Spectroscopic analysis of the pyrimidine(6-4)pyrimidone photoproduct: Insights into the (6-4) photolyase reaction

We synthesized a dinucleoside monophosphate of the ¹⁵N-labeled (6-4) photoproduct, which is one of the major UV-induced lesions in DNA, to investigate the (6-4) photolyase repair mechanism, and characterized its protonation state by measuring ¹⁵N NMR spectra as a function of pH. We expected that chemical-shift changes of the pyrimidone ¹⁵N3, due to protonation, would be observed at pH 3, as observed for the ¹⁵N-labeled 5-methylpyrimidin-2-one nucleoside. Interestingly, however, the changes were observed only in alkaline solutions. In UV absorption spectroscopy and HPLC analyses under acidic conditions, a change in the maximum absorption wavelength, due to the protonation-induced hydrolysis, was observed at and below pH 1, but not at pH 2, whereas the protonation of 5-methylpyrimidin-2-one occurred at pH values between 2 and 3. These results indicated that the pK_a value for this N3 is remarkably lower than that of a normal pyrimidone ring, and strongly suggest that an intramolecular hydrogen bond is formed between the N3 of the 3′ base and the 5-OH of the 5′ base under physiological conditions. The results of this study have implications not only for the recognition and reaction mechanisms of (6-4) photolyase, but also for the chemical nature of the (6-4) photoproduct.