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 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.     

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.     

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.     

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.     

Direct demonstration of the monomerization of thymine-containing dimers in U.V.-irradiated DNA by yeast photoreactivating enzyme and light

Abstract— Ultraviolet-irradiated E. coli DNA (³H-thymine-labelled) was mixed with un-irradiated E. coli DNA (¹⁴C-thymine-labelled) and exposed to light in the presence of purified yeast photoreactivating enzyme. As the ³H-thymine-containing cyclobutane dimers disappeared during the photoreactivation, there was a stoichiometric increase of monomeric ³H-thymine as determined from the ³H/¹⁴C ratio in thymine. This is the first direct demonstration that thymine-containing dimers in u.v.-irradiated DNA are monomerized by yeast photoreactivating enzyme in the presence of light.     

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.     

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Abstract— Photoreactivation in the extreme halophilic archaebacterium Halobacterium cutirubrum was studied both in vivo and in vitro. Cells irradiated with ultraviolet (UV)-fluences up to 350 J/m² could be completely photoreactivated, indicating very efficient repair of pyrimidine dimers in UV-irradiated DNA. Dark repair is apparently absent in Halobacterium since liquid holding under non-growth conditions did not influence the survival of UV-irradiated cells, while cells remained completely photoreactivable with no change in the kinetics of photoreactivation. Experiments with Halobacterium isolates of different carotenoid content indicated that carotenoids do not influence either UV-inactivation or photoreactivation. Small differences in the rates of UV-inactivation and photoreactivation could be assigned to the occurrence of gas vesicles. Flash experiments and the temperature dependence of photoreactivation indicated an enzymatical reaction. This was confirmed by in vitro experiments with partially purified photoreactivating enzyme. The in vivo action spectrum of photoreactivation showed a main band in the 400-470 nm region with a maximum at 440 nm. Comparison with action spectra of other microorganisms classified the Halobacterium enzyme as a 8-hydroxy-5-deazaflavin type photoreactivating enzyme.     

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.     

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.

     

PHOTOREACTIVATION OF KILLING INE. coliK–12 phr- CELLS IS NOT CAUSED BY PYRIMIDINE DIMER REVERSAL

Abstract— Escherichia coli K–12 strains deleted in the phr gene which encodes the apoenzyme of photolyase show significant residual photoreactivation activity after exposure to high photoreactivating light doses. We show that this photoreactivation of killing is not associated with reversal of pyrimidine dimers and conclude that the effect is probably attributable to the direct photoconversion of the6–4 photoproducts to their Dewar isomers (Type III photoreactivation).     

PHOTOREACTIVATING ENZYME FROM ESCHERICHIA COLI

Abstract— Escherichia coli photoreactivating enzyme (PRE) has been purified in large amounts from an E. coli strain lysogenic for a defective Λ bacteriophage carrying the phr gene. The resulting enzyme has a pH optimum of 7.2 and an ionic strength optimum of 0.18. It consists of an apoprotein and cofactor, both of which are necessary for catalytic activity. The apoprotein has a monomer molecular weight of 35 ,200 and shows stable aggregates under denaturing conditions. The amino acid analysis of the E. coli enzyme is very similar to that of the photoreactivating enzyme from orchid seedlings ( Cattelya aurantiuca ). Both have arginine at the amino terminus. The cofactor, like the holoenzyme, shows absorption, magnetic circular dichroism, and emission properties indicative of an adenine moiety. Although the isolated enzyme has an action spectrum which peaks at about 360 nm, neither the cofactor, apoenzyme nor holoenzyme shows any detectable absorption between 300 and 400 nm.     

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.     

Absence of photoreactivation of pyrimidine dimers in the epidermis of hairless mice following exposures to ultraviolet light

Abstract—The influence of photoreactivating light on the fate of UV-induced DNA damage has been measured in the epidermis of hairless mice using damage-specific endonuclease from Micrococcus luteus. Groups of mice were exposed to varying fluences of UV at 297nm or from an FS40 fluorescent sun lamp to induce UV photoproducts. The same fluence-dependent DNA damage was observed in high molecular weight epidermal DNA regardless of whether the mice were killed immediately, or maintained in the dark or under photoreactivating light for 20 h after UV. Thus, no detectable photoreactivation of UV-induced pyrimidine dimers could be demonstrated in mouse epithelial cells in vivo.     

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.     

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.     

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.     

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.     

Thymine-dimer and action-spectrum evidence for indirect photoreactivation in Escherichia coli

Abstract— Cells of Escherichia coli B and B phr- were labeled with tritiated thymidine, exposed to inactivating ultraviolet radiation at 254 nm, given photoreactivation (PR) treatment immediately thereafter, and then immediately hydrolyzed and assayed for thymine-containing dimers. It was found that (1) PR treatment of strain B phr- does not split thymine dimers and (2) the amount of splitting of thymine dimers in strain B at 334 nm is only 45 per cent of the amount of splitting observed at 405 nm for the same amount of biological PR. These findings show that all of the PR in E. coli B phr-, and part of the PR at 334 nm in E. coli B, is indirect (does not use PR enzyme) and is not due to thymine-dimer splitting. Action spectra for PR in Escherichia' coli strains B_s-1 and B/r were obtained. At wavelengths below 366 nm (the indirect PR region), PR is relatively much more efficient in strain B/r in logarithmic phase than in strain B/r in stationary phase or in strain B_s-l. This is consistent with the expectation that indirect PR would not be exhibited by strains, such as B_s-1 that lack dark-repair ability, or by certain strains in the stationary phase, such as B/r, which, upon plating, have a ‘built-in’ growth delay that can permit optimal dark repair, but indirect PR would be exhibited by intermediate cases, such as in strain B (which is less resistant to u.v. than strain B/r) or in strain B/r in logarithmic phase. These findings support the hypothesis that indirect PR results from enhancement of dark repair.     

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.