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Boundaries of the pSC101 minimal replicon are conditional
C A Miller1, H Ingmer, S N Cohen
1Department of Genetics, Stanford University School of Medicine, California 94305, USA.
Journal of Bacteriology
|September 1, 1995
Summary
The minimal replicon of plasmid pSC101 is not absolute; host and plasmid factors influence its replication and partitioning efficiency. These findings redefine the core replicon concept and highlight the dual role of the origin region complex.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- The core replicon is traditionally defined as the minimal DNA segment essential for plasmid replication.
- Plasmid pSC101 replication and partitioning are complex processes involving multiple host and plasmid factors.
- Understanding these factors is crucial for comprehending plasmid stability and inheritance.
Purpose of the Study:
- To investigate the factors external to the core replicon that influence plasmid pSC101 replication and partitioning.
- To reassess the definition of a core replicon based on conditional sequence requirements.
- To explore the role of specific proteins and DNA elements in plasmid DNA replication and segregation.
Main Methods:
- Analysis of autonomous replication of modified pSC101 DNA fragments.
- Introduction of missense mutations in the plasmid-encoded RepA protein.
- Mutation of the Escherichia coli topoisomerase I gene (topA).
- Investigation of the roles of integration host factor (IHF), partitioning (par) locus, and inverted repeat (IR) sequences.
- Assessment of RepA and DnaA protein concentrations and their interactions.
Main Results:
- Autonomous replication of a minimal pSC101 fragment is achievable with specific mutations (repA or topA).
- In the absence of these mutations, host factor IHF and cis-acting elements (par locus, IR sequences) are required.
- The IR sequences' effect is mediated through RepA binding, while the par locus promotes negative supercoiling.
- RepA and DnaA protein concentrations and their interactions significantly impact both replication and partitioning.
- Sequence requirements for pSC101 replication are conditional, not absolute.
Conclusions:
- The definition of a core replicon needs reassessment due to conditional sequence requirements.
- Factors external to the minimal origin significantly determine replicon competence and partitioning efficiency.
- The RepA-DnaA-DNA complex at the origin initiates replication and also mediates plasmid partitioning during cell division.