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The ColE1 unidirectional origin acts as a polar replication fork pausing site
E Viguera1, P Hernández, D B Krimer
1Departamento de Biología Celular y del Desarrollo, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Velázquez 144, 28006 Madrid, Spain.
The Journal of Biological Chemistry
|September 13, 1996
Summary
Replication origins in plasmids usually align. However, pPI21 plasmid DNA replication initiates at only one origin, with forks pausing at the second origin, explaining why head-to-head plasmid multimers are less advantageous.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Multimeric plasmids commonly exhibit co-oriented replication origins.
- Exceptions include Streptococcus pyogenes plasmid pSM19035 and Escherichia coli plasmid pPI21.
- pPI21 contains two inverted repeats, each with a potential ColE1 origin.
Purpose of the Study:
- To investigate the replication mechanism of the pPI21 plasmid.
- To understand the functional implications of inversely oriented origins in plasmid replication.
Main Methods:
- Analysis of replication intermediates (RIs) using two-dimensional agarose gel electrophoresis.
- Electron microscopy of pPI21 replication intermediates.
Main Results:
- A specific RI with a single internal bubble accumulated.
- DNA replication initiated at a single origin in pPI21.
- Replicating forks were transiently stalled at the inversely oriented silent ColE1 origin.
- Accumulated RIs showed stereoisomers with varying knot numbers.
Conclusions:
- Provides a functional explanation for the disadvantage of head-to-head plasmid multimers compared to head-to-tail ones.
- Demonstrates a unique replication control mechanism involving origin stalling.