantly larger (30%) than unstressed cells and had a lower growth yield. Furthermore, efflux pump activity was diminished in adapted cells. In a second irradiation period (after omitting UVA irradiation for 70h) adapted cells were able to trigger the adaptive response twice as fast. Additionally, this study shows that continuous cultivation with direct stress application allows reproducible investigation of the physiological and possibly also molecular mechanisms during adaptation of E. coli populations to UVA light.     

INTRAMOLECULAR ENERGY TRANSFER IN NATIVE tRNA AT ROOM TEMPERATURE

Abstract— The odd nucleoside 4-thiouridine, which is present in position 8 of 70% of E. coli tRNAs, possesses unusual spectroscopic properties which make it suitable for intramolecular energy transfer studies. Both its luminescence excitation spectrum and the action spectrum (230–380 nm) for the 8–13 link formation have been established in native E. coli tRNA at room temperature. The spectra are identical and present a new unexpected peak around 260 nm. At this wavelength, they are amplified by a factor of nine as compared with the absorption and excitation spectra of the free nucleoside in aqueous solution.
The origin of this new peak is discussed and it is concluded that energy transfer does occur from the common nucleosides to the 4-thiouridine residue. Using the values of the nucleosides to 4-thiouridine distances inferred from the sets of atomic coordinates obtained on yeast tRNA^phe crystals, a satisfactory account of our finding can be obtained assuming singlet-singlet energy transfer. The efficiency of the mechanism is probably favoured by a good overlap between the emission spectra of the common nucleosides and the absorption spectrum of 4-thiouridine.     

ACTION SPECTRUM FOR GROWTH DELAY INDUCED BY NEAR-ULTRAVIOLET LIGHT IN E. coli B/r K*

Abstract— The action spectrum for growth delay induced by near-uv light was determined for Escherichia coli B/r growing in a defined medium. This spectrum agrees with and extends that determined earlier by Jagger and his co-workers for E. coli B growing in nutrient broth. The extended spectrum is indistinguishable from the absorption spectrum for 4-thiouridine above 320 nm, but deviates significantly at wavelengths shorter than this from the spectrum for 8–13 link formation in transfer RNA containing 4-thiouridine at position 8. These results extend the evidence that 4-thiouridine in transfer RNA is the chromophore for near-UV induction of growth delay, but weaken the case for linkage of a pyrimidine at position 13 in transfer RNA as the mechanism of growth delay.     

Attenuation of DNA damage in the dermis and epidermis of the albino hairless mouse by chronic exposure to ultraviolet-A and -B radiation

Mammalian skin is vulnerable to the photocarcinogenic and photoaging effects of solar UV radiation and defends itself using a variety of photoprotective responses including epidermal thickening, tanning and the induction of repair and antiradical systems. We treated Skh-1 albino hairless mice for 60 days with ultraviolet-A (UVA) or ultraviolet-B (UVB) radiation and measured the frequency of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by a single acute sunburn dose of UVB at different stages of the chronic treatment. We found that both UVA and UVB exposure produced a photoprotective response in the dermis and epidermis and that the degree of photoproduct attenuation was dependent on dose, wavelength and the type of damage induced. Although epidermal thickening was important, our data suggest that UV protective compounds other than melanin may be involved in mitigating the damaging effects of sunlight in the skin.     

The Dewar valence isomer of the (6-4) photoadduct of thymidylyl-(3'-5')-thymidine monophosphate: formation, alkaline lability and conformational properties.

The formation of the Dewar valence isomer of the pyrimidine(6-4)pyrimidone photoadduct of thymidylyl-(3'-5')-thymidine monophosphate (TpT) was investigated under different irradiation conditions. This photoproduct was generated on exposure of TpT to far-UV radiation. However, no detectable amount of the Dewar isomer or its precursor (pyrimidine(6-4)pyrimidone photoadduct) was observed following acetone photosensitization of TpT. The Dewar valence isomer was much more unstable than the pyrimidine(6-4)pyrimidone photoproduct when treated with hot piperidine. A detailed conformational analysis of the TpT Dewar isomer photoproduct is reported as inferred from extensive one- and two-dimensional 300 and 620 MHz proton nuclear magnetic resonance (1H NMR) measurements and molecular mechanics calculations.     

DNA SYNTHESIS-KINETICS, CELL DIVISION DELAY, AND POST-REPLICATION REPAIR AFTER UV IRRADIATION OF FROZEN CELLS OF E. COLI B/r

Abstract— Previous studies have shown that the relative yields of photoproducts produced in the DNA of Escherichia coli cells UV irradiated at -79°C differ from those produced at +21°C; the yield of DNA-protein cross-links was markedly enhanced at -79°C while the yield of thymine dimers was reduced. In the present studies, cells of E. coli B/r thy were frozen at -79°C, and then UV irradiated (254nm) while frozen(4.7 J m^-2), or after thawing (22 Jm^-2). Essentially the same survival, cell division delay, and DNA synthesis kinetics were observed for these two samples after irradiation, even though the UV fluence differed by a factor of ˜5. This supports previous observations that a correlation exists between the magnitude of the effects of UV radiation upon DNA synthesis kinetics and on cell survival. The weight average molecular weight of the pulse labeled DNA in the sample irradiated at +21°C was one-half that of the sample irradiated at -79°C, and complete repair of daughter-strand gaps was observed in both cases. Thus, UV-induced lesions produced in cells at -79°C (i.e. DNA-protein cross-links) appear to be amenable to post-replicational repair. While the overall DNA synthesis kinetics were the same for the two irradiation procedures, the apparent number of lesions produced per unit length of DNA was not. This suggests that each of the lesions produced in frozen cells, although apparently fewer in number, must cause a longer local delay in DNA synthesis than those lesions produced at +21°C.     

A monochromatic action spectrum for the photoinduction of the UV-absorbing mycosporine-like amino acid shinorine in the red alga Chondrus crispus

To determine the action spectrum for photoinduction of the ultraviolet (UV)-absorbing mycosporine-like amino acid shinorine, specimens of the marine red alga Chondrus crispus were irradiated with monochromatic light of various wavelengths using the Okazaki large spectrograph at the National Institute for Basic Biology, Okazaki, Japan. Fluence response curves were determined for the wavelengths between 280 and 750 nm, by irradiating the algae with monochromatic light for 10 h, followed by 4 h of 25 micromol m(-2) s(-1) photosynthetically active radiation and 10 h darkness. Samples were taken after the second exposure interval. A linear correlation between fluence rate and accumulated shinorine concentration was detected for wavelengths between 350 and 490 nm in the fluence rate range of 20-30 micromol m(-2) s(-1), whereas there was no induction above 490 nm. Below 350 nm a decline in shinorine concentration could be observed at fluence rates above 30 micromol m(-2) s(-1), probably due to an inhibition of photosynthetic activity and a subsequent impairment of shinorine biosynthesis. The constructed action spectrum indicated that the photoreceptors mediating shinorine photoinduction might be an unidentified UV-A-type photoreceptor with absorption peaks at 320, 340 and 400 nm.     

INCREASED LEVELS OF UNSCHEDULED DNA SYNTHESIS IN UV-IRRADIATED HUMAN FIBROBLASTS PRETREATED WITH SODIUM BUTYRATE

Pretreatment of growing normal and xeroderma pigmentosum (XP) human fibroblasts with sodium butyrate at concentrations of 5-20 m M results in increased levels of DNA repair synthesis measured by autoradiography after exposure of the cells to 254 nm UV radiation in the fluence range 0-25 J/m². The phenomenon manifests as an increased extent and an increased initial rate of unscheduled DNA synthesis (UDS). This experimental result is not due to an artifact of autoradiography related to cell size. Xeroderma pigmentosum cells from complementation groups A, C, D and E and XP variant cells all exhibit increases in the levels of UV-induced UDS in response to sodium butyrate proportional to those observed with normal cells. These UDS increases associated with butyrate pretreatment correlate with demonstrable changes in intracellular thymidine pool size and suggest that sodium butyrate enhances uptake of exogenous radiolabeled thymidine during UV-induced repair synthesis by reducing endogenous levels of thymidine.     

THE BIOLOGY OF THE (6–4) PHOTOPRODUCT

The (6-4) photoproduct is an important determinant of the lethal and mutagenic effects of UV irradiation of biological systems. The removal of this lesion appears to correlate closely with the early DNA repair responses of mammalian cells, including DNA incision events, repair synthesis and removal of replication blocks. The processing of (6-4) photoproducts and cyclobutane dimers appears to be enzymatically coupled in bacteria and most mammalian cell lines examined (i.e. a mutation affecting the repair of one lesion also often affects the other), although exceptions exist in which repair capacity may be evident for one photoproduct and not the other (e.g. UV61 and the XP revertant cell line). These differences in the processing of the two photoproducts in some cell lines of human and rodent origin suggest that in mammalian cells, different pathways for the repair of (6-4) photoproducts and cyclobutane dimers may be used. This observation is further supported by pleiotropic repair phenotypes such as those observed in CHO complementation class 2 mutants (e.g., UV5, UVL-1, UVL-13, and V-H1). Indirect data, from HCR of UV irradiated reported genes and the cytotoxic responses of UV61, suggest that the (6-4) photoproduct is cytotoxic in mammalian cells and may account for 20 to 30% of the cell killing after UV irradiation of rodent cells. Cytotoxicity of the (6-4) photoproduct may be important in the etiology of sunlight-induced carcinogenesis, affecting mutagenesis as well as tumorigenesis. The intricate photochemistry of the (6-4) photoproduct, its formation and photoisomerization, is in itself extremely interesting and may also be relevant to sunlight carcinogenesis. The data reviewed in this article support the notion that the (6-4) photoproduct and its Dewar photoisomer are important cytotoxic determinants of UV light. The idea that the (6-4) photoproduct is an important component in the spectrum of UV-induced cytotoxic damage may help clarify our understanding of why rodent cells survive the effects of UV irradiation as well as human cells, without apparent cyclobutane dimer repair in the bulk of their DNA. The preferential repair of cyclobutane dimers in essential genes has been proposed to account for this observation (Bohr et al., 1985, 1986; Mellon et al., 1986). The data reviewed here suggest that understanding the repair of a prominent type of noncyclobutane dimer damage, the (6-4) photoproduct, may also be important in resolving this paradox.     

Synthesis of the TT pyrimidine (6-4) pyrimidone photoproduct-thio analogue phosphoramidite building block

The phosphoramidite building block synthesis of the thio analogue at the 5,6-dihydropyrimidine C5 position of the thymidylyl(3'-5')thymidine (6-4) photoproduct 1 is presented. This compound was readily obtained from the appropriately protected dinucleotide P-methyl-5'-O-dimethoxytritylthymidilyl(3' --> 5')-4-thiothymidine 2 after irradiation at 366 nm, then S-sulfenylmethylation of the thiol function of the resulting (6-4) adduct, and phosphitylation of the 3'-hydroxyl group.     

RECOVERY OF Escherichia coli K-12 FROM NEAR-ULTRAVIOLET RADIATION-INDUCED MEMBRANE DAMAGE

Abstract A wild-type Escherichia coli K-12 strain was irradiated with broad-band near-ultraviolet radiation (from Black-Light Blue fluorescent lamps) and after holding at 37°C for various times in a complex recovery medium, was assessed for viability on either complex medium (YENB) or minimal medium containing a high inorganic salt content. A near-ultraviolet radiation fluence was used which reduced the surviving fraction to approximately 10% when assessing for viability on the complex medium plates. A near-ultraviolet radiation induced sensitivity to inorganic salt was observed which was largely recoverable by holding treated cells in a complex recovery medium. The majority of the recovery process occurred in the initial 2 h post-irradiation holding period. No inhibition of the recovery process was produced by adding chloramphenicol (40 μg/m l ) or penicillin G (11 units/m l ) to the recovery medium, indicating that neither protein synthesis nor cell wall synthesis, respectively, were required for recovery. However, by adding bacitracin, an antibiotic which acts in part by inhibiting membrane synthesis, to the recovery medium, an effect on recovery from salt sensitivity was observed. At the concentrations of bacitracin used (0.6 and 0.2 units/m l ), little or no effect was observed on unirradiated cells, but both concentrations decreased the amount of recovery of irradiated cells. These results demonstrate that recovery from near-ultraviolet radiation-induced salt sensitivity occurs, it is independent of cell growth and the effect of bacitracin suggests that membrane synthesis may be required for recovery.