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.     

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.     

EVIDENCE FOR THE FORMATION OF A CHLOROPHYLL a/ZEAXANTHIN COMPLEX IN LECITHIN LIPOSOMES FROM FLUORESCENCE DECAY KINETICS

The interaction of Chi a with zeaxanthin (Zea), which is an analogue of lutein, has been studied in soya bean lecithin liposomes using the fluorescence of Chi as monitor. The fluorescence emission spectrum at 4.2 K of Chi a showed characteristic changes in the presence of Zea: the emission maximum shifted from 688 nm to 680 nm, and a peak at 731 nm appeared. The fluorescence decay kinetics of Chi a alone could be described by the sum of two exponential components (T₁,≅0.8 ns, T₂≅2.5 ns). In the presence of Zea a component with a long lifetime, T≅5 ns, appeared with a large relative amplitude (40%). This indicated the formation of a Chl a /Zea complex, in which Chl a /Chl a interaction is negligible, presumably because of strong interaction between Chl a and Zea. The fluorescence anisotropy decay kinetics supported the hypothesis of the formation of a large Chl a containing complex in the presence of Zea. A rotational correlation time, φ≅14 ns at 4°C and φ≅21 ns at 30°C, was found, which is distinctly larger than for samples containing Chl a only. We interpret these results as further evidence for a strong interaction between Chl a and Zea in the hydrophobic environment of the lecithin liposomes. This interaction may also occur in the Chl-proteins of the Chi alb light-harvesting complex of plant photosynthesis.     

LIGHT-CONTROLLED ADAPTATION KINETICS IN Phycomyces: EVIDENCE FOR A NOVEL YELLOW-LIGHT ABSORBING PIGMENT

When sporangiophores of the fungus Phycomyces blakesleeanus adapt from high to low fluence rate, dark adaptation (sensitivity recovery) can be accelerated by dim subliminal light [Galland et al. (1989) Photochem. Photobiol. 49, 485-491]. We measured fluence rate-response curves for this acceleration under the following conditions. After sporangiophores were initially adapted symmetrically to a fluence rate of 1 W m-2 (447 nm), they were exposed to unilateral subliminal light (subthreshold for phototropism) of variable wavelength and fluence rate, and then to unilateral test light (447 nm) of fluence rate either 10(-3) or 10(-5) W m-2. The duration of the subliminal light was chosen so that phototropism would not occur during this period. Phototropic latencies could be shortened by subliminal light that was less intense than the test light by several orders of magnitude. In experiments with the final unilateral light of fluence rate 10(-3) W m-2, the 447 nm subliminal light had a threshold (for the acceleration effect) of about 10(-11) W m-2. Yellow light of wavelength 575 nm, which itself is extremely ineffective for phototropism was extremely effective in shortening phototropic latencies in response in response to the test light. At 575 nm, the threshold was about 2 x 10(-12) W m-2. Conversely, near-UV light of wavelength 347 nm, which is highly effective for phototropism, was relatively ineffective (threshold approximately 7 x 10(-8) W m-2) in shortening the phototropic latency. Our results suggest the presence of a novel yellow-light absorbing pigment in Phycomyces that specifically regulates dark adaptation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 PYRIMIDINE DIMERS IN DNA FROM THYROID CELLS OF THE TELEOST, POECILIA FORMOSA

Abstract— We have developed and used a simple technique to estimate the quantity of pyrimidine dimers in unlabeled cellular DNA. DNA is extracted from cells, treated with an endonuclease specific for dimers, and its molecular weight estimated by its electrophoretic mobility on alkaline agarose slab gels. The technique is used to show that cells from thyroid tissue of the fish Poecilia formosa have photoreactivating activity towards dimers in the cellular DNA.     

TWO-PHOTON INACTIVATION, PHOTOREACTIVATION AND PHOTOPROTECTION IN YEAST CELLS IRRADIATED BY 266 NM-LASER RADIATION

Abstract— The quadratic dependence of inactivation of yeast cells on laser fluence rate at 266 nm suggests the occurrence of two-photon photochemical reactions in DNA, producing photoproducts similar to those found with ionizing radiation. Observations of a significant diminution of photoreacti-vation and the disappearance of photoprotection at high laser fluence rates provide additional evidence for this.     

PHOTOREACTIVATION IN A phrB MUTANT OF Escherichia coli K-12: EVIDENCE FOR THE ROLE OF A SECOND PROTEIN IN PHOTOREPAIR

Abstract In Escherichia coli , the light-dependent repair of pyrimidine dimers in UV-irradiated DNA is now accepted as being due to enzymatic photoreactivation (PR) by a 50 kDa enzyme, photolyase (EC 4.1.99.3). The gene for this enzyme has been mapped at 16.2 min and designated phr . This gene was earlier described as phr B, another locus phr A having been proposed in association with PR. The relevance of the putative phr A gene has now been placed in doubt. The recent report of the discovery of a photoreactivating enzyme in Drosphila melanogaster . which specifically repairs pyrimidine (6–4) pyrimidone photoproducts ([6–4] photoproducts), and that E. coli does possess a protein with specific affinity for the (6–4) photoproduct, has cast new light on the prospective role of phr A in PR. We have determined the nucleotide sequence of the putative phr A gene, which suggests it codes for a protein of 38 kDa. When the putative phr A gene was cloned into an expression vector and transformed into a phr A phr B mutant of E. coli , a level of photorepair was observed, which could correspond to repair of (6–4) photoproducts.     

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.     

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.     

DNA PHOTOLYASE FROM THE FUNGUS NEUROSPORA CRASSA. PURIFICATION, CHARACTERIZATION AND COMPARISON WITH OTHER PHOTOLYASES

Abstract A phr-gene from the filamentous fungus Neurosporu crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,1O-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurosporu photolyase is shifted from ca 380 nm to 391 nm (t = 34 800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiue photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurosporu photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.     

ACTION SPECTRUM FOR PHOTOREACTIVATION OF ULTRAVIOLET-IRRADIATED MARSUPIAL CELLS IN TISSUE CULTURE

Abstract— An action spectrum has been determined for photoreactivation of the PtK-2 mammalian cell line of Potorous tridactylus . Maximum effectiveness occurs around 366 nm, but appreciable photo-reactivation occurs at wavelengths as long as 546 nm.     

ACTION SPECTRUM FOR PHOTOREACTIVATION OF ULTRAVIOLET-INDUCED MORPHOLOGICAL ABNORMALITY IN SEA URCHIN EGGS

Abstract An action spectrum was obtained for photoreactivation (PR) of morphological abnormality arising from ultraviolet (UV)-irradiation of sea urchin sperm. The wavelength dependence of PR was measured by the restoration of the formation of normal pluteus larvae after the exposure of fertilized eggs to various fluences of monochromatic PR light (313 to 500 nm). The PR action spectrum showed a maximum around 365 nm and a secondary peak somewhere above 400 nm. High PR activity beyond 400 nm wavelengths may reflect an advantageous or adaptational ability to cope with harmful effects of solar UV radiation.     

EVIDENCE AGAINST DNA AS THE TARGET FOR 334 NM-INDUCED GROWTH DELAY IN ESCHERICHIA COLI*

Abstract— Growth delay was induced with near-UV (334 nm) radiation in Escherichia coli K12 bacterial strains followed by attempts at photoreactivation (PR) of this effect at 405 nm. In the UV-sensitive strain AB2480, a small PR of the observed population growth delay occurred after 334 nm irradiation at 35°C and a much larger PR after 334 nm irradiation at 5°C. However, much of the population growth delay in this strain can be explained as being due to killing, and all or most of the observed PR pertains only to this killed fraction of the population. The true cell growth delay (i.e. that of surviving cells) thus appears to be only slightly, if at all, photoreactivable. This conclusion is supported by studies with a wild-type strain KW8, which shows growth delay at non-lethal doses; this growth delay shows no PR, regardless of the temperature during 334 nm irradiation. These findings indicate that photoreactivable lesions (cyclo-butyl pyrimidine dimers) are not an important cause of near-UV-induced growth delay. Strain AB2480 lacks known dark-repair systems for DNA damage induced by far-UV (below 300 nm) radiation, yet shows the same efficiency for 334-nm-induced growth delay as the wild type, which possesses these dark repair systems. This indicates that lesions in DNA that are dark-repairable by the systems not tunctional in AB2480are not responsible for 334-nm-induced growth delay. It is possible, however, that fragmentary repair systems in AB2480 can operate on some DNA lesion that might cause growth delay. Spontaneously decaying lesions are unlikely, since growth-delay damage decays at a very low rate in non-nutrient medium. Since most of the known types of DNA damage and repair are thus eliminated, these considerations suggest that DNA damage is not involved in near-UV-induced growth delay.     

ACTION SPECTRA FOR ULTRAVIOLET KILLING AND PHOTOREACTIVATION IN THE BLUE-GREEN ALGA AGMENELLUM QUADRUPLICATUM

Abstract— The action spectrum for photoreactivation has been determined in a coccoid blue-green alga, Agmenellum quadruplicatum. The spectrum is rather similar to that recorded for Streptomyces griseus conidia, with some suggestion of a little more structure. The action spectrum suggests possible carotenoid involvement; however, no other evidence for this could be found. The action spectrum for u.v. killing is also broad with some evidence of fine structure. The possible implication of tetrahydropteridines or c -phycocyanin as chromophores in the region 240–300 nm, along with DNA, is pointed out.     

THE ACTION SPECTRA FOR PHOTOREACTIVATION AND PHOTOREPAIR IN ICR 2A FROG CELLS

Abstract— Action spectra for photoreactivation (enhancement of colony forming ability) and photorepair (monomerization of pyrimidine dimers in DNA) were obtained for ICR 2A frog cells over the334–577 lira range. These spectra were very similar with peaks at 435 nm and little effectiveness at wavele.     

EVIDENCE FOR HIGH ENERGY STORAGE INTERMEDIATES IN BIOLUMINESCENCE

Abstract— The bioluminescent enzyme from Photobacterium fischeri is normally activated in vifro by reaction with FMNH₂ and O₂. in the presence of a long-chain aldehyde. Emission from enzyme intermediates in this reaction continues for several seconds, and if the mixture is frozen just after initiation of the reaction, this presumptive emission may be delayed until the system is warmed again. Light is then emitted in a fashion analogous to thermo-luminescent emission, with a maximum intensity at — 10°C. The experiments described here show that the total amount of light which is emitted under these conditions no longer depends so much upon aldehyde, a relatively high quantum yield being obtained both with and without aldehyde.
It is further shown that bioluminescence may be activated by light, populating it is believed, the same state which is responsible for the emission in the case of the FMNH₂-induced emission. The light-induced reaction does not depend on flavin in the enzyme preparations, nor does the activation spectrum resemble that of a flavoprotein. Activation may be carried out in the solid state at temperatures down to at least — 100°C, and so does not involve the diffusion of large molecules. It is proposed that energy storage takes place by charge separation, and that the excited state from which emission takes place is associated with charge recombination.