Mechanism of the alkali degradation of (6–4) photoproduct-containing DNA

Abstract

The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5' component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3' component. The sugar moiety of this component was lost, even when a 3'-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3' component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2'-deoxycytidine 5'-monophosphate, while 2'-deoxyguanosine 3'-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3' component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3' component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct.

Using Organic Synthesis and Chemical Analysis to Understand the Photochemistry of Spore Photoproduct and Other Pyrimidine Dimers

“…70 This induced strand cleavage offers a functional assay for the analysis of 6-4PP formed in the genomic DNA, 71 although some studies question whether 6-4PP is truly alkaline labile. 72,73 It was reported previously that alkaline treatment of 6-4PP results in a similar hydration product possible via the gem-diol intermediate at the C4 followed by rupture of the N3-C4 bond. 72,74 Our group, however, demonstrated that the 6-4PP hydrate may not be stable enough to be detected as it undergoes a rapid esterification reaction facilitated by the 5-OH group, resulting in a stable 2-oxazolidinone (5-4) pyrimidone product after eliminating a molecule of ammonia (Scheme 10).…”

An Unexpected Deamination Reaction after Hydrolysis of the Pyrimidine (6-4) Pyrimidone Photoproduct

“…The small mass difference (∼0.02 amu) due to the involvement of various isotopes can be readily resolved by high-resolution mass spectrometry. 22 This observation further suggests that 1 is formed by hydrolysis of 6-4PP (+ 18 amu due to the added water) followed by loss of an ammonia (− 18 amu due to the loss of 15 NH 3 ).…”

“…Typically, a mass of 17 amu corresponds to an ammonia molecule; we thus wonder whether the amino moiety attached to the C2O at the 5′-thymine of the hydrolysis product has been removed. To test this hypothesis, we first incorporated a 15 N label at the N3 position via a N-nitration reaction of the uridine residue 23 and synthesized a dinucleotide TpT with the 5′-thymine containing the 15 N label. The corresponding 6-4PP was then prepared photochemically as described above.…”

“…The corresponding 6-4PP was then prepared photochemically as described above. The labeled 6-4PP exhibits an [M − H] − signal of 546.13, and a single peak at 191.7 ppm in the 15 N NMR spectrum (Figure 2).…”

“…The 15 N label provides a marker for NMR analysis, enabling us to follow the 6-4PP hydrolysis reaction via NMR spectroscopy. During the 4 days of reaction in 0.3 M KOD, a gradual loss of the 15 N signal at 191.7 ppm was observed, in agreement with the decomposition of 6-4PP. This signal loss is accompanied by the growth of a new 15 N peak at 0 ppm (Figure 2).…”