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Nucleic Acids Research, 1994, Vol. 22, No. 11 2065-2070
© 1994

ENZYMOLOGY

Phage P4 DNA replication in vitro

Ramn Díaz Orejas1, Günter Ziegelin, Rudi Lurz and Erich Lanka*,

Max-Planck-lnstitut für Molekulare Genetik, Abteilung Schuster Ihnestrasse 73, Dahlem, D-14195 Berlin, Germany 1Centro de Investigaciones Biologicas (CSIC) C/ Velázquez 144, E-28006 Madrid, Spain

* To whom correspondence should be addressed

Received February 17, 1994. Revised April 29, 1994. Accepted April 29, 1994.

Phage P4 DNA is replicated in cell-free extracts of Escherichia coli in the presence of partially purified P4 {alpha} protein [Krevolin and Calendar (1985), J. Mol. Biol. 182, 507–517]. Using a modified in vitro replication assay, we have further characterized this process. Analysis by agarose gel electrophoresis and autoradiography of in vitro replicated molecules demonstrates that the system yields supercoiled monomeric DNA as the main product. Electron microscopic analysis of in vitro generated intermediates indicates that DNA synthesis initiates in vitro mainly at ori, the origin of replication used in vivo. Replication proceeds from this origin bidirectionally, resulting in{Theta}-type molecules. In contrast to the in vivo situation, no extensive single-stranded regions were found in these intermediates. The initiation proteins of the host, DnaB and DnaG, and the chaperones DnaJ and DnaK are not required for P4 replication, because polyclonal antibodies against those polypeptides do not inhibit the process. The reaction is inhibited by antibodies against the SSB protein, and by ara-CTP, a specific inhibitor of DNA polymerase III holoenzyme. Consistent with previous reports, P4 in vitro replication is independent of transcription by host RNA polymerase. Novobiocin, a DNA gyrase inhibitor, strongly inhibits P4 DNA synthesis, indicating that form I DNA is the required substrate.


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