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Nucleic Acids Research, 1994, Vol. 22, No. 5 767-772
© 1994

MOLECULAR BIOLOGY

Functional difference between the two oppositely oriented priming signals essential for the initiation of the broad host-range plasmid RSF1010 DNA replication

Katsunori Tanaka, Kouichi Kino, Yoshitomo Taguchi, Deng-Ming Miao, Yoichi Honda, Hiroshi Sakai, Tohru Komano* and Michael Bagdasarian1

Laboratory of Biochemistry, Department of Agricultural Chemistry, Kyoto University Kyoto 606-01, Japan 1Department of Microbiology, Michigan State University East Lansing, MI 48824, USA

*To whom correspondence should be addressed.

Received December 9, 1993. Revised February 8, 1994. Accepted February 8, 1994.

The broad host-range plasmid RSF1010 contains two oppositely oriented priming signals, ssiA and ssiB, for DNA synthesis dependent on the origin of vegetative DNA replication (orlV). If either ssiA or ssiB was deleted or Inverted, the RSF1010 miniplasmids containing engineered oriVs were maintained at low copy numbers, replicated abnormally as dimers, and accumulated specific single strands In the Escherichia coll strain supplying the three RSF1010-encoded RepA, RepB', and RepC proteins. Interestingly, an additional Intracellular supply of the Sog prlmase (the sog gene product of plasmid Collb-P9) reversed the replication deficiency of these miniplasmids with respect to all three aspects described above. These were also true for the RSF1010 miniplasmids in which either ssiA orssiB was replaced by the prlmosome assembly site (PAS) or by the G4-type ssi signal (G site). Furthermore, comparative analysis of the functional contribution of the two oppositely oriented ssi signals to the DNA replication of RSF1010 showed that, irrespective of their types, ssi signals conducting the initiation of DNA chain elongation away from the Iterons were functionally more Important than ones in the inverted orientation. We consider that this functional difference reflects the inherent properties of the Initiation mechanism of RSF1010 DNA replication.


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