title: Cnr protein, the negative regulator of bacteriophage P4 replication, stimulates specific DNA binding of its initiator protein alpha.
abstract: Bacteriophage P4 DNA replication depends upon the phage-encoded alpha protein, which has DNA helicase and DNA primase activity and can specifically bind to the replication origin (ori) and to the cis replicating region (crr). The P4 Cnr protein functions as a negative regulator of P4 replication, and P4 does not replicate in cells that overexpress cnr. We searched for P4 mutants that suppressed this phenotype (Cnr resistant [alpha cr]). Eight independent mutants that grew in the presence of high levels of Cnr were obtained. None of these can establish the plasmid state. Each of these mutations lies in the DNA binding domain of gp alpha that occupies the C terminus of the protein. Five different sequence changes were found: T675M, G732V (three times), G732W (twice), L733V, and L737V. A TrxA-Cnr fusion protein does not bind DNA by itself but stimulates the ori and crr binding abilities of alpha protein in vitro. The alpha cr mutant proteins were still able to bind specifically to ori or crr, but specific DNA binding was less stimulated by the TrxA-Cnr protein. We present evidence that Cnr protein interacts with the gp alpha domain that binds specifically to DNA and that gp(alpha)cr mutations impair this interaction. We hypothesize that gp alpha-Cnr interaction is essential for the control of P4 DNA replication.
authors.affiliation: Max-Planck-Institut für Molekulare Genetik, Dahlem, Berlin, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Ziegelin
authors.forename: R
authors.initials: R
authors.lastname: Calendar
authors.forename: D
authors.initials: D
authors.lastname: Ghisotti
authors.forename: S
authors.initials: S
authors.lastname: Terzano
authors.forename: E
authors.initials: E
authors.lastname: Lanka
mesh_terms: D001665:Binding Sites; D003090:Coliphages / Q000235:genetics / Q000502:physiology*; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D005838:Genotype; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018741:Replication Origin; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins; D014779:Virus Replication
chemical_list: C089636:Cnr protein, Bacteriophage P4; D004279:DNA, Viral; D011994:Recombinant Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004265:DNA Helicases