Nuclease P1 Digestion Combined with Tandem Mass Spectrometry for the Structure Determination of DNA Photoproducts

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Abstract
UV irradiation of oligodeoxynucleotides at 254 nm generates several different types of DNA photoproducts, such as cis-syn cyclobutane pyrimidine dimers, pyrimidine[6−4]pyrimidone photoproducts and their Dewar valence isomers, and thymine-adenine photoproducts (TA*). Nuclease P1 degrades the oligodeoxynucleotide photoproducts to small photoproduct-containing trinucleotides which are more amenable to tandem mass spectrometry (MS/MS) and HPLC. Product-ion mass spectra of these digestion products give characteristic fragmentations, allowing us to identify quickly the types of photomodifications. The results also show that mass spectrometry will be a tool for studying enzyme reaction mechanisms because it can determine rapidly and with high sensitivity the structures of the products that are generated.