In vitro DNA replication of plasmid pKYM.

Abstract

DNA of plasmid pKYM replicates in the extract prepared following the method of Fuller et al. (Fuller, R. S., Kaguni, J. M., and Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA, 78, 7370-7374), when it contains the Rep protein of pKYM. Although the synthesis of DNA was completely inhibited by the DNA gyrase inhibitor, a substantial quantity of DNA was synthesized even in the absence of RNA synthesis and the accumulated DNA contained a strand-specific single-stranded circular molecule. The kinetic analysis of the DNA synthesis in the absence of RNA synthesis suggests that the single-stranded DNA appeared after the synthesis of double-stranded molecule. Since the DNA synthesized under the condition which allowed the synthesis of RNA is a fully twisted double-stranded molecule, the synthesis of RNA will initiate the DNA synthesis on the single-stranded molecule. These results support the idea that pKYM replicates via a rolling-circle mechanism.