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Nucleic Acids Research, 1981, Vol. 9, No. 20 5215-5232
© 1981

MOLECULAR BIOLOGY

Rifampicin-resistant initiation of DNA synthesis on the isolated strands of CoIE plasmid DNA

Thomas W. Böldicke, Günter Hillenbrand, Erich Lanka and Walter L. Staudenbauer

Max-Planck-Institut für Molekulare Genetik Abt. Schuster, Ihnestrasse 63–73, D-1000 Berlin 33, GFR

Received August 10, 1981. The opposite strands of the ColE1 and ColE3 plasmids were isolated as circular single-stranded DNA molecules. These molecules were compared with M13 and {varphi}X174 viral DNA with respect to their capacity to function as templates for in vitro DNA synthesis by a replication enzyme fraction from Escherichia coli. It was found for both ColE plasmids that the conversion of H as well as L strands to duplex DNA molecules closely resembles {varphi}X174 complementary strand synthesis and occurs by a rifampicin-resistant priming mechanism involving the dnaB, dnaC, and dnaG gene products. Restriction analysis of partially double-stranded intermediates indicates that preferred start sites for DNA synthesis are present on both strands of the ColE1 HaeII-C fragment. Inspection of the nucleotide sequence of this region reveals structural similarities with the origin of {varphi}X174 complementary strand synthesis. We propose that the rifampicin-resistant initiation site (rri) in the ColE1 L strand is required for the priming of discontinuous lagging strand synthesis during vegetative replication and that the rri site in the H strand is involved in the initiation of L strand synthesis during conjugative transfer.


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