PHOTOREACTIVATING ENZYME FROM Streptomyces griseus—VI. ACTION SPECTRUM AND KINETICS OF PHOTOREACTIVATION

Abstract— A high resolution action spectrum for photoreactivation was determined using purified photoreactivating enzyme from Streptomyces griseus. Conversion of pyrimidine dimers in UV-irradiated DNA, the substrate for photoreactivating enzyme, was measured with a Haemophilus influenzae transformation assay. A high similarity was found between action spectrum (max. at 445 nm) and the long wavelength absorption band (max. at 443 nm)of photoreactivating enzyme. In addition to the400–470 nm region considerable photoreactivation was found with wavelengths between 280 and 320 nm. No evidence was obtained for the presence of nonenzymatic photoreactivation. Comparison of in vitro and in vivo action spectra revealed that the sharp peak at 313 nm found in vivo is probably the result of counteracting photoreactivation and inactivation effects. Comparison of the action spectrum with the absorption spectrum of 8-hydroxy-10-methyl-5-deazaisoalloxazine in an aprotic dipolar solvent (which serves as a model for the 8-hydroxy-5-deazaflavin chromophore in photoreactivating enzyme) indicates the possible presence of other chromophore(s) involved in the photorepair process. From kinetic measurements and flash experiments values were obtained for the rate constants of the photoreactivation reaction. The quantum yield of photoreactivation was estimated to be approximately 1.     

PHOTOREACTIVATION OF Halobacterium halobium: ACTION SPECTRUM AND ROLE OF PIGMENTATION

Abstract— An action spectrum for photoreactivation was measured with Halobacterium halobium R₁m₁ to prove a role of carotenoid pigments in photoreactivation of the bacteria. The action spectrum obtained showed a main peak at 435 nm and a minor peak at about 325 nm. The action spectrum was similar to that of Streptomyces pigment (Eker et al. , 1981) suggesting that the chromophore of the photoreactivating enzyme in Halobacterium halobium is 8-OH-5-deazaflavin. The minor peak may be due to photochemical cleavage of a pyrimidine6–4 hetero adduct. The result indicates that carotenoid pigments do not play a positive role in enhancing photoreactivation. This was confirmed also by comparing the efficiency of photoreactivation at 465 nm among three strains of Halobacterium halobium having different carotenoid pigments; R₁m₁. R₁ and W5002–1.     

DESTRUCTION OF PHOTOREACTIVATING ENZYME BY 365 nm RADIATION*

Abstract— Following the observation that in vivo photoreactivation of 365-nm-induced pyrimidine dimers could not be observed chemically, a study was made of the inactivation of photoreactivating enzyme activity by this near-ultraviolet wavelength. It was observed that: (1) Dimers induced in extracted bacterial DNA by 365 nm radiation are completely photoreactivable and are monomerized as an exponential function of the photoreactivation time. (2) Photoreactivability of 254-nm-induced damage in Escherichia coli B/r Hcr is progressively destroyed in vivo as a function of the dose of 365 nm radiation. (3) The ability of the yeast photoreactivating enzyme to monomerize dimers induced at 365 nm in bacterial DNA is destroyed in vitro as a function of the dose of 365 nm radiation, and at a rate comparable to killing of E. coli. These results are consistent with biological measurements which indicate that photoreactivability of ultraviolet (near and far) lethal damage is reduced by exposure of the bacteria to 365 nm radiation.     

PHOTOREACTIVATION OF ULTRAVIOLET LIGHT-INDUCED DAMAGE IN CULTURED FISH CELLS AS REVEALED BY INCREASED COLONY FORMING ABILITY AND DECREASED CONTENT OF PYRIMIDINE DIMERS

Abstract— Cultured cells derived from a goldfish were irradiated with 254nm ultraviolet light. Cell survival and splitting of pyrimidine dimers after photoreactivation treatment with white fluorescent lamps were examined by colony forming ability and by a direct dimer assay, respectively. When UV-irradiated (5 J/m²) cells were illuminated by photoreactivating light, cell survival was enhanced up to a factor of 9 (40min) followed by a decline after prolonged exposures. Exposure of UV-irradiated (15 J/m²) cells to radiation from white fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence dependent manner, up to about 60% reduction at 120 min exposure. Keeping UV-irradiated cells in the dark for up to 120min did not affect either cell survival or the amount of pyrimidine dimers in DNA, indicating that there were not detectable levels of a dark-repair system in the cells under our conditions. Correlation between photoreactivation of colony forming ability and photoreactivation of the pyrimidine dimers was demonstrated, at least at relatively low fluences of photoreactivating light.     

DNA PHOTOREACTIVATING ENZYME FROM SILKWORM

An ammonium-sulfate-precipitable (33–70%) fraction in extracts from eggs of silkworm Bombyx mori contains photoreactivating enzyme that reactivates the transforming activity of UV inactivated Hemophilus influenzae DNA. The action spectrum for in vitro photoreactivation with the enzyme has a broad peak around 365–385 nm, with a shoulder extending to 460 nm. This relatively higher photoreactivation efficiency at wavelengths longer than 450 nm seems to be a unique feature of DNA photoreactivating enzyme of silkworm. Using gel filtration, a mol wt of 42,000 was estimated for the enzyme. Optimum and isoionic pH of the enzyme were 7.2 and 5.4, respectively. These properties of silkworm enzyme are within the range of variations in reported biochemical characteristics of photoreactivating enzymes from different species.     

Photoreactivation of cyclobutane dimers and (6-4) photoproducts in the epidermis of the marsupial, Monodelphis domestica

Radioimmunoassays were used to investigate the repair of cyclobutane pyrimidine dimers and pyrimidine (6-4)pyrimidone photoproducts ((6-4] photoproducts) in the epidermis of the South American opossum, Monodelphis domestica. In the absence of photoreactivating light, both types of photodamage were excised with similar kinetics, 50% of the damage remaining 8 h after UV irradiation in vivo. Exposure of UV-irradiated skin to photoreactivating light resulted in removal of most of the cyclobutane dimers and an enhanced rate of (6-4) photoproduct repair. Photoenhanced excision repair of non-dimer damage increases the range of biologically effective lesions removed by in vivo photoreactivation.     

PHOTOREACTIVATION OF THYMINE-STARVED ESCHERICHIA COLIBs-1

Abstract —Thymine starvation prior to 254 nm ultraviolet light (UV) exposures has been found to decrease the level of maximum photoreactivation in Escherichia coli B _s-1. The dark equilibrium level of photoreactivating enzyme-substrate complexes was determined from the levels of photoreactivation obtained with exposures to single flashes of high-intensity light. The kinetics indicate that photoreactivating enzyme concentration does not decrease as a result of thymine starvation. The UV sensitivities of normal and thymine-starved cells are found to be the same. Photoreactivation by sequential flashes shows a lesser number of total photorepairable lesions in starved cells. It is concluded that thymine starvation renders a portion of the dimers inaccessible to the photoreactivating enzyme, thus lowering the level of maximum photoreactivation.     

SUBSTRATE SPECIFICITY OF A BACTERIAL UV ENDONUCLEASE AND THE OVERLAP WITH IN VITRO PHOTOENZYMATIC REPAIR

Abstract— The action of an endonuclease from Micrococcus luteus , that operates on ultraviolet (UV) radiation damage, overlaps greatly with that of the yeast photoreactivating enzyme: homo and hetero cyclobutyl pyrimidine dimers in DNA are substrate for both enzymes, but pyrimidine adducts or the 'spore photoproduct' in DNA are not.
As expected from this overlap, the action of the two enzymes is mutually interfering: single-strand nicks introduced by the endonuclease effectively preclude photoreactivation; conversely, formation of a photoreactivating enzyme-dimer complex can prevent nicking by the UV endonuclease. While complex formation between photoreactivating enzyme and dimers in UV-endonuclease-treated DNA is apparently normal, the light-dependent repair step either fails to occur or proceeds at a very low rate. Hence, besides the requirement of a minimum chain length for the function of the photoreactivating enzyme, there is the additional restriction on the position of the dimer in a polynucleotide strand.
Finally, rough approximations of the rate constants, k ₁ and k ₂, for the UV endonuclease indicate that the in vitro UV-endonuclease-dimer complex is relatively unstable, with dissociation of the complex being more probable than hydrolysis of the phosphodiester bond.     

HOST CELL REACTIVATION IN MAMMALIAN CELLS-V. PHOTOREACTIVATION STUDIES WITH HERPES VIRUS IN MARSUPIAL AND HUMAN CELLS

Abstract— The survival of UV-irradiated herpes simplex virus was determined in cultured Potoroo (a marsupial) and human cells under lighting conditions which promote photoreactivation. Photoreactivation was readily demonstrated for herpes virus in two lines of Potoroo cells with dose reduction factors of 0.7-0.8 for ovan cells and 0.5-0.7 for kidney cells. Light from Blacklite (near UV) lamps was more effective than from Daylight (mostly visible) lamps, suggesting that near UV radiation was more efficient for photoreactivation in Potoroo cells. The quantitative and qualitative aspects of this photoreactivation were similar to those reported for a similar virus infecting chick embryo cells. UV-survhal curves for herpes virus in Potoroo cells indicated a high level of "dark" host cell reactivation. No photoreactivation was found for UV-irradiated vaccinia virus in Potoroo cells. A similar photoreactivation study was done using special control lighting (Λ > 600 nm) and human cells with normal repair and with ceils deficient in excision repair (XP). No photoreactivation was found for UV-irradiated herpes virus in either human cell with either Blacklite or Daylight lamps as the sources of photoreacti-vating light. This result contrasts with a report of photoreactivation for a herpes virus in the same XP cells using incandescent lamps.     

BIPHASIC PHOTOCHEMISTRY: THE FIRST ACTINOMETRIC REACTION ON A SOLID SUPPORT

Abstract Measurements were performed to determine the action spectrum and dose dependence for photoreactivation of E. coli B_s-1 cells after γ-irradiation. The similarities between photoreactivation after UV- and after γ-irradiation in action spectra, kinetics, and other characteristics indicate that the increased survival of γ-irradiated cells after illumination with photoreactivating light is the result of true photoenzymatic repair.     

PHOTOREACTIVATING ENZYME FROM NEUROSPORA CRASSA*

Abstract— Extracts of Neurospora crassa contain photoreactivating enzyme by the criteria of ability to split thymine-containing dimers and to increase the transforming ability of u.v.-irradiated Hemophilus influenzae DNA. The latter activity is heat-labile and is destroyed by trypsin. The action spectrum of such in vitro photoreactivation is a simple one (with a single maximum at 405 nm in the range 313 to 436 nm), differing from the more complicated in vitro spectra for yeast and Escherichia coli. However, the in vitro Neurospora spectrum coincides closely with the in vivo spectrum for this organism, suggesting that there is little or no “indirect” photoreactivation in Neurospora. It is concluded that the Neurospora photoreactivating enzyme is probably of a different type than those of yeast and Escherichia coli.     

PHOTOREACTIVATING ENZYME FROM STREPTOMYCES GRISEUS—III. EVIDENCE FOR THE PRESENCE OF AN INTRINSIC CHROMOPHORE

Evidence is presented that DNA photoreactivating enzyme from Streptomyces griseus consists of a high molecular protein part and a low molecular chromophore which is released by denaturation. The free chromophore is highly fluorescent and has an absorption maximum at 420 nm. In native photoreactivating enzyme the chromophore fluorescence is almost completely quenched and there is an additional absorption band at 445 nm. Native photoreactivating enzyme spontaneously looses its chromophore following first order kinetics as measured by the increase of fluorescence intensity. A good correlation was found between the increase of fluorescence intensity and the decrease of biological activity, stressing the importance of the chromophore-protein bond. The presence of DNA greatly retards the spontaneous release of chromophore, and with UV-irradiated DNA the photoreactivating enzyme is almost completely stable. In five different chromatographic systems, cochromatography of biological activity and enzyme-bound chromophore was found, thus ruling out the possibility that the observed chromophore belongs to a contamination in the enzyme preparation. Photoreactivating enzyme binds very strongly to Blue-Sepharose indicating the presence of a positive charge in the polynucleotide binding site.     

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.     

THE ACTION SPECTRUM OF AN IN VITRO DNA PHOTOREACTIVATION SYSTEM

Abstract— The wavelength-dependence of in vitro photoreactivation of transforming DNA by yeast extract has been determined. There is an intensity-dependent lag at the beginning of the biological reaction. There is a similar lag in the splitting of thymine dimers by the yeast extract in the light, a process known to account for most or all of the increase in transforming activity of photoreactivated DNA. The most efficient wavelengths for photoreactivation are around 3550 and 3850 Å. Although the action spectrum is not very similar to flavin absorption, riboflavin at very low concentration inhibits photoreactivation, as it also inhibits a number of flavoenzymes, suggesting that the photoreactivating enzyme might be a flavoprotein.     

LOSS OF PHOTOREACTIVATION IN UV-IRRADIATED CULTURED FISH CELLS UNDER DIFFERENT CONDITIONS

Abstract— CAF-MM1 cells derived from a goldfish have photoreactivability for the damage induced by ultraviolet light (UV). When UV-irradiated cells were incubated in the dark at 26_AoC, the longest interval in which photoreactivation (PR) was observed (i.e. effective time for PR), measured by colony formation technique, was about 30 h after the UV irradiation. However, if the cells were incubated at 20_AoC, the effective time was prolonged. Since each time appeared to correspond to the doubling time of the cells at each temperature, the loss of photoreactivability is suggested to be closely related to cell growth or progression of cell cycle. The loss of PR was not observed in the cells held in confluence up to 48 h after UV irradiation, in support of the above suggestion. Photoreactivating enzyme in growing CAF-MM1 cells incubated in the dark for 24 h after UV irradiation was shown to be active, so that it is not possible that the cause of the loss of PR is change in the activity of photoreactivating enzyme.     

DETECTION OF CYCLOBUTANE THYMINE DIMERS IN DNA OF HUMAN CELLS WITH MONOCLONAL ANTIBODIES RAISED AGAINST A THYMINE DIMER- CONTAINING TETRANUCLEOTIDE

Abstract— A hybrid cell line (hybridoma) has been isolated after fusion between mouse-plasmacytoma cells and spleen cells from mice immunized with a thymine dimer-containing tetranucleotide coupled to a carrier protein. Monoclonal antibodies produced by this hybridoma were characterized by testing the effect of various inhibitors in a competitive enzyme-linked immunosorbent assay (ELISA). The antibodies have a high specificity for thymine dimers in single-stranded DNA or poly(dT), but do not bind UV-irradiated d(TpC)₅. Less binding is observed with short thymine dimer-containing sequences. In vitro treatment of UV-irradiated DNA with photoreactivating enzyme in the presence of light, or with Micrococcus luteus UV-endonuclease results in disappearance of antigenicity. Antibody-binding to DNA isolated from UV-irradiated human fibroblasts (at 254 nm) is linear with dose. Removal of thymine dimers in these cells during a post-irradiation incubation, as detected with the antibodies, is fast initially but the rate rapidly decreases (about 50% residual dimers at 20 h after 10 J/m²). The induction of thymine dimers in human skin irradiated with low doses of UV-B, too, was demonstrated immunochemically, by ELISA as well as by quantitative immunofluorescence microscopy.     

Action spectra for photoreactivation of mutation of prototrophy in strains of Escherichia coli possessing and lacking photoreactivating-enzyme activity

Abstract— The action spectrum and dose-rate dependence for photoreactivation of mutation to prototrophy in late-lag-phase cultures of Escherichia coli H³r30 (which lacks active photo-reactivating enzyme) are roughly similar to those for photoprotection from killing in other strains. It is suggested that photoreactivation of this mutation in H/r30 is an indirect effect, similar in mechanism to photoprotection. The action spectrum and dose-rate dependence for photoreactivation of mutation to prototrophy in late-lag-phase cultures of E. coli H³r30-R (which possesses active photoreactivating enzyme) are roughly similar to those for photoreactivation of killing in most other strains. It is suggested that photoreactivation of this mutation in H/r30-R is a direct effect at long wavelengths, but that there is an indirect component at short wavelengths. A quite different interpretation of these data is noted. Finally, it is found that, under the conditions of these experiments, indirect photoreactivation of killing in H/r30 and H/r30-R is weak or nonexistent.     

PHOTOREACTIVATION OF DNA-CONTAINING CAULIFLOWER MOSAIC VIRUS AND TOBACCO MOSAIC VIRUS RNA ON DATURA

Abstract— Datura stramonium L. is a local lesion host for TMV-RNA and DNA-containing cauliflower mosaic virus (CAMV). Datura can photorepair UV-damaged TMV-RNA and CAMV, giving photoreac-tivation sectors of 0.40 and 0. 33 , respectively. Dose response curves for photoreactivation of TMV-RNA and CAMV show that 4540 min of cool white light (15 W.m^-2) is required for maximum photoreactivation. Blue light and near UV are equally effective in photoreactivating UV-irradiated TMV-RNA, whereas near UV is initially more effective than blue light for the photorepair of UV-inactivated CAMV. Higher doses of near UV apparently inactivate the CAMV photorepair system. In the case of CAMV, photoreactivating light must be applied immediately after inoculation with the virus. Two to three hours of incubation in the dark after inoculation results in complete loss of response to photoreactivating irradiation. In contrast, limited photoreactivation of TMV-RNA occurs even after 4 h of dark incubation after inoculation, although photoreactivating irradiation is most effective when applied immediately after inoculation. Light is required for the maintenance of photoreactivation for both TMV-RNA and CAMV. Daturas placed in the dark for six days lose their ability to photoreacti-vate. Recovery of the TMV-RNA photorepair system is rapid; complete recovery attained with 90 or more min of white light (15 W m-'). Recovery of CAMV photorepair system is slow; 90% recovery attained after only 20 h of light. However, full recovery can be induced by as little as 6h of light when CAMV is inoculated 24 h after the onset of illumination. These results suggest two photorepair systems are present in Datura .     

A sequential repair model of photoreactivation in bacteria

Abstract— Kinetics of photoreactivation were studied in E. coli WP2 hcr^-, a strain deficient in dark repair. Cells in aqueous suspension were subjected to u.v.-irradiation, then exposed to photoreactivating light for different periods. Survival curves, with samples at a minimum of six u.v. doses, were obtained at several periods of photoreactivation ranging from zero to maximum. The surviving fractions do not conform to a dose-reduction model, but instead, they fit a ‘sequential repair’ model that assumes as a limiting condition that the number of active enzyme molecules is small. The model used assumed: (1) a single enzyme molecule is active at any one time; and (2) inactivating events are nullified consecutively around the DNA molecule. The mathematics of the model are derived and presented. Photoreactivation is attributed to the action of two processes. (1) A photochemical process, that is rate limiting below 1000 ergs mnr^-2 sec^-1, was measured at a photoreactivating irradiance of 60 ergs mm^-2 sec^-1. This has a rate constant of 5 × 10^-5‘events’ erg^-1 mm². (2) A dark process, measured at photoreactivating irradiances of 4000 and 6000 ergs mm^-2 sec^-1, has a rate constant of 2.2 ‘events’ min^-1.     

Photoreactivation in the yeast Schizosaccharomyces pombe

Abstract— Visible light (VL) illumination of u.v.-irradiated cells of the fission yeast Schizosaccharomyces pombe does not increase the survival of wild-type cells, but does increase the survival of some specific UVS strains. This photoreactivation has been studied in the U VS 1,₁ mutant in the stationary growth phase.

• 1 It is not dependent on temperature during VL illumination.
• 2 The effect of pre-u.v. or post-u.v. illumination on survival is the same.
• 3 There is an overlap of photoreactivation and liquid holding recovery.
• 4 VL does increase the growth delay after irradiation. It is concluded from these results that the photoreactivation is not due to a photoreactivating enzyme, but to an indirect process. The existence in this yeast of two different repair pathways of u.v. lesions has been demonstrated previously. The study of indirect photoreactivation in different strains, blocked in one or the other repair pathway by mutation or by a repair inhibitor (caffeine), leads to the conclusion that the VL treatment favours only one of these two repair mechanisms, which is presumably the excision-repair pathway. The strain UVS A, which would repair u.v. lesions by a recombinational mechanism, does not show any photoreactivation.