Functional characterization of the P2 A initiator protein and its DNA cleavage site.
The A protein of bacteriophage P2 initiates DNA replication by a single-stranded cut at the origin, and the DNA replication proceeds unidirectionally by a modified rolling circle type of replication. The P2 A protein belongs to a family of proteins involved in the initiation of rolling circle DNA replication, and the prototype for this family is the well-characterized A protein of phage phi X174. One of the common motifs of this family contains two conserved tyrosine residues, which have been shown to be able to alternate in catalyzing the cleavage as well as joining reactions in the phi X174 A protein. We investigated the role of the conserved tyrosine residues in P2 A protein by in vitro mutagenesis. Only one of the two conserved tyrosine residues was found to be involved in the cleavage reaction. The tyrosine residue dispensable for cleavage and ligation is, however, required at some other stage of the P2 growth cycle, since viable recombinants containing this mutation could not be obtained. The sequence requirements for cleavage of the target site were analyzed with a set of oligonucleotides having single base alterations in the nick region, and the results indicate that only five core nucleotides need to be conserved for efficient cleavage.
Liu
Y
Y
Department of Genetics, Stockholm University, Sweden.
Haggård-Ljungquist
E
E
D000595:Amino Acid Sequence; D017901:Bacteriophage P2 / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014443:Tyrosine / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D014764:Viral Proteins; C081601:gpA protein, Enterobacteria phage P2; D014443:Tyrosine