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Nucleic Acids Research, 1983, Vol. 11, No. 14 4957-4975
© 1983

MOLECULAR BIOLOGY

Initiation signals for complementary strand DNA synthesis on single-stranded plasmid DNA

Arie van der Ende1,*, Renske Teertstra2, Harrie G.A.M. van der Avoort2 and Peter J. Weisbeek1,2

1Institute of Molecular Biology Padualaan 8, Utrecht, The Netherlands 2Department of Molecular Cell Biology, State University of Utrecht Padualaan 8, Utrecht, The Netherlands

*To whom correspondence should be addressed

Received May 5, 1983. Revised June 29, 1983. Accepted June 29, 1983.

The bacteriophage ØX174 origin for (+) strand DNA synthesis, when inserted in a plasmld, is in vivo a substrate for the initiator A protein, that is produced by infecting phages. The result of this interaction is the packaging of single-stranded plasmid DNA into preformed phage coats. These plasmld particles can transduce ØX-sensitive cells; however, the transductlon efficiency depends strongly on the presence in the packaged DNA strand of an initiation signal for complementary strand DNA synthesis. A plasmid with the complementary (–) strand origin of ØX inserted in the same strand as the viral (+) origin transduces 50–100 times more efficient than the same plasmld without the (–) origin of ØX. The transductlon efficiency of such a particle is comparable to the infection efficiency of the phage particle.

It is shown that in this system the ØX (–) origin can be replaced by the complementary strand origins of the bacterlophages G4 and M13. We have used this system to isolate sequences, from E. coli plasmids (pACYC177, C1oDF13, miniF and OriC) and from the E. coli chromosome that can function as initiation signals for the conversion of single-stranded plasmld DNA to double-stranded DNA. All isolated origins were found to be dependent for their activity on the dnaB, dnaC and dnaG proteins. We conclude that these signals were all primo-some-dependent origins and that primosome priming is the major mechanism for Initiation of the lagging strand DNA synthesis in E. coli.

The assembly of the primosome depends on the sequence-specific interaction of the n' protein with single-stranded DNA. We have used the isolated sequences to deduce a consensus recognition sequence for the n' protein. The role of a possible secondary structure in this sequence is discussed.


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