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Active site of the replication protein of the rolling circle plasmid pC194
M F Noirot-Gros1, V Bidnenko, S D Ehrlich
1Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Abstract:
Mutation analysis of the rolling circle (RC) replication initiator protein RepA of plasmid pC194 was targeted to tyrosine and acidic amino acids (glutamate and aspartate) which are well conserved among numerous related plasmids. The effect of mutations was examined by an in vivo activity test. Mutations of one tyrosine and two glutamate residues were found to greatly impair or abolish activity, without affecting affinity for the origin, as deduced from in vitro gel mobility assays. We conclude that all three amino acids have a catalytic role. Tyrosine residues were found previously in active sites of different RC plasmid Rep proteins and topoisomerases, but not in association with acidic residues, which are a hallmark of the active sites of DNA hydrolyzing enzymes, such as the exo- and endonucleases. We propose that the active site of RepA contains two different catalytic centers, corresponding to a tyrosine and a glutamate. The former may be involved in the formation of the covalent DNA-protein intermediate at the initiation step of RC replication, and the latter may catalyze the release of the protein from the intermediate at the termination step.
Insights
Mutations in specific tyrosine and glutamate residues of the rolling circle replication initiator protein RepA significantly impair its activity. These findings suggest distinct catalytic roles for tyrosine and glutamate in plasmid replication initiation and termination.
Area of Science:
- Molecular Biology
- Biochemistry
- Plasmid Biology
Background:
- Rolling circle (RC) replication is a key mechanism for plasmid DNA propagation.
- The initiator protein RepA is essential for pC194 plasmid RC replication.
- Conserved residues in RepA suggest functional importance.
Purpose of the Study:
- To investigate the catalytic roles of conserved tyrosine and acidic amino acids (glutamate, aspartate) in RepA.
- To determine the impact of specific mutations on RepA activity and DNA binding.
- To elucidate the functional domains within the RepA active site.
Main Methods:
- Site-directed mutagenesis of RepA targeting conserved tyrosine and glutamate residues.
- In vivo activity assays to assess RepA's replication function.
- In vitro gel mobility assays to evaluate RepA's origin binding affinity.
Main Results:
- Mutations in one tyrosine and two glutamate residues severely reduced or abolished RepA activity.
- These mutations did not affect RepA's affinity for the replication origin.
- Evidence suggests these residues are critical for catalysis, not DNA binding.
Conclusions:
- The identified tyrosine and glutamate residues play essential catalytic roles in RepA function.
- RepA's active site likely possesses distinct catalytic centers for initiation and termination.
- Tyrosine may be involved in covalent intermediate formation, while glutamate facilitates its release.