Cnr interferes with dimerization of the replication protein alpha in phage-plasmid P4.
DNA replication of phage-plasmid P4 in its host Escherichia coli depends on its replication protein alpha. In the plasmid state, P4 copy number is controlled by the regulator protein Cnr (copy number regulation). Mutations in alpha (alpha(cr)) that prevent regulation by Cnr cause P4 over-replication and cell death. Using the two-hybrid system in Saccharomyces cerevisiae and a system based on lambda immunity in E.coli for in vivo detection of protein-protein interactions, we found that (i) alpha protein interacts with Cnr, whereas alpha(cr) proteins do not; (ii) both alpha-alpha and alpha(cr)-alpha(cr) interactions occur and the interaction domain is located within the C-terminal of alpha; (iii) Cnr-Cnr interaction also occurs. Using an in vivo competition assay, we found that Cnr interferes with both alpha-alpha and alpha(cr)-alpha(cr) dimerization. Our data suggest that Cnr and alpha interact in at least two ways, which may have different functional roles in P4 replication control.
Tocchetti
A
A
Dipartimento di Genetica e di Biologia dei Microrganismi, Università di Milano, Via Celoria 26, 20133 Milano, Italy.
Serina
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S
Oliva
I
I
Dehò
G
G
Ghisotti
D
D
D019943:Amino Acid Substitution; D001667:Binding, Competitive / Q000235:genetics; D003090:Coliphages / Q000235:genetics; D004265:DNA Helicases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D005656:Fungal Proteins / Q000235:genetics; D010449:Peptide Mapping; D010957:Plasmids / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D012316:RNA Nucleotidyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins
C089636:Cnr protein, Bacteriophage P4; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D054334:Viral Regulatory and Accessory Proteins; C095083:alpha protein, Bacteriophage P4; C016649:phage repressor proteins; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases