Related Experiment Videos
Mutation in the plasmid pUB110 Rep protein affects termination of rolling circle replication
V E Bidnenko1, A Gruss, S D Ehrlich
1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Journal of Bacteriology
|September 1, 1993
Summary
A single mutation in the pUB110 plasmid replication protein causes toxicity and increased copy number in Bacillus subtilis. This altered protein shows reduced ability to terminate rolling circle replication.
Area of Science:
- * Molecular Biology
- * Genetics
- * Microbiology
Background:
- * Plasmid pUB110 is a widely used vector in Bacillus subtilis.
- * Plasmid replication is tightly regulated to maintain stable copy numbers.
- * Rolling circle replication is a common mechanism for plasmid DNA replication.
Purpose of the Study:
- * To characterize a novel mutant of plasmid pUB110 with altered replication properties.
- * To investigate the functional consequences of a specific mutation in the plasmid replication (Rep) protein.
- * To understand the mechanism of replication termination in rolling circle replication.
Main Methods:
- * Isolation and characterization of a mutant plasmid in Bacillus subtilis.
- * Analysis of plasmid copy number and multimer formation.
- * Complementation assays with a satellite plasmid.
Main Results:
- * A single amino acid change (Gly148Glu) in the Rep protein resulted in toxicity in recA and add mutant cells, especially at higher temperatures.
- * The mutant plasmid exhibited a threefold increase in copy number, with amplified copies present as multimers.
- * The mutant Rep protein efficiently complemented replication of a cmp- satellite plasmid, indicating altered replication control.
Conclusions:
- * The Gly148Glu mutation in the Rep protein impairs the termination of rolling circle replication.
- * This impairment may stem from a reduced affinity for the plasmid origin or a defect in recognizing specific DNA conformations required for termination.
- * The findings provide insights into the regulation of plasmid replication and the function of the Rep protein.