The two active-site tyrosine residues of the a protein play non-equivalent roles during initiation of rolling circle replication of bacteriophage p2.
The A protein of bacteriophage P2 initiates rolling circle DNA replication by a single-stranded cut at the origin. Two well-conserved tyrosine residues, interspaced by three amino acid residues, are required for the cleavage-joining activity of the protein. The functional relationship between these tyrosine residues was investigated by site-directed mutagenesis. We found that the two tyrosine residues located in the presumed catalytic site of P2 A play non-equivalent functional roles. Tyrosine residue 454 is superior in nicking single-stranded DNA compared to tyrosine residue 450, while both could promote joining at equal efficiency. Specific peptide-oligonucleotide adducts after cleavage reaction and protease digestion could be observed for both tyrosine residues. We propose that tyrosine 454 initiates replication and that tyrosine 450 is able to cleave the DNA only when tyrosine 454 is covalently joined to DNA, thereby reinitiating replication. Also, the involvement of divalent cations in the catalytic activity of P2 A was investigated. While the cleavage reaction was strongly discriminating between different divalent cations, primarily prefering magnesium, the joining reaction showed the same efficiency independently of what divalent cation was provided. This phenomenon could reflect conformational changes of the protein upon binding to DNA. Finally, we found that a large part of the C terminus but not the N terminus is dispensable for initiation of replication both in vivo and in vitro.
Odegrip
R
R
Department of Genetics, Stockholm University, Stockholm, S-106 91, Sweden
Haggård-Ljungquist
E
E
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D017901:Bacteriophage P2 / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics / Q000821:virology; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002413:Cations, Divalent; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C081601:gpA protein, Enterobacteria phage P2; D014443:Tyrosine