Abstract
The (6-4) photoproduct formed by ultraviolet light is known as an alkali-labile DNA lesion. Strand breaks occur at (6-4) photoproducts when UV-irradiated DNA is treated with hot alkali. We have analyzed the degradation reaction of this photoproduct under alkaline conditions using synthetic oligonucleotides. A tetramer, d(GT(6-4)TC), was prepared, and its degradation in 50 mm KOH at 60 degrees C was monitored by high performance liquid chromatography. A single peak with a UV absorption spectrum similar to that of the starting material was detected after the reaction, and this compound was regarded as an intermediate before the strand break. The formation of this intermediate was compared with intermediates from the degradation of other alkali-labile lesions such as the abasic site, thymine glycol, and 5,6-dihydrothymine. The results strongly suggested that the first step of the alkali degradation of the (6-4) photoproduct was the hydrolysis between the N3 and C4 positions of the 5'-pyrimidine component. Analyses by NMR spectroscopy and mass spectrometry supported the chemical structure of this product. Assays of the complex formation with XPC.HR23B and the translesion synthesis by DNA polymerase eta revealed that the biochemical properties are indistinguishable between the intact and hydrolyzed photoproducts.
Highlights
UV light in solar radiation is absorbed by nucleobases in DNA and induces photochemical reactions
We expected that small changes in the chemical structure of the photoproduct could be detected by using a short oligomer and that the fragments after the strand break could be identified by HPLC using a photodiode array detector if the nucleotide attached on the 5Ј side of the photoproduct had a different base moiety from that attached on the 3Ј side
One reason was that a tetramer could not be the substrate for uracil DNA glycosylase in the preparation of the AP site-containing oligonucleotide, and the other reason was that relatively long sequences were favorable at the deprotection step in our synthesis of the thymine glycol-containing oligomers (26)
Summary
UV light in solar radiation is absorbed by nucleobases in DNA and induces photochemical reactions. While the reactions of other alkali-labile DNA damages such as the apurinic/apyrimidinic (AP) site (15) and the bleomycin-induced lesion (16) have been analyzed in detail, the mechanism of the strand break caused at the (6-4) photoproduct by the alkali treatment has not been elucidated so far.
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