Helga Harm

ANALYSIS OF PHOTOENZYMATIC REPAIR OF UV LESIONS IN DNA BY SINGLE LIGHT FLASHES. II. IN VIVO STUDIES WITH ESCHERICHIA COLI CELLS AND BACTERIOPHAGE.

Abstract Intracellular complexes of UV lesions in DNA with molecules of photoreactivating enzyme (PRE) in E. coli can be photolysed with a probability close to 1 by single light flashes of about 1 millisecond duration. The observed photoreactivation (PR) effect permits the number of PRE-substrate complexes present at the time of the flash to be determined on the following basis: ( a ) The number of PRE-substrate complexes equals the number of lesions repaired; ( b ) The number of photorepairable UV lesions present in DNA equals the number of pyrimidine dimers recoverable from acid hydrolysates; a UV dose of 1 erg·mm −2 of 2537 A radiation causes the formation of approximately 6.5 dimers per E. coli chromosome; ( c ) The observed PR effect can be expressed by the formal “dose-reduction”, i.e. by the reduction in the number of dimers. The kinetics of intracellular complex formation can be followed by varying the time interval between UV irradiation and flash reactivation. Stationary phase B s−1 cells, irradiated with 4.8 erg·mm −2 form a maximum number of complexes within approximately 5 min at room temperature, 50% of them being formed within the first 10–15 sec. For greater UV doses (6.4–24 erg·mm −2 ) the maximum number formed reaches a constant limiting value of 20, indicating that this is approximately the number of PRE molecules in these cells. Experiments with sequential flashes at various temperatures between 2° and 44° show that both the maximum extent of complex formation and the photolytic rate constant are temperature-independent in this range. Hence, the usually observed temperature-dependence of PR under conditions of continuous illumination reflects the temperature-dependence of the complex formation only. Extensive PR effects with single light flashes were also found in UV-irradiated phage T1 after injection of unirradiated B s−1 cells. The effects are much smaller in irradiated host cells due to competition by the bacterial DNA. Creating the competitive substrate after the phage DNA has reacted with the PRE results in a time-dependent decrease of the PR effect in phage as the enzyme equilibrates between the host-cell and phage DNAs. The slow rate of equilibration indicates a relatively high stability of the complexes in the dark. A heterogeneity of the photoproducts is evident with regard to both complex formation and stability. Comparative experiments with phage infecting other host strains indicate that the number of PRE molecules in strain B/r equals that in B s−1 , but is lower in the K12 derivatives AB 1157, AB 2437 and AB 2480.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes: I. In vitro studies with haemophilus influenzae transforming DNA and yeast photoreactivating enzyme

Abstract The photoenzymatic repair of ultraviolet (UV) lesions in DNA is a photochemical reaction which occurs in an enzyme-substrate complex between these lesions and photoreactivating enzyme (PRE). It is shown here that high intensity flash illumination (duration of the order of 1 millisecond) causes this repair in essentially all enzyme-substrate complexes present at the time. This fact allows several kinds of studies. (1) By applying the flash at timed intervals after mixing PRE and UV-irradiated bacterial transforming DNA, the formation of enzyme-substrate complex can be observed directly. The process takes of the order of minutes for its completion at the usual reaction concentrations, and the effect of changing concentration shows that most of this time is required for the extremely dilute reactants (∼ 10−9M) to encounter each other in solution. (2) When a sequence of flashes is applied with UV lesions in excess, the resulting stepwise repair permits complex formation to be studied at each level of recovery, from the first set of lesions erased to the last. The result shows that the lesions are heterogeneous with respect to rate of complex formation. (3) The rate of complex formation depends on temperature, but once a comples has been formed at 37° it does not rapidly dissociate on shifting the temperature to 2°. Repeating the experiments at lower light intensity, where not all the complexes present are repaired at each flash, then shows that the actual photochemical process in the complex is temperature-independent over the 2–37° range—at least for the most rapidly complexing lesions. (4) When PRE is allowed to complex with irradiated transforming DNA in the dark, and irradiated non-transforming DNA is subsequently added flashes applied at various times after this addition allow the dark dissociation of enzyme-substrate complexes to be followed. Comparison with the converse experiment, in which PRE is first complexed with lesions on non-transforming DNA, shows that some complexes are extremely stable, the enzyme preferentially remaining on the first DNA with which it forms a complex even after 2 h. Complexes with UV lesions on the synthetic polydeoxynucleotides dA:dT and dG:dC are more stable than most of complexes with natural DNA. (5) Measurement of complex formation under conditions of DNA excess, where essentially all the enzyme is bound, permits determination of the number of enzyme molecules relative to the number of UV lesions. The reasonable assumption that the number of lesions equals the number of pyrimidine dimers recoverable from the DNA then gives an absolute count. Provided the molecular weight of 3·104 given by Muhammed is approximately correct, the proportion of pure enzyme to totat protein in crude yeast extract is about 2·10−6.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VII. Photolysis of enzyme-substrate complexes in vitro ☆

Abstract The extent of complexing of the repairable ultraviolet lesions in Haemophilus influenzae transforming DNA with yeast photoreactivating enzyme in the dark can be determined by applying intense light flashes, which cause repair of essentially all lesions complexed. With full complexing of repairable lesions the kinetics of photoreactivation by steady illumination reflect only the photochemical reaction occurring in the enzyme-substrate complex. Complexes are heterogeneous with respect to their rates of photolysis, described by the first-order rate constant k 3 . At early stages of repair k 3 = k p I , where I is the light intensity and k p is the photolysis constant. The measured value of k p permits calculation of the product ϵΦ, where ϵ is the molar extinction coefficient for the complex, and Φ is the quantum yield for photolysis, giving an absolute action spectrum. In the region of most effective wavelengths (355–385 nm), ϵ > 10 4 liter·mole −1 ·cm −1 , and Φ > 0.1, with the possibility that Φ ∼ 1. k p is independent of temperature and ionic strength over a range producing large effects on the rate of complex formation, but does increase with pH over the range 6.7–7.4. For the slower repair of a minority of lesions the measured value of k p depends somewhat on intensity and continuity of illumination, suggesting some differences not yet understood. At the usual illumination intensities used for photoreactivation k 3 is greater than k 2 the rate constant for dark dissociation of complexes.

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 it 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 degrees C than at 5 degrees 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 degrees C) than untreated enzyme.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes VI. Rate constants for enzyme-substrate binding in vitro between yeast photoreactivating enzyme and ultraviolet lesions in Haemophilus transforming DNA ☆

Rate constants k 1 for formation, and k 2 for dissociation of the enzyme-substrate complexes of yeast photoreactivating enzyme and ultraviolet lesions in Haempphilus influenzae transforming DNA, have been measured as functions of temperature, pH and ionic strength in vitro . The measurements employed intense light flashes which cause repair of essentially all lesions complexed, to determine the number of complexes existing at any moment. Absolute concentrations of substrate were referred to the known numbers of pyrimidine dimers in irradiated DNA, while enzyme concentrations were determined by titration against substrate in a concentration range where the binding was essentially complete. The results reported permit a choice of optimum conditions for photoreactivation under various conditions of illumination. UV lesions are heterogeneous with respect to both formation and dissociation of complexes, and the values determined for the constants are weighted averages. The two rate constants depend on temperature as described by the Arrhenius expression with activation energies of about 9.3 and 5.5 kcal·mole −1 , respectively. This particular difference between the two activation energies indicates that the binding energy in the complex is small. Both constants increase with pH over the range 6.0–7.7, but their ratio, which governs the equilibrium complexing in the dark, is higher at the lower pH. k 1 depends critically on the salt concentration, the position of the sharp maximum varying with the particular ions present, while k 2 increases generally with salt concentration over the range studied. Simple empirical expressions have been found which quantitatively describe the behavior of both constants as a function of ionic strength.

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

Abstract 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.

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.

Analysis of photoenzymatic repair of UV lesions in DNA by single light flashes. 3. Comparison of the repair effects at various temperatures between plus 37degrees and minus 196degrees.

Abstract UV-irradiated transforming DNA of Haemophilus influenzae can be repaired in vitro in the presence of yeast photoreactivating enzyme (PRE) by an intense light flash of about 1 msec duration. Such a flash repaires a fraction > 0.9 of enzyme-complexed photorepairable lesions at all temperatures between +37° and −40°, but only a fraction of about 0.25 or 0.1 at −78° or −196° respectively. A similar decline below −40° is observed for photoenzymatic repair (PR) of E. coli B s−1 cells and for PR of transforming DNA by continuous illumination with white light. The decreased effects at low temperatures result neither from damage to the reaction mixture by freezing or by the illumination itself, nor do they result from dissociation of the PRE-substrate complexes in the dark. The drastic reduction in the extent of PR is observed already below −2° if the photolytic reaction is limited by using a less intense light flash. These results are best explained by assuming that the relative repair efficiency of incident photons drops substantially below −2°, but this is not noticed with a very intense flash, where the number of incident photons is excessive. It remains open whether the decreasing repair efficiency with decreasing temperature reflects reduced absorbance or a reduced quantum yield of the absorbed photons. Increased PR effects at −78° and −196° can be obtained by sequential flashes or by increased periods of illumination with white light, but the kinetics indicate the presence of 2 or more fractions of PRE-substrate complexes, differing in their probabilities of being repaired. At low temperature as well as at room temperature flashes photolyse a majority of the complexes more efficiently than equivalent doses of continuous illumination, suggesting an enhancement of PR by absorption of 2 or more photons within a limited time interval.

Dependence of the U.V. Survival of transforming DNA on the amount of DNA uptake per cell

SummaryUV irradiation of transforming DNA from Haemophilus influenzae, carrying a streptomycin resistance marker (Sr), results in decreased transforming activity. At high DNA concentration the “marker survival” is lower than it is at low concentration. The transition from high to low survival occurs at concentrations ranging from 2.5×10-3 to 2.5×10-2 μg/ml; in this range the probability that transformed cells take up DNA fragments in addition to the marked one increases rapidly. A similar effect of DNA concentration on the percentage of transformants is observed for a mixture of unirradiated and irradiated DNA, where virtually all of the transformants originate from the unirradiated component. This eliminates the possible explanation that the concentration dependence of UV survival of a marker reflects increasing competition for a cellular repair system.It is concluded that the lower marker survival obtained at high DNA concentration involves lethality due to UV lesions present in the additional irradiated DNA taken up by the cell. Thus the steeper marker survival curve is due to the increasing UV dose which the additional DNa necessarily receives when a marker survival curve is being established. Intergration of UV lesions rendering a chromosomal DNA strand inviable is suggested by a slight delay in cell multiplication after uptake of irradiated and — to a lesser extent — unirradiated DNA. Acriflavine at a concentration of 0.5μg/ml enhances the effect of DNA concentration on marker survival. Similarly the number of transformants obtained with unirradiated DNA in the presence of acriflavine is more strongly decreased at high than at low DNA concentration. It is suggested that each event of DNA integration involves a small change for lethality, which is enhanced if the DNA carries UV lesions or if acriflavine is present.