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Functional characterization of a replication initiator protein
A E Gammie1, M E Tolmasky, J H Crosa
1Department of Microbiology and Immunology, Oregon Health Sciences University, Portland 97201.
Journal of Bacteriology
|June 1, 1993
Summary
Researchers identified functional domains in the RepI replication initiator protein. Mutations in its DNA binding motif altered plasmid copy number, suggesting separable replication and autorepression functions.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The RepI protein is crucial for plasmid replication initiation.
- Understanding its functional domains is key to controlling plasmid copy number.
- Autorepression mechanisms regulate RepI activity.
Purpose of the Study:
- To identify functional domains within the RepI replication initiator protein.
- To investigate the relationship between DNA binding, autorepression, and plasmid copy number.
- To determine if RepI's initiating and autorepression functions are separable.
Main Methods:
- Classical mutagenesis techniques.
- Site-directed mutagenesis.
- Analysis of deletion clones.
Main Results:
- Mutations in the helix-turn-helix DNA binding motif increased plasmid copy number without affecting autorepression.
- Two classes of mutations diminished autorepression: one with elevated copy number, another with wild-type copy number.
- RepI's autorepression function may depend on intermolecular coupling control.
Conclusions:
- The DNA binding and autorepression functions of the RepI protein are separable.
- Mutations affecting DNA binding motifs can increase plasmid copy number.
- Intermolecular coupling control may regulate RepI's autorepression.