BIOCHIMICA ET BIOPHYSICA ACTA
P Y R I M I D I N E DIMER FORMATION IN POLY (d-dT) AND APURINIC ACID
R. O. RAHN AND L. C. LANDRY Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tenn. 3783 ° (U.S.A.) (Received May 3rd, 1971)
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.
Photodimerization occurs readily between two adjacent thymine residues, linked by a phosphodiester bond T
as in TpT (ref. I), poly(dT) (refs. 2, 3), and DNA (ref. 4). We write for this reaction - T T - ~ - T T - where k1 is the rate constant for dimerization and k 2 the rate constant k2
for photoreversal. Photodimerization also occurs in the dinucleotide TppT (ref. 5) T
S-P-P-S Abbreviations: poly(dA-dT), alternating polymer of dA and dT; poly(d-dT), depurinated poly(dA-dT); TTI, cis-syn, thymine-thymine cyclobutane dimer; TT v trans-syn, thyminethymine cyclobut~ane dimer; CT, cytosine-thymine cyclobutane dimer; UT, uracil-thymine cyclobutane dimer; UU, uracil-uracil cyclobutane dimer; s, deoxyribose; P, phosphate. Biochim. Biophys. Acta, 247 (1971) 197-2o6
R. O. RAHN, L. C. LANDRY
in which the two thymine residues are connected b y a pyrophosphate linkage. Dimerization in T(CH2)3T T
CH~-CH2-CHz in which the thymine residues are connected at the I position b y a trimethylene linkage, has also been observed e. The rate of dimerization for T p p T is about 4 ° ~o less than for T p T (ref. 5), whereas in T(CH2)aT it is 3.5 times that of T p T (ref. 6). This result suggests that the degree of interaction of the thymine residues in T(CH2)zT is considerably greater than in either T p T or TppT. As shown previously ~,s, factors such as base stacking favor dimerization. We were interested in whether dimerization could occur between two thymine residues arranged as T
-S-P-S-P-SSuch an arrangement is obtained upon depurination of either poly(dA-dT) or DNA. DELLWEG AND WACKER9 showed previously that the dimer yield in apurinic acid after 254-nm irradiation is twice that of native DNA. They interpreted this result to mean that in apurinic acid, thymines normally separated b y purines could dimerize. We show in this paper that dimerization does take place between thymines separated b y an additional sugar phosphate linkage, but the rate of dimerization is too slow to account for the enhancement of the dimer yield in apurinic acid.
MATERIALS AND METHODS Materials
Poly(dA-dT) labeled with [3Hlthymine (purchased fiom Biopolymers, Inc.) was mixed with nonlabeled poly(dA-dT) (obtained from Miles Laboratories, Inc.). Poly(dT) labeled with [14Clthymine was a gift from F. J. Bollum of the University of Kentucky. Escherichia coli DNA labeled either with [3Hlthymine or [14Clcytosine was a gift from W. L. Carrier, Oak Ridge. Depurination and deamination
Samples of DNA and poly(dA-dT) were depurinated 1° b y treatment with 67 % formic acid for 24 h at 25 ° and subsequent dialysis against phosphate buffer (I raM, p H 6.8) to remove the purines. For deamination of the DNA, the procedure of ZMUDSKA et al. 11 was followed: the DNA solution was made 3.8 M in NaNO 2 and 4.8 M in acetic acid, heated at 60 ° for approximately I h, and then dialyzed against phosphate buffer. All samples were irradiated in I mM phosphate buffer, p H 6.8. Irradiation
A low-pressure mercury lamp was used for irradiation at 254 nm, while a 5oo-W, high-pressure mercury lamp whose output was passed through a Bausch and Lomb o.5-m grating monochromator was used to obtain radiation at 280 nm. Biochim. Biophys. Acta, 247 (1971) 197-2o6
PYRIMIDINE DIMER FORMATION
A YSI-Kettering model 65 radiometer (Yellow Springs Instrument Co.) was used to measure the intensity of the incident radiation. Dose-response curves were plotted using this measured incident intensity. Samples were irradiated in I-cm cells. The absorbance at 26o nm was never greater than 0.4 for those samples that were ultraviolet irradiated. Hence, the average exposure throughout the cell was never less than 65 % of the incident exposure.
Sensitization Photoproducts were also formed b y triplet-sensitization with acetophenone as the sensitizer 12. Since acetophenone has a triplet state whose energy is greater than thymine or uracil but not cytosine 1~, sensitization with acetophenone leads to the excitation of the triplets of thymine and uracil but not cytosine. A 2oo-W, superpressure mercury lamp whose output was passed through a NiSO 4 solution and a Coming c.s. 0-54 filter was used to obtain wavelengths greater than 300 nm. Sensitization was done on solutions in Pyrex tubes containing 2-4 ° fig of nucleic acid per ml and 2 mlVi acetophenone. Nitrogen was bubbled through the samples before irradiation or else they were degassed b y pumping. Samples were irradiated for times up to 5 h at a distance of 30 cm from the lamp.
Photoproduct analysis The amount of thymine converted into photoproducts was determined either by measuring the optical absorbance change at 260 nm on the intact polymers or b y hydrolyzing the irradiated samples in 98 % formic acid at 175 ° for 30 min and then chromatographing (descending) them on W h a t m a n No. I paper in a solvent mixture of n-butanol, acetic acid, and water (80 : 12 : 30, by vol.). The chromatogram was cut into strips and the radioactivity in each strip was measured with a liquid scintillation counter. The identification of the various photoproducts was based on their known chromatographic mobilities 4. Since acid hydrolysis deaminates cytosine photoproducts, the initially formed CC and CT dimers are isolated as UU and UT. Thymine dimers (TT) are unaffected by the hydrolysis treatment.
Poly(d-dT) and poly(dT) A chromatogram of poly(d~lT) irradiated (~ > 300 nm) in the presence of acetophenone and then subjected to acid hydrolysis is shown in Fig. I. In addition to the thymine peak (Rv = 0.6) there are two peaks corresponding to the photodimers T T t (RF -----0.3) and TT~ (R F ----0.43 ). Both of these dimers are photolabile and, as indicated in Fig. I, are completely reversed upon irradiation of the hydrolysate. The m a x i m u m yields upon acetophenone sensitization are 63 % for poly(d-dT) and 80 % for poly(dT) (Table I). These yields represent the m a x i m u m number of thymines which can dimerize in these polymers since there is no dimer reversal when ~ > 300 nm. Biochim. Biophys. Acta, 247 (1971) 197-2o6
R . o . RAHN, L. C. LANDRY
I t ~o
DISTANCE FROM ORIGIN (cm)
Fig. I. C h r o m a t o g r a p h i c analysis of poly(d-dT) following m a x i m u m acetopbenone sensitization and acid hydrolysis ( O - O ) - The dimers were reversed ( O - - - O ) b y irradiating the h y d r o l y s a t e w i t h 254 nm. TABLE I THYMINE DIMER YIELDS IN P O L Y ( d T ) AND P O L Y ( d - d T )
The dimers were obtained either with the indicated exposure oi 254-nm or 28o-nm radiation or u p o n acetophenone sensitization with ~ > 300 rim. Sample
Incident exposure (ergs/mm 2 • z o -3)
Percentage o / t h y m i n e as dimer A
TT~ 3 6
254 28o 30o
IOO 3o0 29 144 Max
32 6o 2.3 5.8 7°
0. 5 2
T T 1+ T T 2
The m a x i m u m yields of thymine dimers obtained with saturating doses of either 254-nm or 28o-nm radiation were also measured. As shown in Table I, both TT 1 and TT~ were formed, the yields being considerably greater for poly(dT) than for poly(d-dT). The absorbance changes after irradiation were also followed and, Biochim. Biophys. Acta, 247 (1971) 197-2o6
PYRIMIDINE DIMER FORMATION
as indicated in Fig. 2, the rate of change in the absorbance was greater, b y a factor of 4-5, for poly(dT) than for poly(d-dT). In contrast, no difference in the rate of dimerization between these two polymers was observed when acetophenone sensitization was employed (Table I). The quantum yield for dlmerization in poly(dT) has been shown previously* to be approximately 0.02. ^
PbJy(d - dT)
~ o40 k~
°"o.-~,~___.___ o 280
~ 0.20, 0 0
Fig. 2, Absorbance changes at 260 nm after irradiation (254 nm or 280 nm) of poly(dT) and poly(d-dT) in I rnM phosphate buffer (pH 6.8). The absorbances at 260 nm were between o. 3 and 0,37,
As shown in Fig. 2, when poly(d-dT) that has been extensively irradiated at 28o nm is irradiated further at 254 nm, there is an increase in absorbance because of dimer reversal. We also note that the maximum absorbance change following a saturating dose of ultraviolet radiation is considerably greater for poly(dT) than for poly(d-dT). This result is in keeping with the higher dimer yields in poly(dT) as determined chromatographically (Table I). The influence of freezing on the rate of dimerization in poly(d-dT), compared with that in poly(dT), is presented in Fig. 3. As reported previously14, there is a large increase in the rate of dimerization of poly(dT) at --I96°, but only 3o % of the thymines dimerize at this temperature (without intermittent thawing). In contrast, only about 1 % of the thymines dimerize in poly(d-dT) at --196°. We conclude that this 1 % represents the fraction of thymine residues in poly(d-dT) that are trapped at --1960 into an orientation suitable for dimerization. In poly(dT) this fraction is 3 ° %. To test whether poly(d-dT) wotfid interact with poly(dA), we mixed poly(d-dT) with an equivalent amount of poly(dA) in the presence of I miV[ Mg*+. Since no decrease in absorbance was observed upon mixing we rule out the possibility of an interaction between poly(d--dT) and poly(dA) leading to a duplex structure. Attempts to photoreactivate dimers in poly(d-dT) with an extract from yeast were also unsuccessful.
A purinic acid Apurinic acid was irradiated at either 254 nm or 280 nm and the yields of pyrimidine dimers measured as a function of dose. As shown in Fig. 4, the initial rate of thymine dimer formation in apurinic acid is twice that of native DNA. By Biochim. Biophys. Acta, 247 (197I) 197-~o6
R. O. RAHN, L. C. LANDRY
~ - o
IOAPURINIC. & C I D J
EXPOSURE (ergs/mm2x 10.3 )
EXPOSURE (ergs/mrn2 x 10"3)
Fig. 3. F o r m a t i o n of t h y m i n e dimers in poly(dT) and poly(d-dT) b y 28o-nm radiation at 25 ° and -- 196°. Samples were dissolved in ethylene glycol-water (I : I, b y vol.) containing i mM phosp h a t e buffer (pH 6.8). Fig. 4. T h y m i n e dimer f o r m a t i o n in native D N A and apurinic acid as a function of the incident exposure at 254 nm.
comparison, the rate in denatured DNA was only 30 % greater than in native DNA. The yields of both UT and T T 1 obtained with saturating doses of ultraviolet radiation are given in Table II. It is clear that the maximum dimer yields in apurinic acid are twice those obtained for native DNA. These results are in agreement with those of DELLWEG AND WACKER 9, who also found, using 254-nm irradiation, a twofold enhancement in yield upon depurination. TABLE II MAXIMUM YIELDS OF UT
DIMERS IN APURINIC ACID AND NATIVE DNA
The dimers were obtained w i t h s a t u r a t i n g exposures of either 3 " lOS ergs/mm2 at 254 n m or 5 " l°5 e r g s / m m 2 at 280 nm. Percentage o/thymine 254 n m
Native DNA Apurinic acid
as d i m e r 280 n m
0. 7 1.2
The photoproduct yields obtained upon extensive acetophenone sensitization are compared in Table III for a variety of systems. The results for native DNA, 37 % TTi and 1.5 % UT, agree with those of LAMOLAAND YAMANE12 and BEN-IsHAI et al. 15. The total yield of thymine dimer increases to 47 % upon denaturation and
reaches 54 % in apurinic acid. We conclude from this 7 % increase upon depurination (47-54 %) that dimerization occurs in apurinic acid between some but not all of the thymines initially separated by purines. B i o c h i m . B i o p h y s . A c t a , 247 (1971) 197-2o6
PYRIMIDINE DIMER FORMATION TABLE III MAXIMUM
(~t > 300 mn) oF D N A IN VARIOUS STATES (C) i n d i c a t e s c y t o s i n e l a b e l a n d (T) i n d i c a t e s t h y m i n e label.
Native DNA Denatured DNA A p u r i n i c acid Deaminated DNA D e a m i n a t e d a p u r i n i c acid
o~ o/labeled pyrimidine as dimer UU
TTI + TT ~
--3.2 15.3 21
1.9 8.8 17.6 35 41
1.5 7.8 13 21 28
37 41 48 37 38
-6 6 9 7
37 47 54 46 45
Since uracil triplet but not that of cytosine can be sensitized by acetophenone is, we questioned whether the large yield of UT in both denatured and depurinated DNA, as compared with the relatively low yield in native DNA (Table III), resulted from uracil being formed by deamination of the cytosine residues during either denaturation or depurination. We rule out this possibility on the ground that the conversion of cytosine to uracil would also have led to a corresponding increase in the yield of UU. Such an increase was not observed. In denatured DNA the yield of UU was negligible while in apurinic acid there was only 1/6-1/5 as much UU as UT. However, in completely deaminated systems (Table III) the percentage of uracil as UU was about half that of UT. It is possible that the small yield of UU in apurinic acid may reflect some partial deamination, but it is not enough to explain the large yield of UT. As shown in Table III the UT yields with cytosine labeling differ from those with thymine labeling. This result was consistently obtained. Since cytosine and thymine are present in equal amounts in E. coli DNA, the yield of UT should be independent of the label measured. To study the possibility that an additional cytosine photoproduct was formed that chromatographed with UT, we irradiated (at 254 nm) the hydrolysate of an apurinic acid sample that had been extensively photosensitized and found that all the activity which chromatographed as UT had disappeared. So if there is an additional cytosine-containing photoproduct which chromatographs with UT, it too is photoreversible.
From the initial slopes in Fig. 2 it was found that thymine dimerization in poly(dT) is 4-5 times faster than in poly (d-dT). This difference in rate can be calculated from the photosteady dimer yields. For example, with 254-nm irradiation, T T t + T T 2 = 35 % for poly(dT) and IO % for poly(d-dT). Substituting these values into the expression which holds at photosteady equilibrium, kl/k ~ = [ ~ ] / [ T - T J , where T T = TTI+TT~, and assuming that ks, the rate constant for reversal, is the Biochim. Biophys. Acta, 247 (1971) 197-2o6
RAHN, L. C. LANDRY
same for both polymers, we obtain for the ratio of the rate constants for dimerization in these two systems hl(dT) kl(d dT)
__ ° ' 3 5 / ° ' 6 5
where we have assumed that LT-TI + [TT] = I.OO and have used kI to represent the sum of the rate constants for the formation of T T 1 and TT 2. The five-fold difference in k 1 between poly(dT) and poly(d-dT) agrees with the conclusions drawn previously from the initial slopes in Fig. 2. This reduced rate of dimerization in poly(d-dT) relative to poly(dT) is probably due to a weaker stacking interaction between the bases in poly(d-dT) because of the greater separation distance. The importance of stacking in dimerization reactions has been discussed previously 7,s,16. However, stacking appears to be relatively unimportant when dimerization goes through the triplet state. As indicated in Table I, triplet sensitization produces dimers at the same rate in poly(dT) as in poly(d-dT). Since the triplet state is nmch longer lived than the singlet state, the smaller time-averaged interaction between the bases in poly(d
(d-dT) they are - T - - T - - T - - T - - T - - T - - T - . Presumably, the extra sugar-phosphate linkage in poly(d-dT) prevents two dimers from forming next to each other. The formation of TTz is sterically inhibited in native DNA and has been observed previously only in randomly ordered systems such as denatured DNA (ref. 17) and T p T (ref. I). The relative amount of T T 2 to TT~ produced b y acetophenone sensitization of poly(d-dT) is I : 5. This ratio is what JOHNS et al. 1 have found for T p T with direct irradiation. So the relative rates of formation of TT~ relative to TT 2 appears to be independent of whether the initial excited state is a singlet or a triplet. The rate of reversal of TT~ is roughly five times greater than that of TT1 (ref. I8), so at high doses of direct irradiation TT 1 is formed at the expense of TT 2. With sensitization, however, dimer reversal is avoided and the relative amounts of each dimer obtained reflect solely the relative differences in their rates of formation. In DNA, the m a x i m u m percentage of thymines that can dimerize depends upon the probability of a thymine residue having a thymine neighbor with which to dimerize. In E. colt DNA, the nearest neighbor frequency for T - T sequences is o.076 (ref. 19). Hence, the probability that a given thymine will have a thymine on either side with which to dimerize is 2 • ~/o~76 ---- 0.55. Correcting for the fact that only one dimer is formed in sequences of the form T - T - T (by subtracting 0.076 ) we have 47 % for the m a x i m u m percentage of thymines t h a t can dimerize. This value is exactly the same as the value obtained upon photosensitization of denatured DNA (Table III). Hence, complete dimerization b y sensitization of all available thymine pairs in DNA appears to be possible only in denatured DNA, an observation Biochim. Biophys. Acta, 247 (1971) 197-2o6
PYRIMIDINE DIMER FORMATION
made previously by BEN-IsHAI et al. 15 who also obtained 47 ~/o as the maximum yield in denatured DNA. One possible explanation may be that in native DNA, acid-labile dimers are formed between thymines on opposite strands. Formation of such dimers in native DNA would replace the number of thymines available for forming acid-stable dimers. In denatured DNA, interstrand dimers could not form and more thymine would be available for forming acid-stable intrastrand dimers. Clearly, depurination of DNA has led to an increase in the number of thymines dimerizing, as judged by the maximum yields obtained after sensitization. We assume that this increase in T T from 47-54 % has come about because of dimerization between thymines formerly separated b y purines. However, this increase is not sufficient to explain the doubling of the dimer yields in apurinic acid with 254- and 28o-nm irradiation especially since the additional number of thymines which dimerize upon depurination do so at a 5-fold reduced rate, based on the comparison between poly(dT) and poly(d-dT). We propose, therefore, that in native DNA there are excited-state complexes (exciplexes) 2° involving thymines and neighboring purines, and that these exciplexes provide a mechanism which is absent in apurinic acid for deactivating the excited singlet state of thymine. An exciplex formed between two thymines is thought to be a precursor for the dimer ~1 while an exciplex between a thymine and a purine is not likely to lead to a thymine-thymine dimer. Depurination removes this additional mechanism of quenching thymine excited states. Consequently, in apurinic acid more of the energy absorbed by thymine is available for dimerization and this leads to a higher quantum yield of dimerization. The low yield of UT when native DNA is photosensitized makes sensitization a useful means of obtaining TT1 almost exclusively. However, the reason for the increase in the yield of UT when sensitization is done on DNA that has been either denatured or depurinated (Table III) is not clear. One possibilityis that the formation of the precursor of the UT dimer, the CT dimer, is restricted in native DNA by the rigid structure. However, since CT is formed in native DNA upon excitation of the singlet this argument implies that in the rigid double-helical structure the singlet but not the triplet of thymine is capable of interacting with a cytosine neighbor to form a dimer. It should be pointed out that CT dimers are formed after acetone sensitization of DNA (ref. 1 5 ) - - e v e n though with acetone, cytosine is sensitized as well as thymine. So the reason for thymine triplets not forming CT dimers in native DNA may apply as well to cytosine triplets.
ACKNOWLEDGMENTS This work was sponsored by the U.S. Atomic Energy Commission under contract with Union Carbide Corporation. We are grateful to F. J. Bollum for the gift of poly(dT), and to J. K. Setlow for doing the photoreactivation experiments.
Biochim. Biophys. Acta, 247 (1971) 197-2o6
R . O . RAHN, L. C. LANDRY
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