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Nucleic Acids Research, 1993, Vol. 21, No. 23 5495-5499
© 1993

MOLECULAR BIOLOGY

The heat-shock DnaK protein is required for plasmid R1 replication and it is dispensable for plasmid ColE1 replication

Rafael Giraldo-suárez, Elena Fernández-Tresguerres, Ramón Díaz-orejas*, Abderrahim Malki1 and Masamichi Kohiyama1

Centra de Investigaciones Biolgicas (CSIC) C/ Velazquez 144, 28006 Madrid, Spain 1lnstitut Jacques Monod (CNRS) Universite Paris Vll-Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France

*To whom correspondence should be addressed

Received June 16, 1993. Revised October 15, 1993. Accepted October 15, 1993.

Plasmid R1 replication in vitro is inactive in extracts prepared from a dnaK756 strain but is restored to normal levels upon addition of purified DnaK protein. Replication of R1 in extracts of a dnaKwt strain can be specifically inhibited with polyclonal antibodies against DnaK. RepA-dependent replication of R1 in dnaK756 extracts supplemented with DnaKwt protein at maximum concentration is partially inhibited by rifampicin and it is severely inhibited at sub-optimal concentrations of DnaK protein. The copy number of a run-away R1 vector is reduced in a dnaK756 background at 30°C and at 42°C the amplification of the run-away R1 vector is prevented. However a runaway R1 vector containing dnaK gene allows the amplification of the plasmid at high temperature. These data indicate that DnaK is required for both in vitro and in vivo replication of plasmid R1 and show a partial compensation for the low level of DnaK by RNA polymerase. In contrast ColE1 replication is not affected by DnaK as indicated by the fact that ColE1 replicates with the same efficiency in extracts from dnaKwt and dnaK756 strains.


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