Thymine dimerization, quantum

Finally a few sentences are deserved for the vast area of DNA photochemistry. Thymine dimerization is the most common photochemical reaction with the quantum yield of formation in isolated DNA of all-thymine oligodeoxynucleotides 2-3% [3], Furthermore, a recent study based on femtosecond time-resolved transient absorption spectroscopy showed that thymine dimers are formed in less than 1 ps when the strand has an appropriate conformation [258], The low quantum yield of the reaction in regular DNA is suggested to be due to the infrequency of these appropriate reactive conformations. 

Biological, chemical. X-ray diffraction, infrared absorption, e.s.r., n.m.r., luminescence, and quantum studies show that dimer formation is universally observed in irradiated frozen solutions of thymine, thymidine, uridine, thymidylic acid and related compounds, and in DNA [560—576]. The purines of DNA, on the other hand, are little affected [577, 578]. Thymine dimers obtained in frozen solution can be converted to the original monomers by ultraviolet... 

The effect of phase upon the monomer-dimer equilibrium is pronounced. The quantum yields for dimer formation in liquid-aerated water solution are low (from zero for thymine to 10"2 for other pyrimidines) but the quantum yields for dimer formation in frozen aqueous solutions or in single crystals are much higher (reaching unity in frozen water solution for thymine). The quantum yields for monomerization are uniformly high and are about the same in solution or in solid phase. The net result of this phase effect is that even at optimum wavelengths for dimer formation, the yields of dimers are low in solution and high in solid phases, for all the single bases, nucleosides, or nucleotides. 

One obvious reason for the apparent lack of reaction of thymine in water is that the quantum yield for splitting of thymine dimer in solution is 0.5-1.0,28 independent of wavelength, and the probable yield for formation would not be higher, by analogy with other pyrimidines, than... 

Photolysis of a frozen aqueous solution of thymine at 254 nm gives a major product with a reported quantum yield of 1-2,31 and a minor product.32 The abundance ratio of the two products is about 8. The major product is the known thymine dimer.7,10 Identity of the minor product is unknown photolysis of it in solution produces thymine and a dimeric product which chromatographs like the major product. This minor product is alkali labile. Existence of other products in the thawed photolysis solution is suggested by details of the spectrum.32... 

Despite the frequent use of photoreversal as a measure of dimer formation in many biological materials, few detailed studies of this phenomenon have been made. The action spectrum for photoreversal of uracil dimer (and thymine dimer) has been reported by Swenson and Setlow,48 and is shown in Figure 1. Purified, crystalline dimer was used in these studies. The quantum yield for dimer breaking was found to be 0.45, one-half of that for monomerization of thymine dimers.49... 

It should be noted here that thymine photodimerization may occur by a non-concerted mechanism, involving free radical intermediates. Indeed, photoproducts other than cis-syn dimer, such as the next most abundant thymine dimer, so-called 6 4 adduct, were observed in irradiated DNA. However, the quantum yield of cis-syn photodimer formation (r/j 0.02) is more than an order of magnitude higher than that of the 6 4 adduct ( 0.0013) which in turn is an order of magnitude higher than the quantum yields for other thymine isomers [68]. This specificity can lead to the conclusion that the thymine photodimerization occurs predominantly via concerted 2 + 2 cycloaddition mechanism. A time-resolved study of thymine dimer formation demonstrated that thymine cyclobutane dimers are formed on a timescale of less than 200 nsec, while the 6 4 adduct is formed on a timescale of few milliseconds [69]. The delay in the formation of the latter was attributed to the mechanism of its formation through a reactive intermediate. 

There are two different methods of splitting a thymine dimer photochemical and enzymatic. In the photochemical method, the sample containing thymine dimers is irradiated with UV light. Splitting of the thymine cyclobutane dimer follows the same symmetry rules as its formation it is thermally forbidden but photochemically allowed. When a dimer absorbs a photon of suitable wavelength (2 240 nm), it reacts with a quantum yield of nearly 100% forming two thymines [60]. In the enzymatic method an enzyme recognizes thymine dimers and repairs them. It may require the absorption of a photon, or it may happen in the dark. 

Based on the model studies discussed above, an analogous mechanism for the DNA-PL-catalyzed cleavage of pyrimidine dimers would occur via initial photoactivation of one or both of the enzyme cofactors, followed by electron transfer between the activated photosensitizer and enzyme-bound dimer. The studies by Joms indicated that only reduced flavin is required to initiate thymine dimer cleavage in the model reaction 162). This is consistent with the observation that the quantum yield for the blue semiquinone radical form of E. coli DNA-PL in vitro was found to be much lower than the in vivo values. Treatment of the isolated enzyme with dithionite to give fully reduced flavin cofactor then results in a 12- to 15-fold increase in the quantum yield, suggesting that the flavin cofactor is in the reduced oxidation state in vivo 163). 

Weinblum, D., Determination of the quantum yield of reversal in crystalline dimethyl thymine dimer, syn head to head, Photochem. PhotobioL, 12, 509, 1970. 

This apparently happens in the excited singlet dedimerization of thymine photodimer in a rigid matrix as well as subsequent excited singlet dimerization of the pair of thymine molecules formed, since quantum yields of both processes are unity.176)... 

The water lattice may be an important element in forming the ordered thymine structure necessary for dimerization, as pointed out by Beukers and Berends.37 Thymine can crystallize from solution as a monohydrate (a real hydrate)38 in whose crystal lattice one thymine is directly above another. The influence of humidity upon dimer yield in dry films may be connected with monohydrate formation, and monohydrate formation in frozen solutions may be the reason for the almost theoretically maximum quantum yields for dimer formation.31 The possible existence of aggregates in frozen aqueous solutions is supported by a tenfold increase in purine phosphorescence at 44°K produced by the presence of 1% ethanol and by a blue shift of excitation and emission spectra.39... 

Lamola and Eisinger169 have shown that the oriented adjacent pairs of 1,3-dimethyl-thymine monomers formed by photosplitting of the appropriate dimer in frozen solution will photodimerize again with a quantum yield of close to unity, and that the pair of monomers shows no fluorescence, but an exciton splitting in the absorption spectrum. 

Irradiation of frozen aqueous thymine solutions produces the cis head-to-head (chh) dimer, the high quantum yield (0.5-1.0) being attributed to the preferred orientation of adjacent molecules in the microcrystalline thymine hydrate. The gradual isolation of substrate molecules in the photodimer matrix is associated135 with the appearance and increase in intensity of molecular fluorescence as photodimerization proceeds identical behavior... 

The foregoing is of obvious significance in relation to the photochemical behaviour of cyclobutane photodimers of natural pyrimidines such as uracil (III, Scheme 1), thymine, cytosine12,76). Photodissociation of such photodimers to the parent monomers, which proceed with quantum yields ranging from 0.5 to nearly unity, has, indeed, been employed as one of the criteria for identification of such dimers. The validity of this criterion is now, in the light of the behaviour of the dimer electroreduction product of pyrimidone-2, at best somewhat restricted, notwithstanding that the quantum yields for photodissociation of the latter are lower. 

Quantum efficiencies of the thymine derivatives for the photodimer formation were measured in DMSO, DMF and DMSO-EG solution (Table 13). The values for the dimeric model compound (T-T) are almost identical in different solvents. The values for the polymers are higher than that for T-T, and different in these solvents. For the polymers, quantum efficiencies are in the following order in DMSO < in DMF < in DMSO-EG. 

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