3'- and 5'-strand cleavage reactions catalyzed by the Fpg protein from Escherichia coli occur via successive beta- and delta-elimination mechanisms, respectively

The Fpg protein from Escherichia coli is a multifunctional protein that excises damaged purine bases from DNA to generate aldehydic abasic sites and then catalyzes the successive cleavage of the phosphodiester bonds first on the 3'-side and then on the 5'-side of the abasic site to generate 5'- and 3'-phosphate ends, respectively, thereby excising the deoxyribose residue, The mechanisms of the 3'- and 5'-strand cleavage reactions have been studied by nuclear magnetic resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS). The 3'-strand cleavage reaction is a beta-elimination reaction in which the 2'-hydrogen is abstracted and the 3'-phosphate is eliminated. The 5'-strand cleavage reaction is a delta-elimination reaction in which the 4'-hydrogen is abstracted and the S-phosphate is eliminated. Two types of experiments were performed to establish the occurrence of the sequential elimination reactions. First, when the reaction was performed in (H2O)-O-18, P-31 NMR demonstrated that neither phosphate group contained O-18. Second, the five-carbon product derived from the deoxyribose residue was stabilized by reduction with NaBH4 and characterized by GC-MS, The mass spectrum of the reduced product was identical to that of authentic 4-oxo-2-pentenal, the tautomerized product of successive beta- and delta-elimination reactions.