Identification and Characterization of an Intermediate in the Alkali Degradation of (6-4) Photoproduct-containing DNA

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°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 UV light in solar radiation is absorbed by nucleobases in DNA and induces photochemical reactions. The major forms of DNA damage by UV radiation are the cis-syn cyclobutane pyrimidine dimer (CPD) 1 and the pyrimidine(6-4)pyrimidone photoproduct ((6-4) photoproduct) formed between adjacent pyrimidine bases (1). The formation of these lesions not only induces mutations of genetic information (2) but also alters the chemical stability of DNA. In the case of the CPD formed at the sequences containing cytosine, the hydrolysis rate of the amino function is much higher than that of the normal cytosine base (3), which results in the C3 T transition (4 -6). The (6-4) photoproduct was originally identified as a UV-induced alkalisensitive lesion (7,8). Strand breaks occur at the sites of this photoproduct when UV-irradiated DNA is treated with hot alkali, and this procedure has been used to map the (6-4) photoproduct in defined DNA sequences at nucleotide resolution (9 -12). This alkali lability has also been applied for footprinting experiments to analyze protein-DNA interactions in vivo (13,14). 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. Franklin et al. (8) suggested the hydrolysis of the glycosyl bond of the 3Ј component of this photoproduct, but there have been no subsequent reports.The (6-4) photoproduct is reportedly more mutagenic than the CPD (17-19) and blocks replication without being bypassed by error-free translesion synthesis, whereas DNA polymerase correctly incorporates dATP opposite the CPD formed at the TT sequence (20, 21). To avoid mutagenesis and carcinogenesis by the (6-4) photoproduct, this damage must be removed by the nucleotide excision repair (NER) pathway in cells (22)(23)(24). No base excision repair (BER) enzyme is known for the (6-4) photoproduct, but if the photoproduct can be degraded artificially, this DNA may be repaired by the BER pathway, even in xeroderma pigmentosum patients lacking NER function. For such an application, it is important to characterize the chemical properties of the (6-4) photoproduct.In this paper we describe the analysis of the alkali degradation of DNA containing the (6-4) photoproduct using chemically synthesized oligonucleotides. A relati...

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