Pyrimidines, dimeric, absorption

The purified E. coli protein has a molecular weight of 49 kD. It does not require any divalent cation for activity. It contains two different noncovalently bound chromophores that absorb light. One chromophore is flavin adenine dinucleotide (FADH- or FADH2). The other is 5,10-methenyltetrahydrofolyl polyglutamate (MTHF). The absorption of light by the chromophores is essential for the enzymatic reversal of the pyrimidine dimer back to the original pyrimidine monomers. However,... 

An example of direct repair is the photochemical cleavage of pyrimidine dimers. Nearly all cells contain a photoreactivating enzyme called DNA photolyase. The E. coli enzyme, a 35-kd protein that contains bound N lO-methenyltetrahydrofolate and flavin adenine dinucleotide cofactors, binds to the distorted region of DNA. The enzyme uses light energy—specifically, the absorption of a photon by the N, N lO-methenyltetrahydrofolate coenzyme—to form an excited state that cleaves the dimer into its original bases. 

Scheme 34. Mechanism for pyrimidine dimer cleavage by blue photolyase and fully reduced enzyme (163). FIH-, FAD neutral blue radical FIH2, FADH2 SC, second chromophore , excited state due to absorption of light +, radical cation radical anion fT, pyrimidine dimer 2T, repaired pyrimidine monomers.

Ultraviolet (UV) light is another agent that causes damage to DNA. Absorption of UV light by DNA causes adjacent pyrimidine bases to become covalently linked. The product (Figure 24.20) is called a pyrimidine dimer. As a result of pyrimidine dimer formation, there is no hydrogen bonding between these pyrimidine molecules and the complementary bases on the other DNA strand. This stretch of DNA cannot be replicated or transcribed ... 

Potentially deleterious side reactions are avoided. The enzyme itself might be damaged by light if it could be activated by light in the absence of bound DNA harboring a pyrimidine dimer. The DNA-induced absorption band is reminiscent of the glucose-induced activation of the phosphotransferase activity of hexokinase. 

The reaction mechanism can be summarized as follows. In a dark reaction, the enzyme binds to DNA and flips out the pyrimidine dimer from the double helix into the active cavity. After the photochemical repair, the reaction partners are moved out of the cavity. As shown in Scheme 8.7, MTHF (or alternatively 8-HDF) is converted into an excited state, MTHF, upon absorption of a photon. 

The main cause of non-melanoma skin cancers are mutations due to formation of cyclo-pyrimidine dimers in the DNA, which occur after absorption of UVB by the DNA, as the absorption spectrum of DNA shows some overlap with the UVB range. Apart from this direct interaction, UVB and UVA can indirectly induce damage to the DNA via formation of radicals and reactive oxygen species (Fig. 3). 

The first is the formation of cyclobutane pyrimidine dimers (CPDs), accounting for 75 % of direct UV-induced damage to DNA. Absorption of UV-B by thymine or cytosine causes a [2 - - 2] cycloaddition between the carbon-carbon double bonds of neighbouring bases, resulting in a four-membered ring structure, 47 or 52. 

DNA photolyases, which use the energy of blue light to split pyrimidine dimers formed by UV irradiation of DNA, provide other examples of large and variable shifts in the absorption spectmm of a bound chromophore. These enzymes contain a bound pterin (methylenetetrahydrofolate, MTHF) or deazaflavin, which serves to absorb light and transfer energy to a flavin radical in the active site [81]. The absorption maximum of MTHF occurs at 360 nm in solution, but ranges from 377 to 415 nm in the enzymes from different organisms [82]. 

As in the case of the dimeric products, the structure of the hydrates has been demonstrated in only a couple of cases and in fact the hydrates themselves have been isolated in only a few cases. Hydrates are generally characterized by recovery of all or a fraction of the original pyrimidine absorption at 260-280 nm when the solution containing the photoproduct is heated for a few minutes at a low pH. Particularly stable hydrates can often be isolated and warmed in aqueous solution, and the formation of the unhydrated material followed quantitatively with positive identification of the recovered pyrimidine by chromatography. 

Flatta FI, Zhou L, Mori M,Teshima S, Nishimoto S (2001) N(1)-C(5 )-linked dimer hydrates of 5-substi-tuted uracils produced by anodic oxidation in aqueous solution. J Org Chem 66 2232-2239 Flayon E (1969) Optical-absorption spectra of ketyl radicals and radical anions of some pyrimidines. J Chem Phys 51 4881-4892... 

Both the foregoing photoadducts exhibit three diffusion-controlled polarographic waves in aqueous medium in the pH range 1-12 92). The first two waves are due to successive one-electron additions, followed by the third, a two-electron reduction step. The pH-dependence of the initial one-electron reduction wave was found to be similar to that for pyrimidone-2. Following electrolysis, at the crest of wave I, of either of the photoadducts, the two one-electron waves disappeared and the reduction products exhibited UV absorption spectra with a band at 375 nm. A similar absorption band is exhibited following sodium borohydride reduction of Cyt(5-4)Pyo, leading to a product identified as 5-(4-pyrimidin-2-one)-3,6-dihydrocytosine. Mass spectroscopy revealed that the products of reduction on wave I were dimers consisting of two molecules of reduced photoadducts 92). 

It must, nonetheless, be emphasized that the products of reduction of pyrimidine have not been unequivocally identified, largely due to their instability in the presence of air (oxygen). Furthermore, the UV absorption spectra of the reduction products of waves I and II (kmax284 nm, smax 1.5 x 103) are suggestive of rapid conversion (proton-catalyzed hydration ) of the products, since both the dimer and the dihydro derivative possess a reduced system of aromatic bonds relative to the parent pyrimidine, as a result of which the UV absorption maximum should be shifted to the violet, whereas it is, in fact, shifted 44 nm to the red (from 240 nm to 284 nm) for both products. Of possible relevance to this is the fact that the reduced rings of 4-aminopyrimidine 102) and nicotinamide 103) undergo acidic hydration to form products absorbing at 280 to 290 nm. 

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