Enzymatic synthesis of DNA with 4-thio-thymidine triphosphate as substitute for dTTP.

Abstract

When 4‐thio‐thymidine‐5′‐triphosphate is substituted for dTTP in the enzymatic synthesis of poly d(A‐T) by DNA polymerase, formation of a polymer is observed either by incorporation of radioactive labelled deoxyadenosine nucleotide or by a hypochromic effect at 260 mμ and at 335 mμ, the latter corresponding to the ultraviolet absorption of 4‐thio‐thymidine. Both the initial velocity and the extent of polymer synthesis are strictly dependent on the concentration of primer poly d(A‐T). The product for which the designation poly d(A‐4thioT) is proposed contains deoxyadenosine‐ and 4‐thio‐thymidine nucleotide in equal amounts and bears a close resemblance to the parent d(A‐T) polymer with respect to temperature induced helix coil transition. Evidence for enzymatic formation of a hybrid consisting of poly d(A‐BrU) and poly d(A‐4thioT) is given by melting profiles. Poly d(A‐4thioT) is a poor primer for synthesis of poly d(A‐T) and, if dTTP is replaced by 4‐thio‐thymidine triphosphate incorporation of labelled deoxyadenosine nucleotide becomes almost negligible. Moreover, 4‐thio‐thymidine triphosphate cannot replace dTTP when calf thymus DNA or the homopolymer double strands are used as templates. These results cannot be interpreted merely in terms of the classical rules of base pairing, but additional restrictions, e. g. influence of nearest neighbors, might be operating in the enzymatic reaction.