Mapping of the DNA linking tyrosine residue of the PRD1 terminal protein.
DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5' terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (phi 29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (phi 29) and serine-577 (adenovirus-2) are found in beta-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a beta-sheet in a hydrophobic region.
Shiue
S Y
SY
Department of Microbiology and Immunology, College of Medicine, University of Arizona, Tucson 85724.
Hsieh
J C
JC
Ito
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000494:pharmacology; D004247:DNA; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D014443:Tyrosine / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C054298:DNA terminal protein, Enterobacteria phage PRD1; D003848:Deoxyguanine Nucleotides; D014764:Viral Proteins; D014443:Tyrosine; C007257:2'-deoxyguanosine 5'-phosphate; D004247:DNA; D003488:Cyanogen Bromide