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

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.