Direct H atom abstraction from spore photoproduct C-6 initiates DNA repair in the reaction catalyzed by spore photoproduct lyase: evidence for a reversibly generated adenosyl radical intermediate

Spore photoproduct (SP) lyase, which catalyzes the direct reversal of SP (5-thyminyl-5,6-dihydrothymine) to thymine monomers, is the only identified nonphotoactivatable pyrimidine dimer lyase. Unlike DNA photolyase, SP lyase does not contain a flavin cofactor and does not require light for activation. Instead, preliminary studies point to the presence of an iron-sulfur cluster in SP lyase and the requirement for S-adenosylmethionine (AdoMet) for catalytic activity, suggesting that SP lyase belongs to the growing group of iron-sulfur cluster and AdoMet-dependent radical enzymes. Here we provide evidence for the role of AdoMet as a reversible deoxyadenosyl radical generator, which initiates repair by hydrogen atom abstraction from C-6 of SP. Reaction of 6-(3)H-SP, but not methyl-(3)H-SP, with SP lyase and AdoMet results in transfer of (3)H to AdoMet, while no tritiated 5'-deoxyadenosine is observed. When 5'-tritiated AdoMet is used in the reaction with unlabeled SP, transfer of (3)H into the repaired thymine monomers is observed. These results point to the reversible generation of a 5'-deoxyadenosyl radical intermediate, which reacts directly with the DNA lesion to initiate a radical-mediated beta-scission. We also demonstrate that AdoMet is a catalytic cofactor that is not consumed during turnover. Together, these results support a novel radical-based mechanism for the repair of UV-induced DNA damage.     

VARIATION IN THE PHOTOCHEMICAL REACTIVITY OF THYMINE IN THE DNA OF B. SUBTILIS SPORES, VEGETATIVE CELLS AND SPORES GERMINATED IN CHLORAMPHENICOL*,†

Abstract— The chief photoproduct of thymine produced in u.v. irradiated (2537Å) vegetative cells of B. subtilis is the cyclobutane-type dimer while in spores very little of this dimer is produced (maximum yield 2·6 per cent of thymine) but a new photoproduct is produced in high yield (maximum of 28·4 per cent of thymine). This difference in photochemical response appears to be due, at least in part, to a difference in uydration of the DNA. The photochemistry of thymine in isolated DNA irradiated in solution is similar to that of DNA in irradiated vegetative cells, but differs markedly from that of isolated DNA irradiated dry. The yield of cyclobutane-type thymine dimer is much reduced in isolated DNA irradiated dry but a new photoproduct of thymine. is produced which is chromatographically similar to the spore photoproduct. The yield of this photoproduct, however, is never as great as that obtained in irradiated spores. The photochemistry of the DNA thymine of spores germinated in the presence of chloramphenicol is very similar to that of normal vegetative cells. Except for hydration, the physical state of the DNA is probably not otherwise altered by germination in the presence of chloramphenicol since DNA replication is prevented by the presence of chloramphenicol. These results are also consistent with the hypothesis that the unique photochemistry of spores is due, at least in part, to the hydration state of the DNA. The acid stability of the spore photoproduct is indicated by the fact that it is isolated from irradiated spores after hydrolysis in trifluoroacetic acid at 155°C for 60 min. It still contains the methyl group of thymine as judged by the fact that for a given dose of u.v. the same yield of photoproduct was obtained whether the spores were labeled with thymine-2–C-14 or -methyl-C-14. This photoproduct is stable to reirradiation (2537Å) in solution under condiditions where thymine dimers of the cyclobutane-type are completely converted back to monomeric thymine. On a column of molecular sieve material (Sephadex-G10), the spore photoproduct elutes in a region intermediate between the cyclobutanetype thymine dimers and monomeric thymine. Of the numerous compounds tested by paper chromatography, the spore photoproduct is most similar (but not identical) in several solvents to 5–hydroxyuracil and 5–hydroxymethyluracil. Our data do not allow us to decide if the product is a monomer or a dimer. Although the photochemistry of thymine in the DNA of spores differs markedly from that for vegetative cells, several lines of evidence make it seem doubtful that the enhanced resistance of spores to u.v. relative to that of vegetative cells can be explained solely on the basis of this difference in the photochemistry of DNA thymine.     

Photochemistry and Photobiology of the Spore Photoproduct: A 50‐Year Journey

Fifty years ago, a new thymine dimer was discovered as the dominant DNA photolesion in UV‐irradiated bacterial spores [Donnellan, J. E. & Setlow R. B. (1965) Science, 149, 308–310], which was later named the spore photoproduct (SP). Formation of SP is due to the unique environment in the spore core that features low hydration levels favoring an A‐DNA conformation, high levels of calcium dipicolinate that acts as a photosensitizer, and DNA saturation with small, acid‐soluble proteins that alters DNA structure and reduces side reactions. In vitro studies reveal that any of these factors alone can promote SP formation; however, SP formation is usually accompanied by the production of other DNA photolesions. Therefore, the nearly exclusive SP formation in spores is due to the combined effects of these three factors. Spore photoproduct photoreaction is proved to occur via a unique H‐atom transfer mechanism between the two involved thymine residues. Successful incorporation of SP into an oligonucleotide has been achieved via organic synthesis, which enables structural studies that reveal minor conformational changes in the SP‐containing DNA. Here, we review the progress on SP photochemistry and photobiology in the past 50 years, which indicates a very rich SP photobiology that may exist beyond endospores.     

Structure determination of an interstrand-type cis-anti cyclobutane thymine dimer produced in high yield by UVB light in an oligodeoxynucleotide at acidic pH

UVB irradiation of DNA produces photodimers in adjacent DNA bases and on rare occasions in nonadjacent bases. UVB irradiation (312 nm) of d(GTATCATGAGGTGC) gave rise to an unknown DNA photoproduct in approximately 40% yield at acidic pH of about 5. This product has a much shorter retention time in reverse phase HPLC compared to known dipyrimidine photoproducts of this sequence. A large upfield shift of two thymine H6 NMR signals and photoreversion to the parent ODN upon irradiation with 254 nm light indicates that the photoproduct is a cyclobutane thymine dimer. Exonuclease-coupled MS assay establishes that the photodimer forms between T2 and T7, which was confirmed by tandem mass spectrometric MS/MS identification of the endonuclease P1 digestion product pd(T2[A3])=pd(T7[G8]). Acidic hydrolysis of the photoproduct gave a product with the same retention time on reverse phase HPLC and the same MS/MS fragmentation pattern as authentic Thy[ c,a]Thy. 2D NOE NMR data are consistent with a cis-anti cyclobutane dimer between the 3'-sides of T2 and T7 in anti glycosyl conformations that had to have arisen from an interstand type reaction. In addition to pH dependency, the photoproduct yield is highly sequence specific and concentration dependent, indicating that it results from a higher order folded structure. The efficient formation of this interstrand-type photoproduct suggests the existence of a new type of folding motif and the possibility that this type of photoproduct might also form in other folded structures, such as G-quadruplexes and i-motif structures which can be now studied by the methods described.     

Neutral histidine and photoinduced electron transfer in DNA photolyases

The two major UV-induced DNA lesions, the cyclobutane pyrimidine dimers (CPD) and (6-4) pyrimidine-pyrimidone photoproducts, can be repaired by the light-activated enzymes CPD and (6-4) photolyases, respectively. It is a long-standing question how the two classes of photolyases with alike molecular structure are capable of reversing the two chemically different DNA photoproducts. In both photolyases the repair reaction is initiated by photoinduced electron transfer from the hydroquinone-anion part of the flavin adenine dinucleotide (FADH(-)) cofactor to the photoproduct. Here, the state-of-the-art XMCQDPT2-CASSCF approach was employed to compute the excitation spectra of the respective active site models. It is found that protonation of His365 in the presence of the hydroquinone-anion electron donor causes spontaneous, as opposed to photoinduced, coupled proton and electron transfer to the (6-4) photoproduct. The resulting neutralized biradical, containing the neutral semiquinone and the N3'-protonated (6-4) photoproduct neutral radical, corresponds to the lowest energy electronic ground-state minimum. The high electron affinity of the N3'-protonated (6-4) photoproduct underlines this finding. Thus, it is anticipated that the (6-4) photoproduct repair is assisted by His365 in its neutral form, which is in contrast to the repair mechanisms proposed in the literature. The repair via hydroxyl group transfer assisted by neutral His365 is considered. The repair involves the 5'base radical anion of the (6-4) photoproduct which in terms of electronic structure is similar to the CPD radical anion. A unified model of the CPD and (6-4) photoproduct repair is proposed.     

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.     

PHOTOCHEMISTRY OF 4-THIOURIDINE AND THYMINE

Abstract— When thymine is irradiated in aqueous solution with monochromatic 334-nm UV radiation in the presence of 4-thiouridine a photoproduct of thymine is formed, as shown by thin-layer chromatography and autoradiography. The quantum yield for the formation of thymine photoproduct (θ=0.017) is greater than that for cytosine photoproduct formation (θ= 0.0015). The identity of the photoproduct is not known: one possibility is the formation of an adduct between the sensitizer and the base yielding a pyrimidine-pyrimidone type of photoproduct.     

Role of sequence and conformation on the photochemistry and the photophysics of A-T DNA dimers: an experimental and computational approach

The role of base sequence and conformation on the photochemistry and photophysics of thymidylyl (3'-5')-2'-deoxyadenosine sodium salt (TpdA) and 2-deoxyadenylyl (3'-5')-thymidine ammonium salt (dApT) was studied. To this end, nanosecond transient absorption at 266 nm, steady-state irradiation at 254 nm, and quantum chemical calculations were used. The transient absorption spectra show the solvated electron broad band in the visible region for each dinucleotide. In addition, low-intensity absorption bands are observed in the UV region, which are attributed to the deprotonated and protonated neutral radicals of adenine and thymine bases. Photoionization (PI) occurs by one- and two-photon pathways; the latter accounting for approximately 70% of the net PI yield. A diffusion-limited rate constant of 2.0 x 10(10) M(-1) s(-1) was obtained for the reaction of the neutral molecule with the photoejected electron in both sequences. The photodestruction yield, measured from the chromophore loss at 260 nm, decreases in the presence of well-known electron scavengers. This suggests the participation of base radical anions as one of the photodegradation pathways, which is higher in TpdA than in dApT. The intermediacy of a radical ion pair (charge separated state) between the adjacent adenine and thymine bases is proposed in the formation of the [2 + 2] cycloadduct intermediate. The [2 + 2] cycloadduct intermediate is known to be the precursor of the thymine-adenine eight-member ring photoproduct (TA*). Conformational constrains in the radical ion pair are suggested to explain the absence of the TA* photoproduct in dApT. This hypothesis is supported by semiempirical calculations performed on all relevant reactive intermediates proposed to participate in the mechanism of formation of TA*. Altogether, the results show that sequence and conformation profoundly influence the photochemistry and the photophysics of these DNA model systems.     

Structural factors controlling the action of UV light in nucleic acids

Abstract— UV light induced conformational effects of different deoxyoligonucleotides and deoxypolynucleotides containing thymine and adenine residues are investigated by means of CD measurements and quantum yield calculations. UV-irradiation at the wavelengths 254 , 280 and 313 nm indicate that unsensitized irradiation at low doses leads to thymine photoproduct formation of non-cyclobutane type. In contrast to that irradiation at 313 nm in the presence of acetophenone causes different changes in the CD spectra due to the formation of thymine dimers of the cyclobutane type structure. Quantum yield calculations demonstrate a pronounced dependence of the photoproduct formation on the nucleotide sequence of the oligomers. Thus, clustering of thymine dimer formation can be neglected. Adenine photoproducts in the (A.T) containing oligomers are only formed at higher fluences. > 1.5 × 10⁴ J/m² and are biological less important events.     

INFLUENCE OF pH AND OXYGEN ON 315 mμ INACTIVATION AND THYMINE DIMERIZATION IN MUTANTS OF BACTERIOPHAGE T4

Abstract— 1. Irradiation with 315 mμ light inactivates phage T4v-x C, and T4v^-x- , and forms thymine dimers in their DNA.
2. Both the rates of inactivation and of thymine dimerization depend upon pH and gaseous environment during irradiation. The U.V. sensitivities are: 1 (pH 7, N₂, 03, 2.2 (pH 3.5, Oz), 3.3 (pH 3–5, N₂; and the corresponding rates of thymine dimerization 1: 2.5: 5.2. The number of thymine dimers per lethal hit observed withT4v-x + are: 5.7 (pH 7, N₂, O₂, 5.4 (pH 3.5, O₂, 10.9 (pH 3.5, N₂); and forT4v-x-: 4.6, 3.4, and 7.1 with the same sequence of conditions.
3. Also the photoreactivable sectors depend upon the environmental conditions at 315 mp inactivation. In T4v-x f this sector amounts to about 50 per cent at pH 7, 18 per cent at pH 3.5, O., and 29 per cent at pH 3.5, N, respectively.
4. The molecular basis of these findings is discussed. It is concluded that, besides thymine dimer, at least one other lethal photoproduct (probably a photoproduct of cytosine) is involved in photoreactivation.     

Photochemistry of a naphthalene-thymine dyad in the presence of acetone

Irradiation of dyad 1 in aqueous acetone leads to the introduction of an acetonyl substituent at the naphthalene 5-position, to give photoproduct 2. The proposed reaction mechanism involves electron transfer from the naphthalene excited singlet state to the ketone. Neither thymine dimers, nor acetone photoadducts involving the thymine ring were detected. These photoproducts would arise from the thymine triplet excited state, which in dyad 1 must be efficiently depopulated via a fast intramolecular energy transfer to the naphthalene chromophore, due to the lower energy of its excited triplet state.     

MONOFUNCTIONAL 4',5'-PHOTOCYCLOADDUCT BETWEEN 4,5'-DIMETHYLANGELICIN AND THYMINE

Abstract A new fluorescent compound has been isolated from the products of hydrolysis of DNA irradiated in the presence of 4,5'-dimethylangelicin, a monofunctional photosensitizing furocoumarin. The marked similarity of the UV absorption and fluorescence spectra of this photoproduct to those of 4',5'-dihydro-4,5'-dimethylangelicin, as well as its behaviour on photodissociation (254 nm) yielding thymine and 4,5'-dimethylangelicin in equimolecular amounts, are consistent with a cycloadduct between one molecule of 4,5'-dimethylangelicin and one of thymine, and the C₄-cycloaddition occurs through 4',5'–double bond of the furocoumarin and the 5,6-double bond of the pyrimidine.     

Fragmentation of photomodified oligodeoxynucleotides adducted with metal ions in an electrospray-ionization ion-trap mass spectrometer

We report the effect of metal-ion adduction on the fragmentation of oligodeoxynucleotides (ODNs) bearing DNA photoproducts. When protons on backbone phosphates of ODNs are completely replaced with metal ions, cleavages occur readily within the photoproduct moiety, whereas those cleavages do not occur in photomodified ODNs in which the phosphates are associated with protons. For example, thymine/adenine (TA*) photoproducts revert to their undamaged precursors upon collisional activation, the pyrimidine(6-4)pyrimidone product and its Dewar valence isomer show a characteristic neutral loss of C4H3NO3, and dimeric adenine photoproducts show a distinctive loss of NH2CN from the adenine six-membered ring. The product-ion mass spectra of photodamaged ODNs that are adducted to metal ions are complementary in terms of structure information to those spectra of ODNs in which the phosphates are associated with protons. The results also demonstrate that the energy required for strand cleavages is higher for ODNs adducted with metal ions than that for ODNs bound with protons. Furthermore, the loss of a pyrimidine is more favorable than the loss of a purine in the fragmentation of ODNs associated with metal ions.     

2+2] Photocycloaddition reaction dynamics of triplet pyrimidines

Taking the 266 nm excited pyrimidine (uracil or thymine) with cyclopentene as model reaction systems, we have examined the photoproduct formation dynamics from the [2 + 2] photocycloaddition reactions of triplet pyrimidines in solution and provided mechanistic insights into this important DNA photodamage reaction. By combining two compliment methods of nanosecond time-resolved transient IR and UV-vis laser flash-photolysis spectroscopy, the photoproduct formation dynamics as well as the triplet quenching kinetics are measured. Characteristic IR absorption bands due to photoproduct formation have been observed and product quantum yields are determined to be ～0.91% for uracil and ～0.41% for thymine. Compared to the measured large quenching rate constants of triplet uracil (1.5 × 10(9) M(-1)s(-1)) or thymine (0.6 × 10(9) M(-1)s(-1)) by cyclopentene, the inefficiency in formation of photoproducts indicates competitive physical quenching processes may exist on the route leading to photoproducts, resulting in very small product yields eventually. Such an energy wasting process is found to be resulted from T(1)/S(0) surface crossings by the hybrid density functional calculations, which compliments the experiments and reveals the reaction mechanism.     

Kinetic Isotope Effects and Hydrogen/Deuterium Exchange Reveal Large Conformational Changes During the Catalysis of the Clostridium acetobutylicum Spore Photoproduct Lyase

Spore photoproduct lyase (SPL) catalyzes the direct reversal of a thymine dimer 5‐thyminyl‐5,6‐dihydrothymine (i.e. the spore photoproduct (SP)) to two thymine residues in germinating endospores. Previous studies suggest that SPL from the bacterium Bacillus subtilis (Bs) harbors an unprecedented radical‐transfer pathway starting with cysteine 141 proceeding through tyrosine 99. However, in SPL from the bacterium Clostridium acetobutylicum (Ca), the cysteine (at position 74) and the tyrosine are located on the opposite sides of a substrate‐binding pocket that has to collapse to bring the two residues into proximity, enabling the C→Y radical passage as implied in SPL_(Bs). To test this hypothesis, we adopted hydrogen/deuterium exchange mass spectrometry (HDX‐MS) to show that C74_(Ca) is located at a highly flexible region. The repair of dinucleotide SP TpT by SPL_(Ca) is eight‐fold to 10‐fold slower than that by SPL_(Bs); the process also generates a large portion of the aborted product TpTSO₂^?. SPL_(Ca) exhibits apparent (^DV) kinetic isotope effects (KIEs) of ~6 and abnormally large competitive (^DV/K) KIEs (~20), both of which are much larger than the KIEs observed for SPL_(Bs). All these observations indicate that SPL_(Ca) possesses a flexible active site and readily undergoes conformational changes during catalysis.     

WAVELENGTH DEPENDENCE (150–290 nm) OF THE FORMATION OF THE CYCLOBUTANE DIMER AND THE (6–4) PHOTOPRODUCT OF THYMINE

The action cross sections for the formation of the cyclobutane dimer and the (6-4) photoproduct of thymine as well as the absorption cross sections of thymine were determined in the wavelength region between 150 and 290 nm. Thymine films sublimed on glass plates were irradiated by monochromatic photons in a vacuum; the induced photoproducts were quantitatively analyzed by high-performance liquid chromatography (HPLC). Under our conditions, two major peaks appeared on the HPLC chromatograms of irradiated samples. The two peaks were identified as being the cis-syn cyclobutane dimer and the (6-4) photoproduct, based on their HPLC retention times, absorption spectra in the effluent, and photochemical reactivity. The fractions of the two photoproducts increased linearly with the fluence at low fluences over the entire wavelength range. Their action cross sections were determined by the slopes of the linear fluence response curve at 10 nm intervals between 150 and 290 nm. The two action spectra showed a similar wavelength dependence and had a maximum at 270 nm as well as two minor peaks at 180 and 220 nm, at which wavelengths the peaks of the absorption spectrum of thymine sublimed on a CaF2 crystal plate appeared. The quantum yields had relatively constant values of around 0.008 for the dimer and 0.013 for the (6-4) photoproduct above 200 nm, decreasing to 0.003 and 0.006, respectively, at 150 nm as the wavelength became shorter.     

Characterization of the DNA repair spore photoproduct lyase enzyme from Clostridium acetobutylicum: A radical-SAM enzyme

Spore photoproduct lyase (SPL) is a “Radical-SAM” repair enzyme which catalyzes the cleavage of spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine), a specific lesion found in bacterial spore DNA, to thymine monomers by a free-radical mechanism. The enzyme requires S-adenosyl-l-methionine (SAM) and a [4Fe–4S] cluster for activity. SPL from Bacillus subtilis has been difficult to isolate and characterize due to problems with the solubility and stability of the overexpressed protein in Escherichia coli and the lability of the [Fe–S] cluster, even if the protein was purified under strictly anaerobic conditions. In order to overcome these problems we searched for another SPL enzyme and we found that the recombinant SPL enzyme from Clostridium acetobutylicum, isolated either aerobically or anaerobically from overexpressing E. coli, behaves more stably than the B. subtilis one. We report here a complete spectroscopic and biochemical characterization of this enzyme. In particular we show for the first time that, using HYSCORE spectroscopy, SAM binds to the cluster as observed in the case of other members of the “Radical-SAM” enzyme family such as the activases of pyruvate formate lyase and ribonucleotide reductase.     

Facile synthesis of a cis-syn thymine dimer building block and its incorporation into oligodeoxynucleotides

The synthesis of a building block containing the photobiologically relevant cis-syn thymine cyclobutane photoproduct and its incorporation into oligonucleotides by the phosphoramidite-based solid-phase synthesis is reported. Compared to previous syntheses, this route is extremely short and allows such modified oligonucleotides to be easily available for biological studies.     

Far-UV-lnduced Dimeric Photoproducts in Short Oligonucleotides: Sequence Effects

Cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone adducts represent the two major classes of far-UV-induced DNA photoproducts. Because of the lack of appropriate detection methods for each individual photoproduct, little is known about the effect of the sequence on their formation. In the present work, the photoproduct distribution obtained upon exposure of a series of dinucleoside monophosphates to 254 nm light was determined. In the latter model compounds, the presence of a cytosine, located at either the 5′- or the 3′- side of a thymine moiety, led to the preferential formation of (6-4) adducts, whereas the cis-syn cyclobutane dimer was the main thymine-thymine photoproduct. In contrast, the yield of dimeric photoproducts, and particularly of (6-4) photoadducts, was very low upon irradiation of the cytosine–cytosine dinucleoside monophosphate. However, substitution of cytosine by uracil led to an increase in the yield of (6-4) photoproduct. It was also shown that the presence of a phosphate group at the 5′- end of a thymine-thymine dinucleoside monophosphate does not modify the photoproduct distribution. As an extension of the studies on dinucleoside monophosphates, the trinucleotide TpdCpT was used as a more relevant DNA model. The yields of formation of the thymine-cytosine and cytosine–thymine (6-4) photoproducts were in a 5:1 ratio, very close to the value obtained upon photolysis of the related dinucleoside monophosphates. The characterization of the two TpdCpT (6-4) adducts was based on H NMR, UV and mass spectroscopy analyses. Additional evidence for the structures was inferred from the analysis of the enzymatic digestion products of the (6-4) adducts of TpdCpT with phosphodiesterases. The latter enzymes were shown to induce the quantitative release of the photoproduct as a modified dinucleoside monophosphate in a highly sequence-specific manner.     

2 + 2 Dimerization of cinnamylidene malonic acid: A structural and kinetic study

Cinnamylidene malonic acid was synthesized and its crystal structure obtained. The dicarboxylic acid hydrogen-bonding motif of this structure consists of catemer chains that lie along the c- glide plane. A photo induced 2?+?2 cycloaddition occurred upon exposure of the crystals to UV light which resulted in the destruction of the crystal. The structure of the photo products were determined by IR and NMR analysis. The regio- and stereo-chemistry of the photoproduct can be rationalized by examining the relative orientation and symmetry relating reacting molecules within the crystal structure. A kinetic study demonstrated first order reaction kinetics which is consistent with a reaction occurring under topochemical control.