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

Abstract

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 5'-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 H2(18)O, 31P NMR demonstrated that neither phosphate group contained 18O. 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.