Functional division and reconstruction of a plasmid replication origin: Molecular dissection of the oriV of the broad-host-range plasmid RSF1010

Yoichi Honda, Hiroshi Sakai, Hiroshi Hiasa, Katsunori Tanaka, Tohru Komano, Michael Bagdasarian

Research output: Contribution to journalArticlepeer-review

38 Scopus citations

Abstract

Two single-stranded DNA initiation signals (designated ssi) present in the origin of vegetative DNA replication (oriV) of the broad-host-range plasmid RSF1010 are essential for the priming of replication of each complementary DNA strand of this plasmid in Escherichia coli. Each of the RSF1010 ssi signals, ssiA and ssiB, could be replaced by a primosome assembly site from plasmid pACY184 or from bacteriophage (oøX174. In these chimeric origins, replication of the strand complementary to that containing the primosome assembly site was no longer dependent on the RSF1010 primase, protein RepB′, but required the E. coli primase, DnaG. If both ssiA and ssiB sites of RSF1010 were replaced by primosome assembly sites, protein RepB′ was no longer essential for the replication at this origin, whereas proteins RepA and RepC of RSF1010 were still required. These results strongly suggest that the two ssi sites and the RepB′ protein actually direct the priming of DNA synthesis in the replication of RSF1010, and the proteins RepA and RepC are involved in the preprinting events - i.e., the opening of the DNA duplex at oriV. It is evident that the origin of RSF1010 can be separated into three functional domains and reconstructed by replacing the ssi sites with heterologous elements.

Original languageEnglish (US)
Pages (from-to)179-183
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number1
DOIs
StatePublished - Jan 1 1991
Externally publishedYes

Keywords

  • DNA primase
  • Primosome assembly site
  • Replication fork
  • Single-stranded DNA initiation
  • dnaG

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