R. O. Rahn

Physical studies on the binding of cis-dichlorodiamine platinum (II) to DNA and homopolynucleotides.

The amount of cis-dichlorodiamine platinum (II) bound to DNAs of varying (dA + dT) content was assayed by both ultraviolet absorbance spectrophotometry and the use of the radioisotope 1 9 5 Pt. Radioisotope labeling indicates twice as much bound platinum as do optical measurements. The molar ratio of bound platinum r at saturation is approximately half the sum of the nearest-neighbor frequencies of all base-pairs that do not contain thymine. We therefore conclude that platinum does not bind to thymine in DNA. Chromatographic studies with (14C) purine-labeled DNA indicate preferential binding of platinum to guanine, followed by binding to adenine. The luminescence properties of DNA and of homopolynucleotides are strongly affected by bound platinum as a result of a heavy-atom effect. A plot of the fluorescence-to-phosphorescence ratio as a function of r gives a saturation binding curve similar to that obtained using 1 9 5 Pt. Ultraviolet irradiation of DNA treated with the platinum compound results in a 30% increase in the rate of formation of thymine homocyclobutadipyrimidine. When acetophenone sensitization is employed, platinum binding enhances cytosine homocyclobutadipyrimidine formation 10-fold presumably because the triplet level of cytosine complexed with platinum is lowered below that of acetophenone. The viscosity of DNA decreases sharply upon binding platinum, with half the change occuring when less that 6% of the bases are complexed. From the rate of reaction with formaldehyde, we conclude that binding of the platinum compound to DNA induces small denatured regions that unwind in the presence of formaldehyde with a rate about 40 times slower than that of a single-strand chain break.

ULTRAVIOLET IRRADIATION OF NUCLEIC ACIDS COMPLEXED WITH HEAVY ATOMS-II. PHOSPHORESCENCE AND PHOTODIMERIZATION OF DNA COMPLEXED WITH Ag*

Abstract— We have studied the luminescence (at 77°K) and photochemistry (at 298°K) of a variety of polynucleotides complexed with Ag+. In all cases we observed that Ag+ induces a ‘heavy atom effect’ as indicated by quenching of fluorescence, enhancement of phosphorescence, and reduction of triplet lifetimes. The enhancement by Ag+ of the phosphorescence intensity of poly (dT) and DNA paralleled a 20‐fold enhancement of the rate of thymine dimerization. These results suggest a possible triplet precursor for thymine dimerization in Ag+ complexes. Dimers were also made with a variety of triplet sensitizers, and Ag+ increased the dimer yield two‐ to four‐fold, depending on the sensitizer used.

INFLUENCE OF RELATIVE HUMIDITY ON THE PHOTOCHEMISTRY OF DNA FILMS.

Abstract 1. 1. Films of DNA were equilibrated at various relative humidities and then were irradiated with ultraviolet light at 25° so that changes in their photochemistry might be correlated with known conformational changes. 2. 2. Above 65% relative humidity the photochemical behavior of the films was the same as that in solution, with pyrimidine dimers, but few if any spore photoproducts, being formed. 3. 3. Below 65% relative humidity there was a 2-fold reduction in the yield of thymine dimers and a concomitant increase in the yield of spore photoproducts. 4. 4. These photochemical changes are consistent with the known conformational change in DNA films from the B form to a disorganized form as the relative humidity is reduced.

PHOTOPRODUCT FORMATION IN DNA AT LOW TEMPERATURes.

Abstract— The temperature dependence of thy mine photoproduct formation in Escherichia culi DNA dissolved either in water or in a 50 per cent ethylene glycol solution was studied at temperatures between + 25 and — 196°C. At low temperatures, the formation of thymine dimer was strongly inhibited. A dose of 1 × 104 ergs/mm2 at 280 nm converted 2 per cent of the thymine to dimer at 25°C as compared with 0.2 per cent at — 196°C. In addition, a new thymine photo‐product which was both nonphotoreversible and nonphotoreactivable was found at low temperatures. On the basis of its chromatographic mobility, this new photoproduct was assumed to be the same as that isolated from irradiated spores of Bacillus megaterium. Extensive irradiation at 254 nrn of DNA at — 120°C resulted in a yield of > 23 per cent for the ‘spore‐type’ photoproduct as compared with 6 per cent for the thymine dimer. In poly d(AT), irradiated at low temperature, no spore‐type photoproduct was found; this suggests that adjacent thymine residues are necessary for the formation of the spore‐type photoproduct.

ULTRAVIOLET IRRADIATION OF NUCLEIC ACIDS COMPLEXED WITH HEAVY ATOMS—III. INFLUENCE OF Ag + AND Hg 2+ ON THE SENSITIVITY OF PHAGE AND OF TRANSFORMING DNA TO ULTRAVIOLET RADIATION

Abstract— We have studied the influence of the heavy metal ions Ag+ and Hg2+ on the photoinactivation and photodimerization of transforming DNA and of bacteriophage. The rate of inactivation of Haemophilus influenzae transforming DNA by ultraviolet (UV) radiation was enhanced by a factor of 30 when it was complexed with Ag+. This enhancement was correlated with a comparable increase in the rate of thymine dimerization. In contrast, mercuric ions led to a reduction in the rates of both inactivation and dimerization. When we examined the effects of these metal ions on the photobiology of bacteriophage, we again found that Ag+ enhanced and Hg2+ reduced the rate of ultraviolet inactivation. These results demonstrate that heavy metals may be useful tools for studying the photochemistry and photobiology of nucleic acids.

FORMATION OF CHAIN BREAKS AND THYMINE DIMERS IN DNA UPON PHOTOSENSITIZATION AT 313 nm WITH ACETOPHENONE, ACETONE, OR BENZOPHENONE*

The triplet states of acetophenone, benzophenone and acetone have higher energy than that of thymine (see Table 1). Therefore, the triplet energy of these ketones can be transferred to the triplet of thymine, which in DNA can interact with a neighboring thymine to form a cyclobutane dimer. It has recently been reported that chain breaking occurs during sensitization with either acetophenone (Zierenberg et al., 1971) or benzophenone (Charlier and Helene, 1971), the efficiency being considerably greater with benzophenone. For biological and physical studies on sensitized DNA it is important to know the extent of chain breaking relative to dimerization, and if possible to minimize the chain-breaking process. Since the chain-breaking studies reported above were done under widely varying conditions, it seemed desirable to repeat these measurements, keeping the experimental conditions constant and including acetone as an additional sensitizer. An effort was made to arrive at the conditions most favorable for minimizing chain breaking relative to dimerization.

PHOTOCHEMICAL STUDIES OF THYMINE IN ICE

Abstract— Solutions containing various concentrations of thymine have been irradiated (254 nm) at — 196°, and the corresponding absorbance changes at 315 nm due to thymine‐thymine adduct formation have been followed as a function of dose. The maximum yield of the adduct is about 3–2 per cent. Additional irradiation leads to a reduction in the yield of adduct and to an increase in the yield of the proposed trimer, which shows a sigmoid dose response curve. Not more than 10 per cent of the thymine is converted to trimer. Irradiation of the trimer in solution converts it into adduct plus thymine. Fluorescence due to the adduct does not appear directly after irradiation at — 196°, but comes about upon annealing at elevated temperatures (T > ‐ 80°).

Energy transfer in poly-l-tyrosine as a function of the degree of ionization of the phenolic hydroxyls: I. Room-temperature fluorescence of model compounds and poly-l-tyrosine

Abstract The existence of energy transfer in poly- l -tyrosine is demonstrated by the following observations: (1) The dependence of the neutral fluorescence on the percentage of ionization of the phenolic hydroxyl groups departs considerably from a linear relationship. The initial slope of the quenching curve indicates that one tyrosinate residue quenches approx. 50 tyrosine residues. (2) An enhancement of the tyrosinate fluorescence occurred for isosbestos irradiation which bears a reciprocal relationship to the decrease in the neutral fluorescence. (3) The ionized fluorescence quantum yield was constant throughout the titration, since for direct excitation of the ionized residues the intensity was linearly dependent on the percentage of ionization. (4) No isoemissive point was found for the fluorescence spectral titration. (5) The excitation spectrum of the ionized emission region in a partially ionized polymer is dominated by the neutral absorption profile. Thus, there is considerable evidence for the transfer of electronic energy from tyrosine residues to tyrosinate residues in poly- l -tyrosine. The high efficiency of quenching suggests that there must also be considerable tyrosine-to-tyrosine energy transfer in the polymer.

Nondimer Damage in Deoxyribonucleic Acid Caused by Ultraviolet Radiation

Pyrimidine dimers of the cyclobutane-type are the major photoproducts formed in ultraviolet(UV)-irradiated deoxyribonucleic acid (DNA). [For a review of pyrimidine dimer formation in DNA, see Patrick and Rahn (1976).] The biological consequences of UV irradiation are generally ascribed to the formation of these dimers (see Jagger, 1976), although it is not a simple matter to directly relate pyrimidine dimer concentration with cell killing. Schenley et al. (1976), e.g., show that cells of Escherichia coli surviving UV irradiation excise the same number of dimers as nonviable cells. Hence, studies are needed to trace the sequence of biochemical and physiological events leading from pyrimidine dimers to cell death (see Swenson, 1976). Nevertheless, it shall be assumed that under normal circumstances DNA is the target of UV radiation, that pyrimidine dimers are formed in readily detected quantities, and that cells lacking the capacity to enzymatically recognize and repair pyrimidine damage are more susceptible to UV inactivation. In particular, enzymatic photoreactivation, which acts exclusively on pyrimidine dimers, can be used to demonstrate that pyrimidine dimers account for 50–90% of the UV inactivation of certain bacteria (Harm, 1976).

Pyrimidine dimer formation in poly (d-dT) and apurinic acid

Thymine dimer yields, obtained with either ultraviolet radiation or acetophenone sensitization, were measured in poly(dA-dT) and Escherichia coli DNA after depurination. In both of these systems, dimers were formed between thymine residues previously separated by purine residues. The rate of dimerization for ultraviolet radiation was five times less in poly(d-dT) than in poly(dT). However, with acetophenone sensitization, thymine was dimerized at the same rate in poly(d-dT) as in poly(dT). Sensitization of poly(d-dT) resulted in a maximum dimer yield of 63 %, compared to 80 % in poly(dT). In depurinated DNA, photosensitization gave a maximum thymine dimer yield of 55 %, compared to 37 % in native DNA. We propose that for a thymine pair in native DNA, exciplex formation between thymine and an adjacent purine leads to a reduction in the amount of energy available for thymine-thymine dimerization.