A study of the mechanism of DNA polymerase I from Escherichia coli with diastereomeric phosphorothioate analogs of deoxyadenosine triphosphate.

Abstract

Diastereomeric phosphorothioate analogs of dATP are used to determine the stereochemistry of action of DNA polymerase I from Escherichia coli as well as the active form of the magnesium*dATP complex on the enzyme. DNA polymerase I co-polymerizes the S, diastereomers of 2’-deoxyadenosine 5’-O-(1-thiotriphosphate) (dATP&) or 2’-deoxyadenosine 5’-0-(2-thiotriphosphate) (dATP@?) and dlTP onto a poly[d(A-T)] template. In the presence of M&+ the R, diastereomers of dATPaS and dATPPS are inactive. At low optimum concentrations of divalent metal cation, Mg2+ can be replaced effectively by Co’+, Mn2+, or Zn’+. While Mg+ can complex phosphorothioates only by chelating through oxygen, the latter three cations can chelate through both oxygen and sulfur (Jaffe, E. K., and Cohn, M. (1978) J. Biol. Chem 253, 48234825). When M&+ is replaced in the assay by either of these three cations, stereoselectivity on the Lu-phosphorus is retained, but is lost on the P-phosphate. It is concluded that the divalent metal ion in the active enzyme complex is chelated to the pbut not to the LXphosphate. dATP& (S,) is polymerized onto a poly(dT)-oligo(dA) template-primer to form polydeoxyadenylic acid with phosphorothioate internucleotide linkages. Its rate of hydrolysis catalyzed by snake venom phosphodiesterase is compared with those of oligoand polynucleotides of known absolute configuration (both R, and S,) of internucleotide phosphorothioate linkages. These experiments show that inversion of absolute configuration at the a-phosphorus occurs during internucleotide bond formation. This is compatible with an in-line mechanism. A model for the stereotopical arrangement of the triphosphate moiety of dATP in the active site of DNA polymerase I is proposed.