In vitro system for the replication of the mini R1 factor Rsc11

The in vitro synthesis of the mini R1-factor, Rsc11, was achieved using a soluble Escherichia coli cell-extract system. Triton X-100 lysates of the K12 strain 1101 (Rsc11) fractionated by Sephadex G25 chromatography supported the incorporation of labeled deoxyribonucleotides into covalently-closed circular (30S) and open-circular (25S) plasmid DNA as well as other molecules of various sizes. DNA synthesis required the presence of all four ribonucleotides and was rifampicin sensitive. Pulse-chase experiments indicated that this reaction is discontinuous. A dependence on ATP and sensitivity to nalidixic acid suggested this system capable of the replicative synthesis of Rsc11 DNA. Density-shift analysis confirmed this. In addition to hybrid, fully-heavy plasmid supercoils were synthesized indicating that more than one round of replication was completed. Approximately one-third of the molecules available to the system participate in this reaction.