PHOTOPHYSICS AND PHOTOCHEMISTRY OF PHOTOREACTIVATION

Abstract— Photoreactivating enzyme (PRE) monomerizes cyclobutyl pyrimidine dimers formed in DNA by UV light ( Λ < 300 nm). The enzyme requires near UV and visible wavelengths (300 < Λ < 600 nm) for activity. Possible mechanisms of action of the PRE are suggested by non-enzymatic processes in which pyrimidine dimers are monomerized by UV and visible light. Two such non-enzymatic processes are (a) photolysis of dimers resulting from direct absorption of UV, and (b) sensitized monomerization involving charge transfer complexes. Several lines of evidence suggest that the mechanism of action of the PRE more closely resembles (b) than (a). Recent experiments on the PRE from E. coli reveal the presence of new long wavelength absorption which may indicate the presence of a ground state complex. The known ability of PRE to monomerize dimers of thymine, cytosine and uracil suggests that the carbonyl groups at 2 position of the pyrimidine ring may be important in the interaction between enzyme and dimer.     

LIGHT-MEDIATED REGULATION OF TMV-RNA PHOTOREACTIVATION

Abstract— – Nicotiana tabacum var. Xanthi n.c. plants placed in the dark lose their ability to photoreactivate u.v.-irradiated tobacco mosaic virus RNA over a course of approximately 1 week. When such plants are returned to the light, they recover their photoreactivation ability. The recovery occurs after a lag of at least 3 hr and is complete in 12–24 hr. Three hours or less of cool-white illumination (2400 ft-c) are necessary to induce recovery. Blue light is effective in inducing recovery; green light is less effective; red and near-u.v. are not effective.     

PHOTOREACTIVATION OF CLOVER YELLOW MOSAIC VIRUS

Abstract— Clover yellow mosaic virus, in common with other plant viruses with flexuous, rod-like particles, can be photoreactivated. At lower U.V. doses, photoreactivation is masked by an inhibitory effect on virus infection of visible light alone.     

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.     

KINETICS OF PHOTOREACTIVATION AND LIQUID-HOLDING RECOVERY IN YEAST CELLS

Abstract— A method is presented for analyzing data on the kinetics of photoreactivation and liquid-holding recovery in microorganisms. The method extracts, from measurements on survival, the number of repairable lethal hits per cell remaining after a period of photoreactivation or liquid-holding recovery. A semilogarithmic plot of this quantity as a function of the duration of the survival-enhancing treatment reveals the nature of the kinetics of inactivation of the lethal hits.
The method has been applied to photoreactivation and liquid-holding recovery in yeast cells with wild-type radiation resistance. In the case of photoreactivation of ultraviolet-damaged cells, the number of lethal hits per cell is found to decrease exponentially with the length of exposure to a continuous source of photoreactivating light. Liquid-holding recovery in cells exposed to ultraviolet light or X-rays also results in exponential decrease of lethal hits with increasing time.
The method is compared with the fluence-decrement method and Dulbecco's treatment of photoreactivation data.     

OVERLAPPING ACTION OF HOSTCELL REACTIVATION AND PHOTOREACTIVATION IN BACTERIA

Abstract— The photoreactivation rate of U.V. irradiated phages is decreased in u.v. irradiated bacteria. In contrast, the normal photoreactivation rate is observed if the irradiated bacteria are photoreactivated before phage infection. The decrease of the photoreactivation ratc is understood as a competing effect of the u.v. lesions in the bacterial nucleic acids for the photoreactivation enzyme. This competitive inhibition can be diminished not only by photoreactivation of the bacteria before phage infection but also by hostcell reactivation of the u.v. lesions in the bacterium. The results provide strong evidence that hostcell reactivation and photoreactivation revert the same u.v. photoproducts in bacterial nucleic acids. The experiments show that the hostcell reactivation enzyme is not induced by phage infection or by irradiation, but is normally present in the bacterial cell.     

EVIDENCE AGAINST A PHOTOPROTECTIVE COMPONENT OF PHOTOREACTIVATION IN SACCHAROMYCES CEREVISIAE

Abstract— Photoreactivation-deficient ( phr ⁻) mutants of Saccharomyces cerevisiae were shown to lack in vitro DNA-photolyase activity. A phr ⁻ mutant was then compared with a phr ⁺ strain for near-UV induced photoprotection from far-UV irradiation. Neither strain exhibited a photoprotective effect.     

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 OF ULTRAVIOLET LIGHT-INDUCED SISTER CHROMATID EXCHANGES IN POTOROUS CELLS

Abstract— Exposure to visible light after UV-irradiation showed a remarkable effect on UV-induced sister chromatid exchanges (SCEs). After 6-h exposure to visible light (3 × 10⁵ J/m²), two-thirds of the UV-induced SCEs were prevented, confirming Kato's findings. Exposure to visible light before UV irradiation had no effect. This effect of visible light on UV-induced SCEs was temperature dependent, suggesting the presence of enzymatic photoreactivation.     

EVIDENCE FROM PHOTOREACTIVATION KINETICS FOR MULTIPLE DNA PHOTOLYASES IN YEAST

Abstract— We have analyzed the kinetics of photoreactivation of ultraviolet-light-irradiated cells of two strains of the yeast Saccharomyces cerevisiae . The analysis yields plots of h(t ), the number of photorepairable lethal hits remaining in the cell as a function of the time t of exposure to a continuous source of photoreactivating light. In the case of one of the strains studied, an excision-deficient haploid strain, it is found that the time-dependence of h(t ) is given by the sum of three decreasing exponential functions of t for stationary phase cultures grown at 23 or 30°C. When the culture is grown at 37°C. the fastest component is absent and the intermediate component is reduced in importance relative to the remaining slow component. In the case of the other strain, a diploid of wild-type radiation resistance, h(t ) is found to contain just one decreasing exponential for cultures grown at 23, 30, and 37°C. The rate constant depends on the growth temperature of the cells, decreasing with increasing temperature. The results are interpreted as evidence for multiple DNA photolyases in the sensitive haploid strain.     

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.     

ULTRAVIOLET-INDUCED APOCHLOROSIS AND PHOTOREACTIVATION IN TWO STRAINS OF EUGLENA GRACILIS*

Abstract— Very low doses of ultraviolet irradiation result in a complete loss of the ability to synthesize chlorophyll in two closely related strains of Euglena gracilis (var. hacillaris and strain Z). Both strains are equally sensitive in this response under non-photoreactivating conditions. The ability to synthesize chlorophyll is completely photoreactivated by visible light in E. grncilis var. bacillaris , even after lethal doses of u.v.: the Z strain. however, while photoreactivable following low doses of u.v., remains bleached after doses adequate to kill only 10–20 per cent of the cells.     

PHOTOREACTIVATION OF ULTRAVIOLET RADIATION-INDUCED RELEASE OF ARACHIDONIC ACID FROM MARSUPIAL CELLS

Exposure of an established marsupial cell line, PtK2 (Potorous tridactylus), to ultraviolet radiation (UVR) from an FS-40 sunlamp (280-400 nm) resulted in a fluence-dependent release of radiolabeled arachidonic acid (AA) from cell membranes. Post-UVR, but not pre-UVR, exposure to photoreactivating light reversed UVR-induced pyrimidine dimers in DNA and suppressed the UVR-induced release of AA. These data indicate that DNA damage contributes to the release of AA from membrane phospholipids.     

IN VITRO PHOTOREACTIVATION OF ULTRAVIOLET-INACTIVATED RIBONUCLEIC ACID FROM TOBACCO MOSAIC VIRUS

Abstract— Treatment of ultraviolet-inactivated tobacco mosaic virus ribonucleic acid (TMV–RNA) with extracts obtained from the local lesion host, Nicotiana tabacum var. Xanthi , n.c., and simultaneous illumination at 365 nm results in up to a four-fold increase in infectivity over non-illuminated controls. The active material in the extract appears to be associated with protein, based on its inactivation by boiling, precipitation with ammonium sulfate, and exclusion from Bio-Rad P100 polyacrylamide. Partially purified DNA photoreactivating enzyme from yeast or pinto bean has no activity on ultraviolet-irradiated TMV–RNA.     

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.