Rolling-Circle Plasmid pKYM Re-initiates DNA Replication

It is believed that rolling-circle plasmids are incapable of re-initiation since they have to maintain their copy number and this is one of the differences between plasmids and phages as phi-x174. To examine whether a rolling-circle plasmid pKYM is incapable of re-initiating DNA replication, we constructed a plasmid that carries both the pKYM origin (fragment 13, 173 bp) and its truncated origin (fragment 32, 56 bp) in the same orientation. This plasmid yielded two smaller plasmids in the presence of RepK, an initiator protein. We showed that RepK can bind to the fragment 13 but not to fragment 32 which lacks the 3'- moiety of fragment 13. These results imply that RepK initiates DNA replication from fragment 13 and terminates at fragment 32, then the same RepK is used for re-initiation of replication from the fragment 32 region. pKYM is likely to be a unique plasmid that re-initiates DNA replication like a phase phi-x174.