3'-STRAND AND 5'-STRAND CLEAVAGE REACTIONS CATALYZED BY THE FPG PROTEIN FROM ESCHERICHIA-COLI OCCUR VIA SUCCESSIVE BETA-ELIMINATION AND DELTA-ELIMINATION MECHANISMS, RESPECTIVELY

The Fpg protein from Escherichia coli is a multifunctional protein tha t excises damaged purine bases from DNA to generate aldehydic abasic s ites 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 cleav age reactions have been studied by nuclear magnetic resonance spectros copy (NMR) and gas chromatography-mass spectrometry (GC-MS). The 3'-st rand cleavage reaction is a beta-elimination reaction in which the 2'- hydrogen is abstracted and the 3'-phosphate is eliminated. The 5'-stra nd cleavage reaction is a delta-elimination reaction in which the 4'-h ydrogen is abstracted and the S-phosphate is eliminated. Two types of experiments were performed to establish the occurrence of the sequenti al elimination reactions. First, when the reaction was performed in (H 2O)-O-18, P-31 NMR demonstrated that neither phosphate group contained O-18. Second, the five-carbon product derived from the deoxyribose re sidue was stabilized by reduction with NaBH4 and characterized by GC-M S, The mass spectrum of the reduced product was identical to that of a uthentic 4-oxo-2-pentenal, the tautomerized product of successive beta - and delta-elimination reactions.