Unambiguous determination of the stereochemistry of nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli.
Nucleotidyl transfer catalyzed by DNA polymerase I from Escherichia coli proceeds with greater than 97% inversion of configuration at P alpha of the alpha-phosphorothioate analogue of dATP. This is shown by experiments in which dAMPS,18O2 is stereospecifically phosphorylated to (Sp)-dATP alpha S, alpha 18O2, which is then copolymerized with dTTP by DNA polymerase. The product of the polymerization is degraded to dAMPS,18O by methods that do not affect the configuration of the phosphorothioate. After the dAMPS,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 non-bridging in (Sp)-dATP alpha S, alpha 18O2, the phosphorothioate configuration must have been inverted in the polymerization reaction. This confirms the determination of P. M. J. Burgers & F. Eckstein [(1979) J. Biol. Chem. 254, 6889-6893], who used kinetic correlations based on the stereoselectivity of snake venom phosphodiesterase to deduce the stereochemistry of this reaction.
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D002250:Carbon Radioisotopes; D004256:DNA Polymerase I / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D010103:Oxygen Isotopes; D013873:Thionucleotides / Q000138:chemical synthesis*
D002250:Carbon Radioisotopes; D010103:Oxygen Isotopes; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D000255:Adenosine Triphosphate; D004256:DNA Polymerase I; D004259:DNA-Directed DNA Polymerase