Enzymatic synthesis, ligation, and restriction of DNA containing deoxy-4-thiothymidine

Phage fd RF I DNA1 about 90% substituted by deoxy-4-thiothymidine (s4Td) in the codogenic strand was synthesized by the simultaneous actions of DNA polymerase I and DNA ligase. While the rate of DNA synthesis was considerably reduced, the yield the rate of DNA synthesis was considerably reduced, the yield was not affected in the presence of s4TdTP. The conversion of RF II to RF I DNA by DNA ligase was even improved. This effect seems to be related with an altered ratio of affinity of polymerase and ligase for the s4Td-containing substrate. The presence of the base analogue in the DNA was verified independently by chromatographic and spectroscopic methods. The modified genome could be cleaved by restriction endonucleases Hpa II (C/CGG)d and Taq I (T/CGA)d. A number of the fragments produced showed altered mobilities under the conditions of polyacrylamide gel electrophoresis.