Influence of highly curved DNA segments on in vivo topology of plasmids

Abstract

Recombinant plasmids carrying a highly curved DNA structure are sometimes unstable in Escherichia coli. In order to know the underlying mechanism, several plasmids carrying one or two highly bent DNA segment(s) from the human adenovirus type 2 (Ad2) enhancer and/or origin region of phage lambda replication were systematically constructed and propagated in E. coli. The highly bent DNA segments disturbed the action of DNA topoisomerases: i.e. they were shown to be able to produce an anomalously wide spectrum of linking number topoisomers that tails toward lower supercoiling with a little of the DNA actually positively supercoiled. Furthermore, bent DNA caused multimeric plasmid formation. The linking number topoisomers and multimers seemed to be intermediate topological states of the bent DNA-containing plasmids that would lead to the deletion occurring in them. The nucleotide sequence of a deletion product of a bent DNA-containing plasmid showed that the putative source of the severe topological constraint was entirely eliminated from the plasmid.