recent aspects of the photochemistry of nucleic acids and related model compounds

BIOCItlMIE, 1985, 67, ,277-292 Revue

Recent aspects of the photochemistry of nucleic acids and related model compounds*.

J. C A D E T *°, L. VOITURIEZ*, A. G R A N D *a, F.E. H R U S K A *~A, P. VIGNY** and L.-S. KAN***.

* Laboratoires de Chimie, Ddpartement de Recherche Fondamentale, Centre d'Etudes Nucldaires de Grenoble, 85 X, F. 38041 Grenoble Cedex, France.

** Laboratoire de Physique et Chimie Biomoldculaire, Institut Curie, 75231 Paris Cedex 05, France. *** Division of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21205 (USA).

zx Member of LA CNRS n ° 321 and of the Universitd Scientifique et Mddicale de Grenoble. a zx Present address : Department of Chemistry, The University of Manitoba, Winnipeg, Manitoba R3T

2N2, Canada.

(Refue le 9-1-1985, acceptde aprOs r~rision le 20-3-1985).

R~sum~ ~ Une revue des rdsultats rdcents de la photochhnie ultraviolette des acides nucldiques et de certains de leurs composds moddles a dtd effectude. Les principales ldsions photochimiques dtudides concernent les dimOres cyclobutidipyrimidiniques, les adduits pyrimidine-pyrimidone, les photoadduits pyrimidine-purine et les produits de pontage entre acides aminds et bases pyrimidini- ques. Les aspects particuliers de la photochimie des ddrivds d'acides nucldiques, lids h l'utilisation de lasers de haute intensitd sont aussi discutds.

Mots-cl~s : photol~sions du DNA / dim~res pyrimidinlques / adduits pyrimidine-pyrimidone / photoadduits des acides amines avec le DNA / photochimie du laser.

S u m m a r y - - This survey focuses on recent developments in the far ultraviolet photochemistry of nucleic acids and related model compounds. The photoproducts discussed are the cyclobutidipyrimidines, the pyrimidine-pyrimidone adducts, the purine-pyrimidine adducts and the addition products of amino acids to pyrimidine bases. The specific aspects of the high-intensity laser photochemistry of nucleic acid components are also briefly reviewed.

Key-words : DNA photolesions / cyclobutidipyrimidines / pyrimidine-pyrimidone adducts / amino acid adducts to DNA / laser photochemistry.

* This paper is dedicated to the memory of Dr. S.Y. Wang. 0 To whom all correspondence should be addressed.

Abbreviations : P)'r < > Pyr : Thy< > Thy : Thy < > Cyt : Cyt < > Cyt : ~'r-l~'o : NMR : PDMS : FABMS : d(Tp T) :

cyclobutidipyrinfidine thymine o'clobutane dimer thyndne-cytosine cyclobut.vl dimer eyclobutidicytosine pyrimidine-pyrimidone adduct nuclear magnetic resonance plasma desorption mass spectrometry fast atom bombardment mass spectrometry thymidylyl(3'-5')thymidine

d(Tp(CE)T) : cyanoethyl ester of thymidylyt (3'-5') thymidine d(T[p](CE)T) : photodimer of the cyanoethyl ester of tlo'midylyl

(3'-5') thymidine Cyt(5-4)Pyo : 5:t4'-p)'rimidin-2'-one] cytosine poly U : polyuridylic acid poly C : polyo'tidylic acid pot)" A : polyadenylie acid poll" G : polyguanylic acid l.rs-trp-lys : L-lysyl-L-tr)ptophyl4--lysine

278 J. Cadet and coll.

1. Introduction

The deleterious effects of far ultraviolet light on living systems are at least partly explained in terms of photo-induced base lesions in nucleic acids [1]. The characterization of cyclobutidithymine Thy < > Thy as the predominant pyrimidine photoproduct in DNA [2] has given impetus to the development of photobiology at the molecular level. Particularly important in this respect was the discovery of a deficiency in the repair of thymine dimers in Xeroderma pigmentosum cell lines [3] which are highly sensitive to ultraviolet light. In addition, several important classes of nucleobase photolesions noted in model systems, have been partly identified in naked and cellular DNA. Extensive work has been done on the characterization of the pyrimidine "photohydra- tes" [4, 5], the addition products of amino acids to pyrimidine bases [6], the pyrimidine adducts including the spore photoproduct [7], and the pyrimidine-pyrimidone adducts [8]. Most of the chemical information and the biological implica- tions of these lesions have been discussed in the two volumes "Photochemistry and Photobiology of Nucleic Acids" [9]. More recently several excellent reviews on one or several topics of the photochemistry of nucleic acids have appeared in the literature [10-16].

In this survey, we focus on recent data concer- ning the far ultraviolet induced modifications in DNA and related model compounds which may have some biological relevance. Progress in the field is mostly due to improvements in analytical separation procedures and in the development of new powerful spectroscopic measurements as well as to the introduction of molecular biology techniques. The main photolesions discussed in this review are the cyclobutidipyrimidines Pyr< >Pyr, the pyrimidine-pyrimidone adducts Pyr-Pyo, the purine-pyrimidine adducts and the amino acid addition products to pyrimidine DNA components. In addition, a short survey on the laser photochemistry of nucleic acids is presented.

2. Cyclobutidipyrimidines Pyr < > Pyr

Pioneering photolysis experiments on frozen aqueous solutions of thymine [17, 18] have greatly facilitated the identification of the cis-syn isomer of the cyclobutidithymine Thy< >Thy in UV irradiated DNA. There is still a need for structural and conformational information on related

Pyr < > Pyr derivatives which may be obtained by using more complicated DNA model compounds.

2.1. DINUCLEOSI DE-M ONOPH OSPHATES

Dinucleoside-monophosphates may be considered to be the smallest representative DNA fragments. Several of these short oligonucleotides have been used as DNA model compounds in recent photolysis experiments.

2.1.1. Structure analysis

The main cis-syn and trans-syn photodimers of the 6yanoethyl ester of thymidylyl(Y-5') thymidine d(T[p](CE)T) have been prepared by acetone photosensitization (J. Cadet, L. Voituriez and L.-S. Kan, unpublished) for incorporation into oligonucleotides of defined sequences. In the course of these investigations, the cis-syn diastereoisomer having a S(P) stereochemistry has been obtained as orthorhombic crystals and its structure has been determined by X-ray analysis [19]. The stereoconfiguration of this cyclobutyl dimer is illustrated in Figure 1. The cis-syn diastereoisomer which is formed as the main photoproduct upon far ultraviolet irradiation of frozen aqueous solution of d(TpT) (vide infra) has the same cyclobutyl stereochemistry. It is likely that this stereoisomer is preferentially produced over the second possible cis-syn cyclobutane dimer Thy< >'Flay in ultraviolet irradiated DNA.

Mass spectrometry is considered a sensitive analytical method which has been widely used in nucleic acid chemistry. However, this technique has been seldom used for the analysis of Pyr< > Pyr until recently, due to the easy splitting of the cyclobutane ring under electron bombardment [20]. Interesting structural information has been obtained on various cyclobutyl dimers of nucleosides and dinucleoside-monophosphates by using soft ionization techniques, such as fast atom bombardment mass spectrometry (FABMS) and 252Cf plasma desorption mass spectrometry (PDMS). In particular, the PDMS mass spectra of the cis-syn and trans-syn internal dimer of d(T[p]T) exhibit predominant pseudomolecular ions at m/z 591 ( M + 2 N a - H ) and at m/z 569 (M + Na) in the positive mode (Fig. 2). In addition, a characteristic fragment of a cyclobutyl structure is observed at m/z 471. This ion, which corresponds to the loss of the deoxyribose moiety from the pT unit, is not present in the mass spectrum of d(TpT) (A. Viari, P. Vigny, L. Voi- turiez and J. Cadet, unpublished).

FZG. I . - - Stereodiagram of the c i s - s y n photodimer of thymidylyl (3'-5') thymidine cyanoethyl ester.

Photochemistry of nucleic acids 279

U3 I-- Z

o o

2000'

tO00

200

Oi

M- BS~'H~'NI~ 34.5

jl (M3~)BS ÷ 2 N')* (M_S,2H+NI) + 471.

1411 9411

(Mr N=)* 569

11411

h drpdr . . . . .

0 0

NO 0

tl 0

C H A N N E L

FIG. 2 . - - 2J2Californiunz plasma desorption mass spectntm of the c i s - s y n photodimer of thymidylyl (3'-5') thymidine in the positive mode.

2.1.2. Conformational properties

It is assumed that the local distortion of the DNA chain induced by a modification such as a pyrimidine cyclobutane dimer Pyr< > Pyr plays a major role in the recognition of the modified nucleobases by repair enzymes. Radiocristallo- graphy and JH NMR analyzes have been widely used to investigate the conformational changes associated with the dimerization of the pyrimidine bases in dinucleoside-monophosphates. Detailed conformational features have been provided by the X-ray diffraction study of the cis-syn diastereoisomer of d(T[p]T) as its S(P) cyanoethyl ester derivative (vide supra). The cyclobutane ring is puckered and the two pyrimidine bases which deviate from planarity exhibit a half-chair conformation. Striking conformational changes

are also noted in the sugar-phosphate backbone. In particular the 3'-nucleotidyl unit shows very unusual features with the base in a syn orientation whereas the sugar moeity adopts a C'2'e,do conformation. Conversely, the furanose ring of the pT unit prefers the Cr e,~o conformation with the base in the anti orientation.

Proton NMR analysis of the corresponding deprotected cis-syn dimer of d (T[p] T) in aqueous solution also shows the most important changes in the pucker of the sugar moieties to have taken place in the 3'-terminal fragment [22]. More recently attempts have been made to analyse various cis-syn and trans-syn photodimers of dinucleoside-monophosphates [23]. However, in- complete determination of the NMR parameters prevented detailed conformationai studies of


these photoproducts. The majority of these analytical difficulties may now be overcome by using high magnet field in association with two dimensional techniques. This is illustrated in Fi- gure 3 which shows the one dimensional 600.1 MHz ~H NMR spectrum of a trans-syn photodimer of d(T[p]T) as its 3'-O-acetyl and cyano-ethyl ester derivative. A complete assignment of the various protons of both sugar moieties has been made by using the spin echo correlated spectroscopy (SECSY) pulse sequence (Fig. 3). The powerful two-dimensional NMR techniques [24, 251 would be useful for the analysis of longer selectively modified oligonucleotides up to 6-10 mers.

Theoretical calculations provide an interesting approach to investigate the conformational changes which are associated with the dimerization of two adjacent pyrimidine bases in an oligonucleotide chain. The energy minimum process and the method of molecular mechanism

have been used in two interesting studies dealing with the cis-syn photodimer of 2'-deoxyuri- dyl(Y-5)-2'-deoxyuridine d(U[p]U) [26] and a self-complementary dodecanucleotide containing a cis-syn thymine dimer d(CGCGAAT < >TCGCG)--d(CGCGAATTCGCG) [27]. Cal- culations show that the 5'-end of d (U [p] U) or of d(T[p]T) presents the more severe distortion in most of the found conformers of lowest energy. In several models, the 5' and 3'-end bases exhibit high anti, anti or low anti orientation, respectively. The corresponding sugar moiety prefers the C2,~,ao and C~,~.~o pucker, respectively. These results present some agreement with the above conformational features obtained from the X-ray cristallography studies of the cis-syn photodimer of d(T[pl(CE)T). Use of these data for further theoretical calculations could be of interest since the two above theoretical studies were based partly on the refined X-ray structure of pyrimidine dimers only.

i

,~ ,~ 9~iI~. ~

H CH'zOH Oq~,1 N " ~ 0 /---o 1 / c.3

A¢O

I ! !

!

I

I

1' 31

66

il ~'O-C1"t2CH2

~4'

5',5" ]4'

CH3CO..~I CH2C_H2C-N

T

5;5"2' ,z 2" l! 1

2'2'

Ls 2'.s l'.s

CH3 Thy it

to A

FIG. 3. -- 600 MHz IH NMR spectra o f the trans-syn photodinter o f th.rnfidylyl (3'-5') 3'-O-acetyl thymidine cyanoethyl ester in D:O.

A) One-dimensional spectrum. B) Two-dimensional J coupling connectivity spectrum, using the spin-echo correlated spectroscopy (SECSY) -- continuous and broken lines refer to the protons of the Tp and pT units, respectively.


2.2. DNA

Significant progress has been recently made in the quantitative determination and the distribution of Pyr< > Pyr within oligonucleotide chains by using new analytical approaches and biology molecular techniques.

2.2.1. Quantitation o f 13'r ~ > Pyr in DNA

Various assays are currently available to monitor the formation and follow the repair of Pyr< > Pyr in ultraviolet irradiated cellular DNA. One of them involves the quantitative nicking of the DNA chains in the vicinity of a Pyr< > Pyr lesion by specific endonucleases [28,29], and subsequent determination of the resulting strand breaks by velocity sedimentation. These methods which are very sensitive do not allow the quantitation of each of the three cis-syn cyclobutidipyrimidines. The resolution of the mixture of Thy < > Thy, Thy < > Cyt and Cyt < > Cyt has been achieved by chromatography on paper and thin-layer plates of the acidic hydrolysates of irradiated DNA [30,31]. However, the possible tailing of radiolabelled thymine or cytosine which generates high background noise in the dimer containing regions of the chromatogram prevents accurate determination of T h y < > T h y or T h y < > C y t at physiological fluences. These difficulties were largely overcome by the development of a high performance liquid chromatographic method on octadecylsilyl silicagel co- lumns [32]. This analytical system was previously shown to be efficient in resolving the mixture of the four cis-syn, cis-antL trans-syn and trans-anti cyclobutidithymines [33]. Following a similar procedure, the cis-syn isomer of Thy < > Thy was measured in an acidic hydrolysate of [3H] thymidine radiolabeled DNA from human skin fibro- blast exposed to ultraviolet light at doses as low as 10 J /m 2 [34]. A similar approach has been later used for monitoring the formation of Thy < > Thy in ultraviolet irradiated naked DNA [35]. Another interesting application of the reversed phase high performance liquid chromatography was the determination of the nucleosomal distribution of cis-syn Thy < > Thy in normal human skin fibroblasts which were exposed to low doses of far ultraviolet light. In fact, a similar amount of cyclobutidithymine was measured in both nucleosomal core- and chromosomal DNA [36]. Further improvement in the sensitivity of the assay was accomplished by mild reduction of the acidic DNA hydrolysate prior to the chromatography [37]. It was shown, using these new experimental

conditions, that the Cyt < > Thy dimer formed in the DNA of normal skin fibroblasts CRL 1221 and CRL 1222 by exposure to 313 nm light was repaired twice as efficiently as Thy< >Thy [37]. In contrast, the excision repair of both Thy< >Thy and T h y < > C y t follows similar kinetics in the DNA of far ultraviolet irradiated human skin fibroblasts GM 38 [38].

Immunological detection of pyrimidine dimers, which does not require pre-labelling of the DNA, represents an excellent alternative to the high performance liquid chromatography assays. Antisera against Pyr< > Pyr have been obtained by immunizing rabbits with ultraviolet irradiated polynucleotides and DNA [39-44]. In most cases, the antibodies were shown to bind dimer-containing oligonucleotides which are at least three or four units long. This would suggest that conformational changes associated with the formation of Pyr< > Pyr are the likely antigenic determi- nants rather than the photolesions. Similar results were also obtained with monoclonal antibodies [45]. The high sensitivity of the latter assay and of an optimized radioimmunoassay [46] enables detection of the Pyr< >Pyr at doses as low as 2.5 J /m -2. Further improvement in the sensitivity of detection was recently achieved by using an enzyme-linked immunosorbant assay (ELISA) [471. Specific binding of the related antibodies to DNA was shown to occur for a dose of 5.0 J /m 2 with as little as 10 ng of DNA. Effort has also been made recently to increase the selectivity of the radioimmunoassay against cyclobutidipyrimidines. A highly specific detection of Thy< >Thy was obtained by using antibodies raised against the cis-syn cyclobutane dimer of N~-thymine butanoic acid which was further covalently bound to bovine serum albumin [48]. This antiserum was shown to cross react with thymine and U r a < > U r a when they are in 67,000- and 53,000-fold excess, respectively. This enabled accurate detection of Thy< >Thy in human DNA fibroblasts which were exposed at a dose of far ultraviolet light as low as 3 J /m 2. Another interesting application of immunologic assays is the determination of the distribution of photolesions within the cells by immunoautoradiography [49] and immunofluorescent flow cytometry [47].

2.2.2. Distribution o f Pyr ~ > Pyr in DNA

The frequency of the photo-induced formation of P y r < > P y r in defined sequences of DNA fragments has been determined as a function of DNA sequence and of the dose of applied far


ultraviolet light [50]. The elegant approach which was used in these studies involves, in the initial step, incubation of the irradiated DNA with the T4 M. luteus ultraviolet specific endonuclease which cleaves the oligonucleotide chain at sites of cyclobutidipyrimidines [51]. The resulting DNA fragments were separated by electrophoresis on high resolution denaturating polyacrylamide DNA sequencing gels for further comparison of their electrophoretic mobilities with those of fragments subjected to chemical sequencing reactions. The extent of dimer formation depends primarily on the nature of the pyrimidines bases involved in the photoreaction [50]. The steady- state level of Pyr< > Pyr was found to reach 10 % formation at sites of adjacent thymines whereas the plateau value for the dimerization of adjacent cytosines was about 1%. These results could be explained in terms of differences in dimerization and monomerization cross sections of the various dimers. In particular the cross section for dimerization of Thy< >Thy is about two times higher than that for Cyt< >Cyt [52]. In addition, the photoreversion rate of the cytosine photodimer is higher than that of cyclobutidithymine. A similar secondary effect of the fanked nucleoside in the steady-state formation of Pyr< > Pyr was observed for single and double stranded DNA. In particular the formation of Thy< >Thy is favoured by the presence of 2'-deoxyadenosine on both 3' and 5' sides. Another interesting feature of these studies was the comparison of the distribution of photo-induced Pyr< > Pyr in a same defined sequence of oligonucleotide from naked DNA and HeLa cellular DNA, respectively [53]. For this purpose, the 342 base-pairs of the alpha sequence from human DNA was used as the substrate. There was no significant difference in the relative distribution of P y r < > P y r under these two conditions, suggesting that the results obtained on isolated DNA could be further extrapolated at the cellular level. However a two-fold decrease in the yield of dimer formation was observed in cellular DNA as the probable result of shielding effects due to other cell components [53].

2.2.3. Model systems for photoreactiving enzymes

One of the two major mechanisms of repair of the Pyr< > Pyr dimers involves the photosplitting of the cyclobutane ring by specific photoreactive enzymes (PRE), the so-called DNA photolyase, EC 4.1.99.3. Two chromophores, which are res-

pectively associated with two different photolya- ses, have been characterized as derivatives of riboflavin [54] and 8-hydroxy-5-deazaflavin [55]. The E. coil DNA photolyase was shown to contain a chromophore which was identified as the blue neutral flavin adenine dinucleotide radical [56,57]. In a model system study it was recently shown that the cis-syn cyclobutidithymine was photoreversed by exposure to visible light in the presence of various flavin derivatives, including 5-deazariboflavin, lumoflavin and 8-methoxy-7,8-didemethyl-Nt°-ethyl-5-deazaflavin [58]. The triplet state of the flavin derivatives is a likely intermediate in the photo-sensitized process since oxygen and diazabicyclo [2.2.2] octane (Dabco), two well-known triplet quen- chers, were found to inhibit thymine formation. A reasonable mechanism for the cleavage of the cyclobutane ring of Thy< >Thy would involve electron transfer from the dimer to the excited triplet flavin in the early stage of the reaction. The electron abstraction from Thy < > Thy is expected to be facilitated in the pH range 10-12 since the pK~ of the cyclobutidithymine is 10.7. This would also explain, at least partly, the increase in thymine production by raising the pH of the irradiated solution [58]. It should be noted that in an earlier proposed repair model system involving the tripeptide lys-trp-lys the electron transfer was shown to occur from the excited indole moiety of the tryptophan amino acid residue to the cyclobu- tidipyrimidine [59].

3. Pyrimidine-pyrimidone adducts

In the course of the investigations on the relative distribution of Pyr< > Pyr in far ultraviolet irradiated DNA, a second class of lesions, which are alkali labile, was detected [51]. This observation and the possible high mutagenicity of these lesions [60, 61], which were indirectly identified as pyrimidine-pyrimidone adducts, explain [60] the recent regrowth of interest in these compounds.

3.1. DINUCLEOSIDE-MONOPHOSPHATES

To investigate further the chemical structure of the above alkali labile lesions, the four DNA pyrimidine dinucleoside-monophosphates have been used as model systems [62]. Far ultraviolet irradiation of aqueous solutions of each of the diriucleoside-monophosphate generates among other photoproducts a fluorescent pyrimidine


adduct which exhibits a red shift in its electronic absorption spectrum. These compounds which were tentatively assigned as pyrimidine-pyrimidone adducts (vide infra) were quantified after being separated on a reversed-phase octadecylsilyl silicagel column. The yields of the (6-4) adducts were shown to decrease in the following order : pdTpdT- pdTpdC > pdCpdC > pdCpdT [62]. Figure4 illustrates the high performance liquid chromatography elution profile of the far ultraviolet d(TpT) photoproducts, as obtained on a Whatman ODS-3 octadecylsilyl silicagel column (L. Voituriez and J. Cadet, unpublished). This analytical method enables a complete separation of the (6-4) d(TpT) adduct from a trans-syn photodimer of d(TpT) which was not reported in previous investigations [62, 63]. In an earlier study [60] the ultraviolet spectrum of this fluorescent adduct was shown to be similar to that of the well characterized oShThy(6-4)mSPyo [64]. Further support for a pyrimidine-pyrimidone structure was provided by the acidic conversion of the (6-4) d(T[p]T) adduct to 6-4'-[5'-methyl-pyrimidin-2'- one]-thymine which is the dehydration product of

3 2

L

_J 1'5 1'0 5 0

Time (rain.)

FIG. 4. - - Reversed phase HPLC separation of the far ultraviolet photoproducts of thymidylyl (3'-5') thyn~idine in aqueous sohttion on a tVhatnzan ODS-3 octadeo'lsilyl silicagel cohtmn.

Eluen t : 0.1 M ammonium acetate + 5% acetonitrile; flow-rate gradient : i to 3 m l / m i n over a period of 10 min. 1 : unknown compound; 2 : cis-syn photodimer of d(T[plT); 3 : (6-4) d(TpT) fluorescent photoproduct; 4 : trans-syn photodimer of d(T[plT); 5 : d(TpT).

the expected precursor oShThy(6-4)mSPyo [8]. Recently complementary structural information was obtained from the fast atom bombardment mass spectrum analysis of the cyanoethyl ester derivative of the (6-4)d(TpT) photoadduct (Fig. 5). Besides a notable pseudo-molecular ion at m/z 622, we may observe a characteristic fragment at m/z 582 which corresponds to the loss of a water molecule (J. Ulrich, L. Voituriez and J. Cadet, unpublished). The IH NMR analysis of this adduct in D20 shows the presence of two singlets at 8.13 and 2.34 ppm in a ratio I/3 which are assigned to the resonance signals of the H-6 and methyl protons on the 5,6-unsaturated thymine derivative (J. Cadet and L. Voituriez, unpublished). In addition, the two related signals which appear at higher field (55.20 and 1.79 ppm) are characteristic of H-6 and CH3 protons of a 5,6-dihydrothymine compound. Another interesting feature of this spectrum is the low magnitude of the trans Jv,r coupling constant which is lower than 1.2 Hz for the two sugar moieties. This is indicative of the unusual pre- ponderance of a Cre,ao or 01"e,ao conformation for the two sugar units.

Structural information regarding the fluorescent adduct of the three other pyrimidine dinucleoside-monophosphates is scarce at the present time. Indirect assignment of the (6-4) adduct of d(TpC) has been made on the basis of its conversion by acidic hydrolysis to the previously characterized 6-(4'-pyrimidin-2'-one)thymine, Thy(6-4)Pyo [65, 66]. The situation is more confu- sed for the fluorescent adduct of d(CpC). In

o

H O - - ~ O ~ j ~ CH3

o N-C-CH2-CH20-~ =O /

o - ~ _ ~ (M+Na) + 20 622

HO

329 363 / 10 I I (M+H'HtO)

0 . . . . . . . . . . . . . . . . . . . . . . , ' , , ; . . . . . . . "'~ " " " " ~ " l ' " : : ' " : ~ . . . . . . . l ' l : " ' " ~ ' l " ~ ' ' ' " ' ' ' 300 400 500 600 FIG. 5. -- Fast atom bombardment mass spectrum of the

o'anoethyl ester of the (6-4) d(TpT) adduct in the positive mode.


earlier studies, the main product of the ultraviolet photolysis of either cytidine, 2'-deoxycytidine or polycytidylic acid, which exhibits a red shift in its ultraviolet absorbance, has been assigned as 5-[4'-pyrimidin-2'-one] cytosine, Cyt (5-4) Pyo [67, 68]. The trifluoroacetic acid hydrolyzed fluorescent adduct of d(CpC) has different ultraviolet and fluorescence features to those of Cyt (5-4) Pyo. Therefore, it was suggested that this photoproduct has a 6-4 linkage between the two pyrimidine rings [69]. Definite assignment of this adduct and of the above (6-4) pyrimidine photoproducts awaits further investigations. Another important point concerns with the unstability of some of the (6-4) adducts. In particular, the (6-4) pyrimidine adduct which is isolated by high performance liquid chromatography analysis of a far ultraviolet irradiated solution of thymidine undergoes a fast conversion at neutral pH to a still fluorescent derivative.

Attempts have been made to gain an insight into the mechanism of the alkali induced breakage of the phosphodiester bond at the 3' end of pyrimidine (6-4) adducts by using dinucleoside monophosphates as model compounds. Treat- ment of the (6-4) adduct of either d(TpT), d(CpC) or d(TpC) with 0.1 M KOH for 30 min at 90°C leads to the formation of a compound more polar than the parent molecule [62]. It is likely that this product bears a free phosphate group which can be removed by enzymatic hydrolysis with the non specific bacterial alkaline phosphatase. The loca- tion of the phosphate group on the sugar moiety attached to the 5,6-dihydro-2,4-dioxopyrimidine moiety was deduced from the observation of the enzymatic digestion of the above phosphomo- noester derivative by the T4 polynucleotide ki- nase. This enzyme contains a phosphatase activity that is specific for 3'-phosphoryl groups.

3.2. DNA

The alkali labile lesions in far ultraviolet irradiated DNA were found to be located at the 3' position of adjacent pyrimidines, using a DNA sequencing analytical method [70]. The formation of these lesions which give rise to oligonucleotide strand breakage under hot alkali treatment depends upon the base sequence. Most of the alkali labile sites were shown to occur preferentially at the T-C sequence. The rate of formation of the T-T (6-4) photoproduct, measured after enzymatic digestion of irradiated DNA, was about three times lower than that of the T-C (6-4) adduct [63]. It is interesting to note that neither the CC nor

the CT (6-4) adducts were detected in the enzymatic DNA hydrolysates. This contrasts with the observed formation of fluorescent adducts in irradiated solution of d(CpC) and d(CpT) (vide supra). The reduced rate in the formation of TT (6-4) adducts and the lack of detectable photoreaction at CT sequence within oligonucleotide chains could be explained in terms of steric hindrance by the methyl group of the 5'-thymine [63]. This would prevent, at least partly, the formation of the oxetane or azetidine intermedia- tes in a DNA chain. The overall formation of the (6-4) fluorescent lesions is at least ten times lower than that of the P y r < > P y r in DNA [62,70]. However, the yield of (6-4) pyrimidine photoadducts could be much higher than that of Pyr< >- Pyr in some sequences [621. Correlation has been found between the formation of such hot spots for (6-4) pyrimidine-pyrimidone adducts and the induction of ultraviolet non-sense mutation in a region of lacI gene of E. coli [71]. Recently, (6-4) photoproducts were shown to be excised from E. coli [72] DNA, according to a repair process which requires the uvr ABC enzyme complex [73].

4. Pyrimidine-purine adducts

Purine nucleobases and nucleosides appear to be more resistant than pyrimidine components to the effects of far ultraviolet light. A major exception is the degradation of 2'-deoxyguanosine in neutral aqueous aerated solution when exposed to 254 nm light [74]. 2-D-deoxy-erythro-pentose and two partly assigned nucleoside derivatives, which result from the splitting of the purine moiety, have been isolated as the main decomposition products.

An adenine photoproduct, which remains to be characterized, was shown to be formed with a quantum yield higher than O = l0 -3 in d(APA ) [75] and polydeoxyadenylic acid [76]. This photoreaction which occurs to a considerably lower extent in the corresponding oligoribonucleotides , is favoured by base stacking and has a singlet excimer as the precursor [77]. Occurrence of hydrogen bonding to a pyrimidine base was shown to quench the yield of the adenine photoproduct when polyd(A) was complexed to poly U [77].

4.1. PURINE-PYRIMIDINE DINUCLEOTIDE ANALOGS

More recently dinucleotide analogs with a trimethylene bridge replacing the sugar-phoshate


backbone have been used as model systems to investigate the photoreactions between purine and pyrimidine bases [78]. The far ultraviolet photolysis of various 5-alkyluracil-adenine dinucleoside analogs generates three main classes of photolesions (Fig. 6). Intramolecular photocycloaddition involving the 5,6-pyrimidine and the 7,8-purine ethylenic bonds is preponderant with respect to the well-known photodealkylation of the 5-substituted uracil derivatives and the photo- hydration of the uracil moiety. Unambiguous structure assignment of the azacyclobutane derivatives was provided by the decrease in the maximum of its ultraviolet absorbance and by careful tH NMR analysis [79]. Particularly, relevant was the observation of only one singlet which was assigned as the resonance signal of adenine H(2) in the aromatic region of the ~H NMR spectra. In addition, 5-alkyluracil H(5) and adenine H(8), which appeared as two doublets in a higher field region (8 4.2-5.7 ppm), are characteristic of an AB system. A novel photocycloadduct was isolated and characterized from the far ultraviolet irradiated aqueous solution of a dinucleotide analog in which a hypoxanthine moiety is linked to a thymine base by a trimethylene chain [80]. The formation of this photoproduct is accounted for by initial cycloaddition of the thymine C(5)-C(6) ethylenic bond to the hypoxanthine C(4)-C(5) double bond.

N

= 0 ¢ l o NH2 / O Ih,) NE2

(R:HIoL~& J

o k=J-k.JL.J FIG. 6. -- Main photoreactions of 5-alkyhwacil-adenine

dinucleoside analogs [78, 79].

4.2. ADENINE-THYMINE PHOTOADDUCT

Evidence for the formation of a photoadduct between adjacent thymine and adenine bases has been obtained from irradiation of thymidylyl (Y~5')-2'-deoxyadenosine d(TpA), of larger oligo- deoxynucleotides and of DNA itself [81]. The

main photoproduct of the far ultraviolet irradiation of neutral aqueous solution of d(TpA) has been isolated and tentatively assigned as an intramolecular adduct. Mass spectrometric measurements of the trimethylsilyl derivative of the photoproduct are indicative of an elemental composition which is identical to that of the starting d(TpA). Other interesting spectroscopic features deal with the disappearance of the maximum of absorption in the ultraviolet spectrum and the upfield shift of the non exchan- geable protons of the thymine and adenine bases in ' the ~H NMR spectrum. These data would suggest a saturation of the 5,6 double bond of the thymine moiety as the result of its photoaddition across one of the double bonds in the pyrimidine ring of the adenine. One possible site of the photocycloaddition would be the 5,6 bond of the adenine moiety, as suggested recently [82]. Howe- ver, additional experimental data are required to further substantiate this tentative assignment. Acidic hydrolysis of the adenine-thymine photoadduct generates the fluorescent 6-methylimi- dazo[4, 5,b]pyridin-5-one which contains a CH3- C= H fragment derived from the thymine moiety [82]. This fluorescence property has been used to monitor the formation of the adenine-thymine adduct in various oligodeoxyribonucleotides and in DNA [81]. The quantum yield of this photoproduct was close to 5 x 10 -4 mole/einstein upon far ultraviolet irradiation of d(pTpA), d(TpApT) and d(TpApTpA). The adenine-thymine photoadduct was shown to be also formed in denaturated and native calf thymus DNA with quantum yields of - 5 x 10 -s and N 1 x 10 -s mole/einstein, respectively. Yield of formation of the photocycloadduct was also found to be about four times higher in single stranded E. coli DNA than in its native form [83]. The possible formation of this minor photolesion in cellular DNA and its biological significance remain to be determined.

5. Amino acid photoadducts to purine and pyrimidine bases

Photo-lnduced DNA-protein cross-linds represent a major class of far ultraviolet damage in living systems [15]. Histone adducts to DNA have been found to be formed in various eukaryotic nuclei and chromatin [84-86]. It is likely that the formation of covalent linkages between proteins and DNA in these organized structures is inde- pendent of the amino acid residues and nucleoti-


des at the interaction sites [87]. Therefore, far ultraviolet light has been used as a suitable probe to investigate histone-DNA interactions in chromatin, isolated nucleosomes or nuclei. However, the chemical structure of the histone-DNA adducts remains for the most part unknown owing to the difficulties encountered in the isolation and the characterization of such lesions. The most significant information on proteins-nucleic acids cross-links comes from studies of model systems including amino acids and DNA components. The related available data for the period 1950-1980 have been presented and critically discussed in a comprehensive survey [151. More recently, several systematic studies of the overall photoreactivity of various aminoacids and short peptides towards synthetic polynucleotides and DNA have appeared [88-90]. Cysteine, lysine, phenylalanine, tryptophan and tyrosine were shown to be the most reactive amino acids with denaturated calf thymus DNA when exposed to 254 nanometer light in oxygen-free aqueous solutions [88]. On the other hand alanine, aspartic acid, glutamic acid, serine and threonine did not exhibit significant photobinding ability under the same experimental conditions. Incorporation of a given amino-acid into a di- or tripeptide leads in most cases to an enhancement of the photoreactivity. Higher binding constants for the formation of DNA-peptide complexes and the involvement of peptide linking in the photoaddition reaction have been proposed as explanations for the increased photoreaction of peptides with respect to amino acids. The presence of oxygen was shown to decrease the quantum yield of photoaddition of amino acids or peptides to DNA with a major exception in the case of glycyl-L-phenylalanine. During the last four years emphasis has been placed on the isolation and characterization of the far ultraviolet irradiation induced photoadducts of the most reactive amino acids to DNA and related model compounds.

5 . 1 . T R Y P T O P H A N - P Y R I M I D I N E P H O T O A D D U C T S

Experiments have shown that ultraviolet B light is able to induce significant binding of ['4C] tryptophan to various polyribonucleotides in deaerated aqueous solutions with reactivities in the order p o l y U > p o l y C = p o l y A > p o l y G . A similar trend was observed when the various purine and pyrimidine nucleobases were used as substrates. Under steady-state photolysis conditions adenine and guanine are resistant whereas thymine, uracil and, to a lesser extent cytosine

undergo partial decomposition. Two classes of photoproducts isolated by anion exchange and thin-layer chromatography were tentatively assigned as 5,6-dihydro-2,4-dioxopyrimidines [91], and tryptophan-nucleobase adducts, respectively [92]. The two tryptophan-thymine and the four tryptophan-uracil adducts were shown to be formed with a 1-1 molecular stoichiometry. Ultraviolet absorption and fluorescence features of the related adducts suggest that the indole moiety of tryptophan is not the site of the photobinding reaction. This received further support from the positive reaction given by the tryptophan-pyrimidine adducts with Erlich's reagent. The disappearance of the maximum of absorbance around 260 nm is an indication of the saturation of the 5,6-pyrimidine bond of these adducts. Another pertinent piece of structural information was deduced from the absence of reaction of the tryptophan adducts with the ninhydrin reagent. This observation may be'related to the lack of an ct amino group as the result either of photo-induced deamination or involvement of the amino group in the covalent bond formation to the pyrimidine base. Several possible structures of the tryptophan-thymine adducts [93] are illustrated in Figure 7. Experimental data, and in particular fluorescence quenching results, are consistent with the involvement of the excited singlet state of tryptophan in the initial step of the reaction sequence leading to the formation of the tryptophan adducts to thymine or uracil. Satura- tion of the ultraviolet irradiated solution of tryptophan and thymine or uracil with NzO or 02 which are efficient electron scanvengers inhibits the formation of the tryptophan-pyrimidine ad-

H Trp

o o II -H3 II

o~"N~" "N'A"~'~"-.-'~-i~ _ _ o j L ;

H H

O O

H H

COzH CO'~

FIG. 7. - - Possible products of the uhraviolet photolysis of tryptophan and thymine aqueous sohttion [92, 93].

Photochemistry of nucleic acids

ducts. Therefore, it is likely that the photoaddition mechanism would involve electron as the reactive species [93]. It has been proposed that electron release could result from the deactivation of the triplet excited state rather than from a biphotonic process. The photo-ejected electron is expected to react at a diffusion control rate limit with thymine or uracil generating a 5,6-dihydro- pyrimidin-6-yl radical after protonation. Dismuta- tion of the latter radical would give rise to the 5,6-dihydro derivative of uracil or thymine and the corresponding unsaturated nucleobase. A likely mechanism for the formation of the thymine or uracil-tryptophan adduct would involve recombination between the pyrimidine radical and a neutral radical derived from tryptophan.

287

o

HN"F"~, ell3 Hfi l '~] CH3 O~LJ~254nm R"N/J%O ILN/H O~Nif

HO H HO C02H N_ .,.~ ~L" IL-Arginin¢ ) H2N

CO2 H H2N {L-Lysine )

FIG. 8. - - Photoexchange reactions o f th)midine with L- arginine and ', -lysine [95].

5.2. P H O T O E X C H A N G E R E A C T I O N S O F P Y R I M I -

D I N E W I T H A M I N O A C I D S

Emphasis has been placed during the past three years on the photoreaction of L-lysine and of t.-arginine with pyrimidine components of nucleic acids, and related model compounds.

5.2.1. Pyrimidine nucleosides

A novel photoreaction has been shown to occur in alkaline oxygen-free aqueous solutions (pH 9.4) of thymidine when exposed to far ultraviolet light in the presence of L-lysine, one of the main amino acid components of eukaryotic chromatin. The main photoproduct separated by reversed phase high performance liquid chromatography was characterized as 2-amino-6-(l-thyminyl) hexanoic acid (Fig. 8) on the basis of 'H and '3C NMR spectroscopic analyses [94]. A

precursor of the lysine-thymine adduct was isolated in a 70 % yield when the steady-state photolysis experiments were carried out at 0°C [95]. This photoproduct was assigned as a pyrimidine ring opened derivative of thymidine with a L-lysine residue attached through its amino group at the C-2 position of the thymine moiety (Fig. 8). Heating of the aqueous solution containing this unstable compound leads to its quantitative conversion to the above thymine-lysine adduct with a concomittant release of free 2-deoxy-D-erythro pentose. Two other stable photoadducts have been recently isolated and characterized, the predominant one resulting from a photochemical exchange reaction of the u-amino group of LAy- sine [96]. This thymine-lysine adduct was found to undergo further decarboxylation to generate l-amino-5-(l-thyminyl)pentane (Fig. 9). These

cr ~ H

FIG. 9. - - Photoreactions o f thymine and L-lysine [96].

~> 254nm ~ '~ It-Lysme 1 " ~

C02H HN~," CH3

O HN/I~, O'13

HOOC~~ 2

O

" HOOCINH2 o


photoreactions involving lysine are selective for thymidine and uridine [96] whereas under similar experimental conditions the other purine and pyrimidine nucleosides did not exhibit any detectable reactivity. Similar photochemical exchange reactions of thymidine were also observed with alanine and glycine [96].

The mechanism of the photoexchange reactions has been investigated in more detail using tertiobutylamine, methylamine and n-butylamine as amino acid model compounds [97, 98]. The photoalkylation of thymine is favoured by increasing the pH to a value between 8.0-11.0, suggesting that a photoexcited state of thymidine anion is involved in the formation of l-alkylthymine. Thetriplet excited state of thymidine was ruled out since acetone sensitization of thymidine in the presence of alkylamine did not give rise to detectable amounts of methyl or butyl photoadducts. Therefore, it is likely that the lowest excited state or a vibrationally excited state of thymidine anion should be the reactive intermediate of the photoreaction. Nucleophilic addition of the alkylamine to the C(2)-N(I) double bond of the thymidine anionic species would generate an adduct with subsequent ring opening of the thymine moiety. The resulting ring opened intermediate would undergo ring intramolecular cycli- zation and subsequent base catalyzed I]-elimina- tion to generate l-alkylthymine [98].

Irradiation of thymidine and L-arginine under basic pH gives rise to a photoexchange reaction which involves the a-amino group and not the guanidino function of the amino acid [951. The related pyrimidine ring opened derivative and the corresponding N - I thymine adduct have been isolated and characterized. This model experi- ment suggests that photo cross-links involving arginine in DNA-histone complexes are probably of minor importance [98].

5.2.2. Lysine-DNA photoadducts

UItraviolet photolysis of aqueous solutions of calf thymus DNA (pH 9.8) in the presence of free L-lysine, followed by heating at 70°C, led to the release of the previously characterized 2-amino-6-(l-thyminyl) hexanoic acid from the oligonucleotide chains [95]. Nucleohistones represent a relevant model to investigate the possible occurrence of photo-induced cross-linkage between the lysine amino group of histones and thymine residues at sites of close interaction. Calf thymus nucleohistone has been irradiated in buffered aqueous solution (pH 10.5) and subse-

quently heated at 70°C for 2 h in order to convert the thymine ring opened compound (vide supra) into 2-amino-6-(l-thyminyl) hexanoic acid. Acidic hydrolysis of the irradiated nucleohistone was found to release the expected above lysine-adduct [99] which was further characterized by comparison of its chromatographic mobility, ultraviolet absorbance and 400 MHz ~H NMR features with those of the authentic sample [95]. The formation of 2-amino-6-(l-thyminyl) hexanoic acid is pH dependent as observed previously with DNA model compounds, the yield of the photoadduct being optimum under alkaline conditions. A similar photoaddition of lysine to thymine was also found to occur in far ultraviolet irradiated chicken erytlu'ocyte nuclei [95].

The selective photoreaction of thymine with m~thylamine [96] may represent an interesting alternative to the tedious thymine removal procedure used in DNA sequencing methods [99]. Alkali treatment was found to induce phosphodiester breakage in photo-modified d(TpA) by exposure to far ultraviolet light in the presence of methylamine [98]. Irradiation of DNA fragments with alkylamines was reported to induce strand breaks at the thymine residues [98, 100]. In addition, removal of guanine was observed to a smaller extent. This could be explained in terms of photooxidation reactions which have been found to involve guanine as the most reactive DNA substrate [101]. Confirmation of the occurrence of a photodynamic effect was provided by the inhibition of the photodegradation of the guanine residue by adding diazabicyclo[2.2.2] octane (Dabco), a quencher of singlet oxygen and triplet excited state, to the irradiated solution of DNA [98]. Under these conditions, a selective methylamine photo-induced removal of thymine was observed.

6. Laser photochemistry of nucleic acids

Growing interest is displayed in the non-linear laser photochemistry of nucleic acid and related model compounds. A detailed survey on the laser photophysics, photochemistry and photobiology of nucleic acids has recently appeared [102]. We focus in the present review on the chemical modifications induced in DNA components upon exposure to high intensity ultraviolet irradiation. One of the main features of powerful lasers is their ability to deliver high energy in a short pulse ranging from the nano-, pico- and even the femto second duration of time. In the ultraviolet irradia-


tion of purine and pyrimidine DNA constituents with high intensity picosecond laser (10%1013 J /m 2) two quantum excitation to a high lying electronic singlet SN or triplet T.~ level was found to occur through corresponding S~ or T~ states [103, 104]. The successive absorption of two quanta of ultraviolet light via the S~ or the more likely T~ state is likely to reach the ionization potential of DNA base components. This is illustrated by the photoionization of thymine upon exposure to the 266 nm laser pulses. The total energy which results from the absorption of a second photon by thymine in its T~ or S~ state is higher that the ionization potential threshold of the pyrimidine base in aqueous solution (E--6.17 eV). The bulk of the final degradation products arising from the high intensity picosecond laser ultraviolet irradiation of thymine have been separated by two-dimensional thin-layer chromatography [105]. Most of these compounds were found to exhibit chromatographic behaviour similar to those of radiation-induced decomposition products of thymine in aqueous aerated solutions [106]. The formation of the thymine degradation compounds may be explained in terms of reaction of a water molecule with the initially produced thymine radical cation, generating the 6-hydroxy-5,6-dihydro thyminyl radical (Fig. 10). Subsequent fast reaction of this intermediate with molecular oxygen would give rise to the cis and trans 5,6-dihydroxy-5,6-dihydrothymine, Nl-formyl-N2-pyruvylurea and 5-hydroxy-5-methylhydantoin as the main products through the intermediary of thymine hydroxyhy- droperoxides [106]. This decomposition scheme receives indirect support from recent detailed investigations of the menadione photosensitized reaction of thymine [107, 108]. Similar product distribution was found to result from the fate of the transient thymine radical cation which is initially formed by an electron transfer reaction. In addition the thymin-l-yl radical, which results from the competitive deprotonation of the cation radical, is able to bind to thymine in the ground state giving rise to several isomeric adducts [108]. It would be of interest to search for the presence of such compounds in high intensity laser irradiated solutions of thymine. This would better substantiate the importance of photoionization processes in the laser induced decomposition of pyrimidine nucleobases. Indeed, hydroxyl radicals [109] which are produced by two-quantum photolysis of water molecules may also be involved in the formation of the first class of thymine degradation products including 5,6-dihy-

o II

HN~,I~. CH3

H

H 2 0

hY - e - Thy Thy" ~ Thy*" , Thy + °

I _H'I-

o II

H N ,,,",,,~ C H3

oJ-.,J FIG. 10. - - Degradation pathways of thymine upon exposure

to high imensiO" picosecond laser [11)2, 105].

droxy-5,6-dihydrothymine and NX-formyl-N2-pyruvylurea [110]. The importance of the latter process is expected to increase with the intensity of the laser pulse. Further experiments are required to determine the relative contribution of direct photolysis of DNA components through photoionization process and indirect effects which are mediated by water radical species. In particular, the release of free bases from laser irradiated nucleosides [110] which is unlikely to involve the transient formation of a pyrimidine radical is probably mediated by hydroxyl radicals through initial hydrogen abstraction within the sugar moiety [111].

7. Conclusions

The data reported in the present review illus- trate the significant progress which has been recently accomplished in several fields of the far ultraviolet photochemistry of nucleic acids and related compounds. Further emphasis should be put in the near future on the development of specific and sensitive assays which would enable the determination of DNA base lesions in cells exposed to low doses of far ultraviolet light. Immunological [112] and DNA labelling methods [113, 114] appear to be very promising in this respect. Another approach, which could provide relevant information on the biological significance of DNA photolesions, is the directed mutagenesis [115, 116]. This would require incorporation of a specific modification in an oligo- deoxyribonucleotide of defined sequence. An important area of research to be considered deals


with the determination of cellular D N A base lesions induced by near ultraviolet light. Only a few modified bases, including mostly the thioura- cil-cytosine adduct [117] and the P y r < > P y r [118-120], have been characterized under these conditions. There is also a need for information on the chemical changes induced in D N A upon exposure to vacuum ultraviolet radiation. Under these conditions, the expected chromophore would be the sugar-phosphate backbone rather than the purine or the pyrimidine bases. Photons from the now available synchrotron radiation [121] would be a very convenient source of vacuum ultraviolet light.

It is also interesting to note the increasing trend in the use of photoreactions involving nucleic acids as a tool in molecular biology. Selective photodegradat ion of the thymine or the guanine bases has been used as an alternative to chemical procedures in DNA sequencing analysis [99, 100, 122]. Another interesting application is the "photofootpr int ing" technique which has been recently developed for the detection of interactions between the lac repressor and the lac Operator in E. coli cells [123]. The photo-induced formation of DNA-amino acid cross-links has been also used to determine the distribution of a protein on specific segments of chromosomal DNA in vivo [124].

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