Damage and repair in mammalian cells after exposure to non-ionizing radiations : I. Ultraviolet and visible light irradiation of cells of the rat kangaroo (Potorous tridactylus) and determination of photorepairable damage in vitro

Cornea cells of the rat kangaroo or “potoroo” (Potorous tridactylus) were exposed to far-UV (254 or 302 nm) radiation, with or without subsequent illumination by near-UV or visible light. The DNA of these cells was extracted and tested for the presence of photoproducts binding yeast photoreactivating enzyme (PRE). The criterion for the latter was competitive inhibition of an in vitro photorepair system, consisting of UV-irradiated transforming DNA of Haemophilus influenzae and an extract containing yeast PRE. The effects on repair kinetics of the transforming DNA indicate that in UV-irradiated potoroo cornea cells up to approximately 90% of photorepairable DNA damage can be photorepaired within 15 min. However, the extent of cellular photorepair, assessed by the reduction in competitive inhibition of the in vitro repair system depends appreciably on experimental parameters during photoreactivating treatment. Control experiments with non-UV-irradiated cells indicated that, depending on specific conditions, the photoreactivating treatment itself produces a varying amount of DNA damage, which reacts with the PRE in vitro. To avoid most of this kind of damage, cells are nitrogen-gassed and kept at 5°C during illumination, and the photoreactivating light must not contain wavelengths shorter than 380–400 nm. Our results show that wavelengths >470 nm are still very effective, whereas wavelengths >555 nm are ineffective in photorepairing potoroo DNA. For unknown reasons, one particular strain of potoroo cornea cells lost its potential for photorepair. Treatment of unirradiated potoroo cells, or their extracted DNA, with hydrogen peroxide also results in competitive inhibition of photorepair in vitro, resembling that observed after near-UV illumination. Because of the occurrence of synergistic effects it is not clear whether the damage only interacts with PRE or can actually be photorepaired under appropriate conditions.

The results presented in this paper suggest that the expression of photorepair in mammalian cells, unlike that in prokaryotes, greatly depends on a number of experimental parameters, including the spectral composition of photoreactivating light. Apparently superposition of damage by the photoreactivating treatment itself is the critical factor. This may explain experimental discrepancies existing in different laboratories studying photorepair in UV-irradiated cells of placental mammals.     

Analysis of photoernzymatic repair of UV lesions in DNA by single light flashes. XI. Light-induced activation of the yeast photoreactivating enzyme

Photoreactivating enzyme (PRE) from yeast (as semi-crude extract, or in highly purified form) shows increased activity if its is illuminated with near UV or short wavelength visible light prior to its use for photoenzymatic repair of UV-induced pyrimidine dimers in transforming DNA in vitro. This effect results from an alternation in PRE molecules changing those with low activity in the light-dependent step of the reaction to a higher activity. Light-induced activation of PRE preparations is slowly lost by dark storage for several hours to 1 day (faster at 23°C than at 5°C), but can be recovered repeatedly by renewed preillumination. The action spectrum for these preillumination effects generally resembles that for the photoenzymatic repair reaction itself, having its maximum in the same 355–385 nm region as the latter, but light of somewhat longer wavelengths (546 nm) is still effective. Preilluminated PRE is also more stable to thermal inactivation (65°C) than untreated enzyme.     

Evidence that the phr+ gene enhances the ultraviolet resistance of Escherichia coli recA strains in the dark

An Escherichia coli recA phr+ purA strain was more resistant to ultraviolet radiation than its isogenic derivative recA phr+ purA+ in the absence of photoreactivating light, whereas their nearly isogenic derivative recA phr showed most UV-induced lethality. The amounts of photoreactivating enzyme (PRE) per cell in the recA phr+ purA was higher than in the recA phr+ purA+. The recA phr is defective for photoreactivation. Thus, in the recA strain, UV resistance in the dark increased in proportion to the amounts of PRE per cell, suggesting that PRE participates in the process of dark repair of UV-damaged DNA.     

Photoreactivating enzyme from Escherichia coli: appearance of new absorption on binding to ultraviolet irradiated DNA.

The photoreactivating enzyme, PRE, monomerizes pyrimidine dimers in DNA in a light requiring reaction (lambda greater than 300 nm). However, the purified PRE from E. coli has no well-defined absorption band for lambda greater than 300 nm. Using absorption difference spectroscopy, we show that when PRE is mixed with ultraviolet-irradiated DNA, new absorption appears in the spectral region required for catalysis. There is a concomitant decrease in the absorption of the mixture for wavelength less than 300 nm. The hyperchromicity for lambda greater than 300 nm is true absorption, not an artifact due to light scattering. Both the hyperchromicity (lambda greater than 300 nm) and hypochromicity (lambda less than 300 nm) can be reversed by irradiation of 365 nm with identical first-order kinetics. We estimate the molar extinction coefficient of the new absorption to be 6900 +/- 1400 at 350 nm. We conclude that the PRE from E. coli does not possess a distinct "chromophore" which by itself is entirely responsible for the absorption of photoreactivating light. Instead, new absorption results when PRE binds its substrate, dimer-containing DNA.     

The kinetics of photoreactivation in the ultraviolet-sensitive mutant Escherichia coli K12 AB2480

An investigation of the kinetics of photoreactivation (PR) in stationary phase cells of the UV-sensitive Escherichia coli mutant K12 AB2480 revealed an initial fast rate of PR which can be attributed to the photolysis of photoreactivating enzyme-substrate complexes already in existence at the start of the illumination period. The initial fast rate could be eliminated by administering a single high intensity flash at the start of continuous illumination. This experimental technique allowed the recognition of 2 additional rate processes during continuous illumination. The first order rate constant for the faster rate process (kf) leads to a value of 4–5 photoreactivating enzyme (PRE) molecules per cell for 16 h nutrient broth grown cells. A photoreactivating irradiance of approx. 60 erg mm^?2 sec^?1 saturates the reaction and under saturating conditions we obtain a value for the activation energy of the fast rate of 9 kcal mole^?1.     

Cloning of the phr gene and amplification of photolyase in Escherichia coli.

A new technique is developed for physically enriching recombinant DNA molecules in an in vitro recombination mixture. UV-irradiation of the donor DNA before recombination enables photoreactivating enzyme (PRE) (deoxyribodipyrimidine photolyase, EC 4.1.99.3) to attach to the donor segments in recombinant molecules. This attached protein causes retention of the recombinant molecules on a nitrocellulose filter, while molecules not containing donor DNA pass through. The bound DNA is repaired of its UV damage and released for insertion into cells by exposure to photoreactivating light in situ, yielding approx. 350-fold enrichment. Although applicable to any gene, this procedure has been used in cloning the Escherichia coli phr gene itself, permitting 100-fold amplification of the gene product in vivo.     

Light-flash analysis of the photoenzymic repair process in yeast cells III. The photoenzymic repair of respiratory-sufficient and respiratory-deficient cells cultured aerobically and anaerobically

In the previous papers, determinations of the number of deoxyribodipyrimidine photolyase or photoreactivating enzyme (PRE) molecules per cell and of the rate constant for the formation of the PRE-pyrimidine dimer complex, k₁, for yeast cells cultured aerobically, were described. In the present work, the number of PRE molecules and ther ate constant k₁ for respiratory-sufficient (RS) and respiratory-deficient (RD) cells of yeast cultured anaerobically were determined. The numbers of PRE molecules in both RS and RD cells cultured anaerobically were larger than those in cells cultured aerobicaly, whereas the rate constant k₁ (nuclear volume). (PRE molecules)^?1 sec^?1 showed a reverse relationship. The numbers of PRE molecules in cells cultured aerobically in various media were also determined.     

Unscheduled DNA synthesis in xeroderman pigmentosum cells after microinjection of yeast photoreactivity enzyme

Photoreactivating enzyme (PRE) from yeast causes a light-dependent reduction of UV-induced unscheduled DNA synthesis (UDS) when injected into the cytoplasm of repair-proficieint human fibroblasts (Zwetsloot et al., 1985). This result indicates that the exogenous PRE monomerizers UV-induced dimers in these cells competing with the endogenous excision repair. In this paper we present the results of the injection of yeast PRE on (residual) UDS in fibroblasts from different excision-deficient XP-strains representing complementation groups A, C, D, E, F, H and I (all displaying more than 10% of the UDS of wild-type cells) and in fibroblasts from two excision-proficient XP-variant strains.In fibroblasts belonging to complementation groups C, F and I and in fibroblasts from the XP-variant strains UDS was significantly reduced, indicating that pyrimidine dimers in these cells are accessible to and can be monomerized by the injected yeast PRE. The UDS reduction in the XP-variant strains is comparable with the effect in wild-type cells. In cells from complementation groups C, F and I the reduction is less than in wild-type and XP-variant cells. Fibroblasts belonging to groups A, D, E and H did not show any reduction in UDS level after PRE injection and illumination with photoreactivating light. These result give evidence that the genetic repair defect in some XP-strains is probably due to an altered accessibility of the UV-damaged sites.     

Studies on the reactivation of bacteria photodamaged by an angular furocoumarin: Angelicin

Summary The angelicin-thymine photoadduct formed by irradiation (365 nm) of an aqueous solution of angelicin and tritiated thymine was isolated by preparative paper chromatography. Reirradiation of this photoadduct at wavelengths shorter than 334 nm splits the adduct, forming again the two parent compounds. A DNA-angelicin combination (8.30 g angelicin per mg of DNA) was prepared by irradiating (365 nm) an aqueous solution of DNA with³H-angelicin. Reirradiation of this combination at wavelengths shorter than 312 nm releases³H-angelicin.The above mentioned conditions were employed to reactivate the photodamaged bacterial cells by angelicin. No reactivation was observed at shorter wavelengths; on the contrary, the lethality was higher after reirradiation. We conclude therefore, that the damage produced directly by the shorter wavelength radiations (formation of pyrimidine dimers) is greater than the small repair produced under our experimental conditions.Reirradiation of bacterial cells with visible light is a condition which activates the photoreactivating enzymes, which are able to provoke the cleavage of pyrimidine dimers. The inability to repair the photodamage caused by furocoumarins under these conditions suggests that this enzyme is highly specific for pyrimidine dimers. Though in both cases,i.e. pyrimidine-pyrimidine and pyrimidine-furocoumarine dimers a cyclo-butane ring is involved, the latter is not recognized by the photoreactivating enzyme.     

Synthesis of photoreactivating enzyme in synchronous and asynchronous cultures of Escherichia coli

The technique of flash photolysis was used to study cellular variations in the number of photoreactivating enzyme (PRE) molecules during the cell division cycle of the UV-sensitive E. coli strain B_S?1. No variations in the number of PRE molecules per genome were observed throughout the cell division cycle when synchronized cells cultured in either glucose-minimal or succinate-minimal medium were used. This is interpreted to mean that PRE synthesis is continuous throughout the cell cycle for glucose-grown cells, but may stop at the time chromosome replication ceases prior to division, in succinate-grown cells. The effect of growth rate and stage of growth on cellular PRE content in asynchronous cultures was also determined. Variations in the number of PRE per genome were observed for both synchronous and asynchronous cells cultured in different media and occurred in a manner that suggested a dependence on growth rate. PRE per genome increased with generation time. Stationary phase cells from each culture medium (nutrient broth, glucose-minimal, succinate-minimal) had more PRE per genome than did respective log phase cells. It is suggested that PRE synthesis may be controlled by some aspect of chromosome replication.     

Binding of Escherichia coli DNA photolyase to UV-irradiated DNA

Escherichia coli DNA photolyase is a flavoprotein which catalyzes the photomonomerization of pyrimidine dimers produced in DNA by UV irradiation. In vivo, the enzyme acts by a two-step mechanism: it binds to dimer-containing DNA in a light-independent reaction and upon exposure to 300-500-nm light breaks the cyclobutane ring and dissociates from the substrate. Using photolyase purified to homogeneity, we have investigated in vitro the first step of the reaction, DNA binding; enzyme-DNA complex formation was quantitated by the nitrocellulose filter binding assay. We find that the enzyme binds specifically to UV-irradiated DNA regardless of whether the DNA is in the superhelical, open circular, or linear form or whether the DNA is single or double stranded. The binding reaction is optimum at a NaCl concentration of 125 mM and at pH 7.5. Although photolyase is retained by the nitrocellulose filters with near 100% efficiency, the binding efficiency of a single enzyme-substrate complex is about 0.34. The complexes can be dissociated by exposing them to photoreactivating light either in solution or on the filter.     

Photoreactivation in phr mutants of Escherichia coli K-12.

We have investigated the genetics of photoreactivation in Escherichia coli K-12. We found that strains with point mutations or deletions in the phr gene showed a significant residual level of photoreactivation after exposure to large fluences of photoreactivating light. It had been previously proposed that a gene in the gal-att lambda interval is also involved in photoreactivation and that the residual photoreactivating activity might be due to this so-called phrA gene located at this interval. We found that deletions of the gal-att lambda region had no effect on either the rate or the final extent of photoreactivation observed in phr+ cells or phr mutants; however strains carrying the delta (gal-att lambda) deletions displayed increased sensitivity to near-UV radiation.     

An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis.

The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.     

Effect of the structure of oligonucleotide substrates on interaction with methylase Eco dam

The interaction of Eco dam methylase with various synthetic oligonucleotide substrates was investigated. The "imperfect" duplexes contained a normal GATC recognition sequence in one chain of the enzyme recognition site and had some defects in the complementary chain, i.e., the absence of one or several nucleotide residues or the presence of S-methyl thiophosphate groups at the 3'-termini. The 3'-S-methyl thiophosphate residue has the same effect on the methylation of oligonucleotide complexes as does the absence of internucleotide phosphate in the analogous complexes. The presence of both GA dinucleotides in the recognition site is necessary for a productive enzyme-substrate interaction. The experimental data suggest that Eco dam methylase does form a symmetrical enzyme-substrate complex which is similar to that formed by type II restriction enzymes.     

Brownian dynamics simulation of substrate motion near active site of enzyme entrapped inside reverse micelle

Brownian dynamics simulation has been applied to analyze the influence of the electrostatic field of a reverse micelle on the enzyme-substrate complex formation inside a micelle. The probability that the enzyme-substrate complex will form from serine protease (trypsin) and the specific hydrophilic cationic substrate Nα-benzoyl-l-arginine ethyl ester has been studied within the framework of the encounter complex formation theory. It has been shown that surfactant charge, dipole moments created by charged surfactant molecules and counterions, and permittivity of the inner core of reverse micelles can all be used as regulatory parameters to alter the substrate orientation near the active site of the enzyme and to change the probability that the enzyme-substrate complex will form.     

Lethal cellular changes induced by near ultraviolet radiation.

There is clear evidence that significant quantities of lesions are induced in DNA by near-UV radiation and that these lesions, although susceptible to repair, may lead to cell death because of the simultaneous disruption of DNA repair systems by the same wavelengths. No particular DNA lesion can be linked to cell death in wild type strains. However, there are good grounds for speculating that a type of near-UV lesion exists which is rapidly "fixed" as a lethal event in cells as a result of the oxygen-dependent disruption of repair. There is a strong indication that the relative ability of various near-UV wavelengths to sensitize cells to heat, chemicals or other radiations is directly related to their efficiency in disrupting DNA repair systems in general. Some important specific questions remain. For example, it is important to ask why breaks formed at 365 nm and 405 nm, although apparently requiring a pol dependent pathway for their repair, do not produce the predicted lethal biological action in the strains tested. In general terms it is hoped to provide more comprehensive physico-chemical data in support of, or contradicting, the proposed model.     

The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro

Escherichia coli DNA photolyase is a flavoprotein that when purified is blue in color and contains a stable neutral radical FAD (E-FADH). In the presence of a suitable electron donor (i.e., thiols, tyrosine, or NADH) the radical FAD adsorbs visible light and undergoes photoreduction to the fully reduced FAD (E-FADH2). The in vitro quantum yield of dimer repair for E-FADH is 0.07 while that of E-FADH2 approaches the in vivo value of 1. Electron paramagnetic resonance studies on whole cells indicate that the in vivo form of photolyase is E-FADH2 with enzyme containing radical FAD generated predominantly during the ammonium sulfate precipitation step of the purification. Activity measurements of E-FADH using long-wavelength photoreactivating light indicate that enzyme containing FAD in the radical form is not active in dimer repair. Dimer repair observed with E-FADH at shorter wavelengths is probably photoreduction of E-FADH followed by dimer repair by E-FADH2.     

Relationship between the lifetime of an enzyme-substrate complex and the properties of the molecular environment.

A theoretical discussion of the decomposition rate constants of enzyme substrate complexes is presented, based upon an enzyme model published earlier (Damjanovich &; Somogyi,1973). These rate constants are expressed by the aid of molecular parameters characteristic for the enzyme-substrate complexes and the molecules in the surrounding liquid phase.Both the exponential and pre-exponential factors of the expressions describing the composition rate constants contain parameters depending on the mass distribution of the reaction mixture in a specific way which is characteristic for the enzyme-substrate complex. The findings suggest a new kind of the enzyme regulation generated by the surrounding medium.     

Damage and repair in mammalian cells after exposure to non-ionizing radiations III. Ultraviolet and visible light irradiation of cells of placental mammals,including humans,and determinations of photorepairable damage in vitro

Cultured cells of placental mammals (including human skin fibroblasts) as well as fresh cornea tissue from oxen have been UV (254 nm)-irradiated and either kept dark or exposed to photoreactivating light (wavelengths >375 nm) only prior to extraction of their DNA. The latter was added to an in vitro photorepair system consisting of UV-irradiated DNA from Haemophilus influenzae and yeast-photoreactivating enzyme, illuminated with broad-spectrum white fluorescent light. The extent of competitive inhibition of the in vitro photorepair of Haemophilus-DNA, resulting from the addition of mammalian DNA, has been taken as a measure of mammalian DNA lesions capable of reacting with photoreactivating enzyme. In most cases the amount of these DNA lesions was reduced if the UV-irradiated mammalian cells had been light-exposed prior to DNA extraction, indicating photoenzymatic repair of up to 90% of the lesions. DNA damage by the photoreactivating light itself was observed at varying degrees in human cells, where this effect presumably masks some of the photorepair.     

Charge accumulation and recombination in Photosystem II studied by thermoluminescence. II. Oscillation of the C band induced by flash excitation

The characteristics of the thermoluminescence band appearing at +50°C in the glow curve (C band) was investigated in maize chloroplasts. The C band, which had a half-time of 10 min, could be charged in the presence of DCMU, and its amplitude significantly increased if preilluminated chloroplasts were reexcited after DCMU addition. Inactivation of the water-splitting system by hydroxylamine- or Tris-treatment did not abolish the C band. In chloroplasts subjected to various numbers of flashes before DCMU addition, the amplitude of the C band exhibited oscillation patterns which were markedly dependent upon dark adaptation of chloroplasts. Flash excitation of chloroplasts preilluminated by continuous light for 30 s prior to 5 min dark adaptation resulted in a period-4 oscillation with maxima occurring at flash numbers 0, 4, 8, 12. After a 6-h dark-adaptation of chloroplasts the period-4 oscillation was superimposed with a period-2 oscillation. The oscillatory patterns were simulated by model calculations and the possible origin of the C band is discussed.