Stereochemical course of nucleotidyl transfer catalyzed by bacteriophage T7 induced DNA polymerase

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Abstract
The bacteriophage T7 induced DNA polymerase, consisting of the phage specified gene 5 protein associated with Escherichia coli thioredoxin, catalyzes the copolymerization of the S epimer of 2''-deoxyadenosine 5''-O-(1-thiotriphosphate) with dTTP, producing the alternating copolymer of 2''-deoxyadenosine 5''-O-phosphorothioate and 2''-deoxythymidine 5''-phosphate by a mechanism involving inversion of configuration at P.alpha.. Degradation of this alternating copolymer by the nucleolytic action of E. coli DNA polymerase I produced the dinucleotide 3''-O-(5''-phospho-2''-deoxythymidyl)-5''-O-(2''-deoxyadenosyl) phosphorothioate, whose configuration at the phosphorothioate diester was assigned as R. by comparison of the 32P NMR chemical shift (55.0 ppm downfield from H3PO4) with that of an authentic sample. Further degradation by alkaline phosphatase to the R epimer of 3''-O-(2''-deoxythymidyl)-5''-O-(2''-deoxyadenosyl) phosphorothioate (55.6 ppm downfield from H3PO4) confirmed the configuration. The stereochemistry provides no evidence of a double displacement mechanism.