Nucleic Acids Research, 1991, Vol. 19, No. 6 1203-1211
© 1991
ENZYMOLOGY |
Plasmid RSF1010 DNA replication in vitro promoted by purified RSF1010 RepA, RepB and RepC proteins
Max-Planck-Institut für Molekulare Genetik Abteilung Schuster, Ihnestraße 73, D-1000 Berlin 33, FRG
Received January 16, 1991. Revised February 19, 1991. Accepted February 19, 1991.
We have constructed and analyzed an in vitro system that will efficiently replicate plasmid RSF1010 and its derivatives. The system contains a partially purified extract from E.coli cells and three purified RSF1010-encoded proteins, the products of genes repA, repB (or mobA/repB), and repC. Replication in this system mimics the in vivo mechanism in that it (i) is initiated at oriV, the origin of vegetative DNA replication, (ii) proceeds in a population of plasmid molecules in both directions from this 396-base-palr origin region, and (iii) is absolutely dependent on the presence of each of the three rep gene products. In addition, we find that E.coli DNA gyrase, DnaZ protein (
subunit of pollll holoenzyme) and SSB are required for in vitro plasmid synthesis. The bacterial RNA polymerase, the initiation protein DnaA, and the primosomal proteins DnaB, DnaC, DnaG and DnaT are not required. Furthermore, the repllcative intermediates seen in the electron microscope suggest that replication in vitro begins with the simultaneous or non-simultaneous formation of two displacement loops that expand for a short stretch of DNA toward each other, and form a theta-type structure when the two displacing strands pass each other.
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