Active site of the replication protein of the rolling circle plasmid pC194

• Published in: The EMBO journal Vol. 13; no. 18; pp. 4412 - 4420
• Main Authors: Noirot‐Gros, M.F., Bidnenko, V., Ehrlich, S.D.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 15.09.1994; EMBO Press
• Subjects: Amino Acid Sequence; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites; Biological and medical sciences; Cellular Biology; Deoxyribonucleases - metabolism; DNA Helicases; DNA Mutational Analysis; DNA Replication; DNA Topoisomerases, Type I - metabolism; DNA-Binding Proteins; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Life Sciences; Microbiology; Models, Chemical; Models, Genetic; Molecular Sequence Data; Plasmids - metabolism; Protein Binding; Proteins; Replicative cycle, interference, host-virus relations, pathogenicity, miscellaneous strains; Sequence Homology, Amino Acid; Trans-Activators; Virology; Plasmid; Structure activity relation; Escherichia coli; Active site; Site directed mutagenesis; DNA binding protein; Bacteria; Replication; Models; Mechanism of action; Enterobacteriaceae
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1002/j.1460-2075.1994.tb06761.x

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.