Mechanistic aspects of DnaA-RepA interaction as revealed by yeast forward and reverse two-hybrid analysis.
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.
Sharma
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Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Kachroo
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Bastia
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D053488:DnaB Helicases; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D016350:Leucine Zippers; D016296:Mutagenesis; D010957:Plasmids; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae; D015534:Trans-Activators; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D011506:Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D000255:Adenosine Triphosphate; D004265:DNA Helicases; D053488:DnaB Helicases