Unambiguous determination of the stereochemistry of nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli

Abstract

Nucleotidyl transfer catalyzed by DNA polymerase I from E. coli proceeds with > 97% inversion of configuration at P.alpha. of the .alpha.-phosphorothioate analog of dATP. This is shown by experiments in which dAMP [2''-deoxyadenosine-5''-O-phosphorothioate], 18O2 is stereospecifically phosphorylated to (Sp)-dATP.alpha.S [2''-deoxyadenosine-5''-O-(1-thiotriphosphate)],.alpha.18O2, which is then copolymerized with dTTTP by DNA polymerase. The product of the polymerization is degraded to dAMP,18O by methods that do not affect the configuration of the phosphorothioate. After the dAMP,18O is stereospecifically phosphorylated, the resulting (Sp)-dATP.alpha.S,.alpha.18O is copolymerized as before with dTTP. The 18O is found in the displaced pyrophosphate by mass spectral analysis and so must have been in the pyrophosphate bridge of (Sp)-dATP.alpha.S,.alpha.18O. Since this 18O was originally nonbridging in (Sp)-dATP.alpha.S,.alpha.18O2, the phosphorothioate configuration must have been inverted in the polymerization reaction. This confirms the determination of Burgers and Eckstein (1979) who used kinetic correlations based on the stereoselectivity of snake venom phosphodiesterase to deduce the stereochemistry of this reaction.