Physical and biochemical characterization of recombination-dependent synthesis of linear plasmid multimers inBacillus subtilis

The synthesis and structure of linear multimeric plasmid molecules (hmw DNA) in Bacillus subtilis were investigated. The replication of covalently-closed-circular supercoiled (form I) DNA requires the rate-limiting plasmid-encoded replication initiation protein. Unlike form I, hmw DNA synthesis is partially resistant to inhibition of cellular transcription or translation and requires the host DnaB protein. In addition, hmw DNA synthesis involves host recombination and repair functions (RecE and Poll). Analysis of hmw DNA by electron microscopy displayed linear DNA molecules up to 100 kb in size, which were either single-stranded, double-stranded or double-stranded with single-stranded ends. Structural features of hmw DNA molecules were mapped by means of heteroduplex studies using defined strand-specific probes. The results suggest that a recombination intermediate, but not plasmid-encoded replication, is involved in the initiation of hmw DNA synthesis.