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Nucleic Acids Research, 1991, Vol. 19, No. 3 497-503
© 1991

MOLECULAR BIOLOGY

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

Heinrich Leonhardt, Rudi Lurz and Juan C. AIonso*

Max-Planck-lnstitut für Molekulare Genetik Ihnestraße 73, D-1000 Berlin 33, FRG

*To whom correspondence should be addressed

Received December 12, 1990. Revised January 14, 1991. Accepted January 14, 1991.

The synthesis and structure of linear multimeric plasmid molecules (hmw DN A) in Bacillus subtilis were investigated. The replication of covalently-closed-circular supercolled (form I) DNA requires the rate-limiting plasm id-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.


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