Claud S. Rupert

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.

Enzymatic Photoreactivation: Overview

Photoreactivation—a reduction in the effect of ultraviolet irradiation by subsequent exposure to longer wavelengths—stems from at least two different kinds of processes. The first is direct, photoenzyme-mediated repair of ultraviolet radiation damage to DNA, while the second (“indirect photoreactivation”) is an enhancement of light-independent repairs due to physiological changes induced in cells by light.1 These two kinds of processes can be distinguished by their different wavelength and temperature dependences (Jagger and Stafford, 1965). Since indirect photoreactivation is merely one aspect of recovery through mechanisms able to act in the dark, it is best discussed in that context. We are concerned in this section only with the direct photoenzymatic process.

Dark Repair of DNA Containing "Spore Photoproduct" in Bacillus subtilis*

SummaryThe major thymine-containing photoproduct—5-thyminyl-5, 6-dihydrothymine, or TDHT—in DNA of UV-irradiated bacterial spores is known to be removed during spore germination. In normal Bacillus subtilis this removal is now shown to occur both by excision and by a second, distinct, “spore repair” process, which changes the photoproduct to a harmless form in situ. An energy source for the cells, suppliable by glucose, is required for excision to function at all, and for the “spore repair” process to proceed beyond a limited photoproduct removal. Both repairs can function fully in spores germinated in the presence of 150 μg-ml chloramphenicol or 5 μg-ml rifampincin. The “spore repair” mechanism does not function in vegetative cells when these are transformed by TDHT-containing DNA extracted from irradiated baceterial spores.

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.

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.

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.

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.

Evidence for the monomerization of spore photoproduct to two thymines by the light-independent "spore repair" process in Bacillus subtilis.

Ultraviolet radiation (UV) induces a unique photoproduct in DNA of bacterial spores (Donnellan and Setlow, 1965) which appears responsible for a major part of the observed killing (Donnellan and Stafford, 1968). The chemical structure of this “spore photoproduct,” after acid hydrolysis of the DNA, has been identified by Varghese (1970) as mostly 5-thyminyl-5,6-dihydrothymine [Thy@-5)hThy, or TDHT]. In Bacillus subtilis this photoproduct may be removed after spore germination by at least two distinct dark repair processes (Munakata and Rupert, 1972, 1974). One of these, which converts TDHT-containing regions of DNA into acid-soluble fragments, has been identified as “excision repair,” controlled by the same genes which regulate the excision of cyclobutadipyrimidines in the vegetative cells. The other process, which destroys the chemical structure of TDHT in situ, is designated as “spore repair,” and is genetically controlled by the ssp, gene. This destruction was originally observed by Donnellan and Stafford in B. megaterium (1968) and, as suggested by these authors, may involve conversion of the “spore photoproduct” back to two adjacent thymine residues. So far, the only evidence for the restoration of “spore photoproduct” to two thymine residues by “spore repair” has been that the radioactive label associated with it disappears from the acid-insoluble fraction after germination, without reappearing either in the acid-soluble fraction or in any identifiable new peak on the paper chromatograms of hydrolyzed acid-insoluble material (Donnellan and Stafford, 1968; Munakata and Rupert, 1974). This remains true regardless of whether the thymine label is located in a methyl hydrogen or in one of the ring carbons (Munakata and Rupert, 1974). It would be entirely possible, however, for the “spore photoproduct” to be metabolized to products lost in the preparation of samples for chromatographic analyses, or to be converted to products which co-chromatograph with thymine in the solvent systems used, thus escaping detection. In the present work, we have further examined these possibilities, and report additional evidence that the ”spore repair” process simply restores TDHT to thymine, leaving the DNA backbone intact at the end of the process.

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.