Incorporation of phosphorothioate groups into fd and φX174 DNA

Abstract

DNA from [phages] fd and .vphi.X174 was synthesized in the presence of 2''-deoxyadenosine 5''-O-(1-thiotriphosphate) (dATP.alpha.S) and the corresponding phosphorothioate derivatives of dCTP and dTTP using ether-permeabilized Escherichia coli cells or crude cell extracts or E. coli DNA polymerase I. Reaction rates of enzymes involved in the formation or breakdown of DNA are decreased in the presence of phosphorothioates. The amount of label incorporated with [35S]dATP.alpha.S suggests that the dAMP was completely substituted by 2''-deoxyadenosine 5''-O-phosphorothioate (dAMPS). The substituted DNA have the same sedimentation coefficients, similar buoyant density, infectivity and thermal stability as the unsubstituted DNA. The procedure therefore allows specific modification at the 5'' position of dA, dC or dT in the DNA. In view of the recent demonstration of specific binding of Pt2+ complexes to the phosphorothioate analog of poly[r(A-U)], the synthesis of phosphorothioate containing DNA may be of use for DNA sequencing by EM.