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Nucleotide sequence analysis and expression of the minimum REPI replication region and incompatibility determinants
J F Perez-Casal1, A E Gammie, J H Crosa
1Department of Microbiology and Immunology, Oregon Health Sciences University, Portland 97201.
Abstract:
We sequenced the minimum REPI replication region and the incompatibility determinants of pColV-K30. The minimum replication region contains an open reading frame which corresponds to a 35-kilodalton (kDa) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis with maxicells transformed with a number of deletion derivatives demonstrated that this replication region encodes a 39-kDa protein and also established the direction of transcription of the RepI protein gene. The 39-kDa polypeptide was identified as the trans-acting factor essential for replication of REPI-containing plasmids. A translated region of the nucleotide sequence of the RepI protein gene showed homology with the helix-turn-helix binding domains of a number of DNA-binding proteins and also with other plasmid replication proteins. Further nucleotide analysis of the REPI region revealed the presence of direct and inverted repeat sequences in the incE, incF, and ori regions. The REPI ori also contained a perfect DnaA-binding site in addition to a high frequency of occurrence of the DNA adenine methylation (dam) site 5'GATC3'.
Insights
Researchers identified the essential 39-kDa RepI protein, crucial for REPI plasmid replication. This protein, encoded by the minimum replication region, features DNA-binding domains and interacts with specific DNA sequences.
Area of Science:
- Molecular Biology
- Genetics
- Plasmid Biology
Background:
- Plasmids are extrachromosomal DNA elements vital for bacterial adaptation.
- Understanding plasmid replication mechanisms is key to controlling their inheritance and function.
- The pColV-K30 plasmid's replication system, REPI, requires detailed characterization.
Purpose of the Study:
- To sequence and analyze the minimum replication region of the pColV-K30 plasmid.
- To identify and characterize the protein essential for REPI replication.
- To elucidate the DNA-binding and regulatory features of the replication initiation.
Main Methods:
- DNA sequencing of the REPI minimum replication region.
- Maxicell analysis using deletion derivatives to identify protein products.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein size determination.
- Bioinformatic analysis of nucleotide and amino acid sequences.
Main Results:
- The minimum replication region encodes a 39-kDa protein, identified as the RepI protein.
- The RepI protein is essential for the replication of REPI-containing plasmids.
- Sequence analysis revealed helix-turn-helix DNA-binding motifs in RepI, suggesting direct DNA interaction.
- The REPI origin of replication (ori) contains a DnaA-binding site and numerous DNA adenine methylation (dam) sites.
Conclusions:
- The 39-kDa RepI protein is the essential trans-acting replication factor for REPI plasmids.
- RepI likely functions as a DNA-binding protein, interacting with specific sequences within the REPI origin.
- The REPI replication mechanism involves recognition of specific DNA sites, including a DnaA-binding site and dam sites.