PMID:11500384
| Citation |
Sharma, R, Kachroo, A and Bastia, D (2001) Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis. EMBO J. 20:4577-87 |
|---|---|
| Abstract |
Using yeast forward and reverse two-hybrid analysis and biochemical techniques, we present novel and definitive in vivo and in vitro evidence that both the N-terminal domain I and C-terminal domain IV of the host-encoded DnaA initiator protein of Escherichia coli interact physically with plasmid-encoded RepA initiator of pSC101. The N-terminal, but not the C-terminal, region of RepA interacted with DnaA in vitro. These protein-protein interactions are critical for two very early steps of replication initiation, namely origin unwinding and helicase loading. Neither domain I nor IV of DnaA could individually collaborate with RepA to promote pSC101 replication. However, when the two domains are co-expressed within a common cell milieu and allowed to associate non-covalently with each other via a pair of leucine zippers, replication of the plasmid was supported in vivo. Thus, the result shows that physical tethering, either non-covalent or covalent, of domain I and IV of DnaA and interaction of both domains with RepA, are critical for replication initiation. The results also provide the molecular basis for a novel, potential, replication-based bacterial two-hybrid system. |
| Links |
PubMed PMC125567 Online version:10.1093/emboj/20.16.4577 |
| Keywords |
Adenosine Triphosphate/metabolism; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Binding Sites; Chromatography, Affinity; DNA Helicases/metabolism; DNA Replication; DNA, Bacterial/biosynthesis; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; DnaB Helicases; Enzyme-Linked Immunosorbent Assay/methods; Escherichia coli/genetics; Leucine Zippers; Mutagenesis; Plasmids; Protein Folding; Protein Structure, Tertiary; Proteins/genetics; Proteins/metabolism; Replication Origin; Saccharomyces cerevisiae; Trans-Activators; Two-Hybrid System Techniques |
| edit table |
Significance
You can help EcoliWiki by summarizing why this paper is useful
Useful Materials and Methods
You can help Ecoliwiki by describing the useful materials (strains, plasmids, antibodies, etc) described in this paper.
Annotations
<annotationlinks/>
EcoliWiki Links
Add links to pages that link here (e.g. gene, product, method pages)
References
See Help:References for how to manage references in EcoliWiki.