Incorporation of 4-thiothymidine into DNA by the klenow fragment and HIV-1 reverse transcriptase

The 5′-triphosphate of 4-thiothymidine (4S–TTP) is an excellent substrate for the Klenow fragment of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase with values of kcat/Km within a factor of 3 of those for TTP. A large UV change (Δε=−9770 M−1 cm−1 at 340 nm) associated with incorporation of 4S–TMP into nucleic acid duplexes makes possible a rapid, continuous spectrophotometric assay of the reaction progress.