Transition metal-ion photosensitized monomerization of pyrimidine dimers

165 Biochimica et Biophysica Acta, 378 (1975) 165--168

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA R e p o r t BBA 91407 TRANSITION METAL-ION PHOTOSENSITIZED M O N O M E R I Z A T I O N OF PYRIMIDINE DIMERS

I. ROSENTHAL, M.M. RAO and J. SALOMON Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot (Israel)

(Received September 10th, 1974) (Revised manuscript received November 6th, 1974)

Summary The photochemical cleavage of free cyclobutane-type pyrimidine dimers, as well as of such dimers in irradiated DNA is sensitized b y K3Fe(CN)6 and UO2 SO4. The mechanism most probably involves, as the determining step, a charge transfer from the ground state dimer to the excited sensitizer.

The lesions induced in D N A b y ultraviolet light can be partially repaired through irradiation of the infected nucleic acid with visible light in the presence of a photoreactivating enzyme. The photochemical reaction involved in this process has been demonstrated to be the monomerization of cyclobutane dimer moieties in the irradiated DNA [1], and it has been assumed that the light-requiring step of the process involves photosensitization through the appropriate system in the enzyme [2]. Indeed, thymine or uracil dimers were split into the monomers u p o n irradiation with ultraviolet light of k > 300 nm or visible light in the presence of a variety of photosensitizers [ 3, 4], among which quinones are b y far the most efficient [5--7]. These reactions can be considered as simple chemical models for the photoreactivation process. 0

0

RN

0 NR

©

0 R

R=N

R

or CH3

RN

hv

0 R

166 A mechanistic hypothesis for the photosensitized cleavage of pyrimidine dimers suggests the transfer of an electron from the ground state dimer to the excited photosensitizer [7, 8] to yield an intermediate which might be the radical cation of the dimer. The latter species subsequently undergoes ring cleavage to the pyrimidine monomers. It follows that two basic conditions should be fulfilled by a compound in order to sensitize this process: 1) to absorb light at long wavelengths, preferably in the visible region and 2) to have a high electron affinity. Salts of transition metals in their oxidized state may satisfy these two requirements. In order to test this assumption, we have investigated the photosensitized cleavage of thymine, 1,3-dimethylthymine and 1,3-dimethyluracil cyclobutane type-dimers, by K3Fe(CN)6 and UO2 SO4 and we have indeed found these salts to be very efficient sensitizers of this reaction. The results are summarized in Table I. TABLE I PHOTOSENSITIZED

CLEAVAGE OF PYRIMIDINE DIMERS

Hanovia 4 5 0 W high pressure m e r c u r y v a p o r lamp p r o v i d e d w i t h an Uranium glass cut-off filter (k > 3 5 0 n m ) w a s used as the light source, cis-syn T h y m i n e dimer w a s prepared b y the irradiation of a f r o z e n a q u e o u s s o l u t i o n of t h y m i n e . 1 , 3 - D i m e t h y l u r a c i l and 1 , 3 - d i m e t h y l t h y m i n e dimers were prepared b y p h o t o d i m e r i z a t i o n in s o l u t i o n [ 9 , 1 0 ] . In a typical e x p e r i m e n t for m e t h y i a t e d dimers, a m i x t u r e o f cis-anti 1 , 3 - d i m e t h y l t h y m i n e d i m e r ( 5 0 r a g ) a n d UO~SO~ ( 5 0 r a g ) in w a t e r ( 1 . 5 m l ) w a s irradiated for 3 . 5 h. The reaction m i x t u r e w a s separated b y preparative thick-layer c h r o m a t o g r a p h y (acetone:CHC13 , 1 : 1 ) to yield 1 , 3 - d i m e t h y l t h y m i n e ( 2 5 r a g ) . S i m i l a r l y , a s o l u t i o n o f cis-syn t h y m i n e dimer ( 2 0 r a g ) and K ~ F e ( C N ) 6 ( 2 0 r a g ) in w a t e r ( 4 0 m l ) w a s irradiated for 3 . 5 h. T h e separation b y thin-layer c h r o m a t o g r a p h y (15% m e t h a n o l in c h l o r o f o r m ) y i e l d e d 6 m g t h y m i n e . Dimer

Chemical yields of cleavage (%) in the presence of K~Fe(CN) 6

UO~SO~

T h y m i n e d i m e r (cis-syn) m ~ ' 3 T h y d i m e r (cis-syn) m 21,s T h y dimer (cis-anti) m ~ ' 3 U r a d t m e r (cis-syn) m ~' 3 Ura dimer (cis-an ti) m~ '~ Ura dimer (trans.syn) m~'3Ura dimer (trans-anti)

30 14 14 20

60 40 50 80

~1 25

<:1 70

The quantum yields for sensitized cleavage of thymine and 1,3-dimethylthymine dimers (cis-syn) in this reaction was found to be 0.6. This value compares very well with the quantum yields 1.03±0.05 and 1 obtained for the splitting of cis-syn 1,3-dimethylthymine dimer by direct photolyses and for the photosensitization by 2-anthraquinonesulfonate, respectively. This value is at least one order of magnitude higher than those reported for other sensitizers

[2]. The major light-absorbing species in these systems are undoubtly the UO22 + and Fe(CN) 3+ salts, due to their absorption bands in the visible region at 415 and 405 nm respectively. The transfer of the excitation energy from the sensitizer to the dimer can be performed by ways which are the best illustrated for UO22 ÷ reaction. Mechanistic considerations of U (VI) photochemical reactions have centred around two theories, i.e. "complex formation" and "kinetic

167

encounter". The first hypothesis is based on the prior existance of a U(VI)dimer complex in the ground state, which undergoes a light induced chargetransfer to metal (CTTM) breakdown to give free radicals: U(VI) + Why[]Thy ~ U(VI).Thy[]Thy U(VI).Thy[]Thy + h v ~ U(V) + Thy + Thy "+ U(V) + Thy -+-* U(VI) + Thy where Thy [ ] Thy is thymine dimer. The second possibility, based on "kinetic encounter" theory, implies the following scheme: U(VI) + h v ~ U(VI)* V(VI)* -~ U(VI) + hvfluorescence U(VI)* ~ T h y [ ] T h y -~ U(V) + T h y [ ] T h y "+ Thy [ ] Thy "+-* Thy + Thy "+ Thy v + U(V) -* Thy + U(VI) The first route which involves the formation of a ground state complex between the uranyl salt and the dimer can be eliminated, since no support from spectroscopic measurements could be provided for the formation of such a complex. It has also been found that the rate of photomonomerization increases with rise of temperature (temperature range 5--50°C). This observation eliminates a mechanism involving a ground state charge-transfer complex, since the association constant of such a complex decreases with a rise in temperature [11] and suggests that the reaction mechanism involves a collisional transfer [12] from the excited uranyl ions to dimer molecules. Experimental support for the "kinetic encounter" theory was provided by Stern-Volmer plots. Fluorescence quenching of U(VI) by various 1,3-dimethyluracil dimers exhibited Stern-Volmer behaviour (Fig. 1). The lower photoreactivity of a n t i - d i m e r s reciprocates the low fluorescence quenching. 6

I

s

-

I

I

_,J®

-

3

2

®

I

I

001

0/32

1

0.025

Fig. 1. T h e S t e r n - V o l m e r p l o t ; r e l a t i v e i n t e n s i t i e s o f t h e u r a n y l f l u o r e s c e n c e at t h e 5 1 6 n m p e a k . 1o/I w e r e m e a s u r e d f o r s o l u t i o n c o n t a i n i n g 0 . 0 5 M u r a n y l s u l f a t e a n d v a r i o u s c o n c e n t r a t i o n s of m~'3Ura d i m e r s w i t h a 4 1 0 n m e x c i t a t i o n b e a m . (A) s y n - d i m e r s a n d (B) anti-dimers.

168

However, since even the dimers which are practically uncleaved show a quenching effect of the uranyl fluorescence, it appears that the extent of cleavage is also related to other factors, presently unknown, in addition to quenching efficiency. Furthermore, evidence of the separate existence of oneequivalent charge-transfer intermediates required by the "kinetic encounter" scheme, i.e. UO2 + [13] and thymine radical anion [8] have been reported. A final c o m m e n t concerns the stereo-selectivity o f cleavage of 1,3-dimethyluracil dimers. The dimers of syn-type are monomerized to a much larger extent then the anti-type. This effect results most probably from steric factors involved in the facility of approach between dimer and excited salt which preceeds the electron transfer step. It is interesting to note that a similar observation has been made in quinone-photosensitized cleavage of these dimers [7]. The results obtained in this molecular study have been extended to the DNA-containing dimers. Thymine-labeled D N A was irradiated in the presence of acetone as photosensitizer (~ > 290 nm) [14] to yield a biopolymer with a high content of thymine dimer. This D N A was irradiated with light of > 300 nm in the presence of such amount of K3 Fe(CN)6 so that all light was absorbed by the latter. Following this irradiation a decrease of up to 60% in the dimer content of D N A was observed (Table II). TABLE II K3Fe(CN)6-SENSITIZED CLEAVAGE OF THYMINE DIMER IN DNA DNA (33 Dg/ml; 104 cpm/Dg) with a high t h y m i n e d i m e r c o n t e n t (37%) w a s prepared b y irradiation of l ~ C - t h y m i n e labeled D N A in the presence o f a c e t o n e [ 1 4 ] . Samples o f this DNA (0.3 ml) were irradiated (k > 300 nm, incident d o s e rate 10 - s Einstein "rain - l ) for various p e r i o d s of time in the presence of K3Fe(CN) 6 in 0.1M p h o s p h a t e buffer (pH 6.8, 2 m l s o l u t i o n in total). A f t e r centrifugation and dialysis the dimer c o n t e n t w a s a n a l y s e d as described [ 15]. Time of irradiation

Ks Fe(CN) 6

(min,)

(mg.)

(%)

0 45 45 75 90 120

5 -5 5 5 5

37 37 30 25 21 17

T h y m i n e dimer

References 1 Setlow, R.B. (1968) Progr. Nucleic A c i d Res. MoL Biol. 8, 257--295 2 Lamola, A.A. (1972) Mol. Photochem., 4(1), 107--133 3 Wacker, A., Dellweg, H., Traeger, L., Kornhauser, A., Lodemann, E., TuerclL G., Selzer, R., Chandra, P. and Ishimoto, M. (1964) Photochem. Photobiol. 3 , 3 6 9 - - 3 9 4 4 Lamola, A. (1966) J. Am. Chem. Soc. 88, 813--819 5 Rosenthal, I. and Elad, D. (1968) Biochem. Biophys. Res. Commun. 32, 599--601 6 Ben-Hut, E. and Rosenthal0 I. (1970) Photochem. Photobiol. 11, 163--168 7 Sasson, 8. and Elad, D. (1972) J. Org. Chem. 37, 3 1 6 4 - - 3 1 6 7 8 Roth, H.D. and Lamola, A.A. (1972) J. Amer. Chem. Soc. 94, 1013--1014 9 Elad, D., RosenthaL I. and Ssason, S. (1971) J. Chem. Soc. C, 2053--2067 10 Morrison, R. and Kleopfer, R. (1968) J. Am. Chem. Soc. 90, 5037--5088 11 Foster, R. (1969) In Organic Charge-Transfer C o m p l e x e s , p. 189, A c a d e m i c Press, N e w Y ork 12 Sakuraba, S. and Matsushima, R. (1970) BulL Chem. Soc. (Japan) 43, 2359--2363 13 Heidt, L.J. (1954) J. Am. Chem. Soc. 76, 5 9 6 2 - - 5 9 6 8 14 Ben Ishai, R., Ben Hut, E. and Hornfeld, Y. (1968) Israel J. Chem. 6, 769--775 15 Setlow, R.B. and Carrier, W.L. (1966) J, MoL Biol. 17, 237--264