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Related Experiment Videos

Rolling circle replication of single-stranded DNA plasmid pC194.

M F Gros1, H te Riele, S D Ehrlich

  • 1Institut Jacques Monod, Paris, France.

The EMBO Journal
|December 1, 1987
PubMed
Summary

Small plasmids like pC194 replicate using a circular single-stranded DNA intermediate. This study identifies a specific origin region and suggests a rolling circle replication mechanism, similar to E. coli phages.

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Area of Science:

  • Molecular Biology
  • Microbiology
  • Genetics

Background:

  • Some small plasmids replicate via circular single-stranded DNA intermediates.
  • Staphylococcus aureus and Bacillus subtilis are common bacterial species harboring such plasmids.

Purpose of the Study:

  • To identify the replication origin of the pC194 plasmid.
  • To elucidate the replication mechanism of pC194.

Main Methods:

  • In vitro complementation assays using a 55 bp region of pC194 and the plasmid replication protein.
  • Analysis of palindromic sequences and nicking sites within the origin.
  • DNA synthesis initiation and termination studies.

Main Results:

  • A 55 bp region of pC194 exhibits origin activity when complemented in trans.

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  • This region contains two palindromes and a replication protein nicking site.
  • DNA synthesis initiated at the nick can be terminated by a homologous sequence, suggesting rolling circle replication.

    • Conclusions:

    • The pC194 plasmid likely replicates via a rolling circle mechanism.
    • Homology exists between pC194 replication origins/proteins and the phage phi X174.
    • This mechanism is similar to that of single-stranded DNA phages in E. coli.