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 phenylalanlne and the other tyrosine residue with asparaglne. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amlno acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amlno acid residue of the PRD1 terminal protein. Our results further show that tyrosine residues at both the amino-terminal and the carboxyl-termlnal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amlno acid residues were compared in three terminal proteins (ø29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (β29) and serine-577 (adenovirus-2) are found in β-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a β-sheet in a hydrophobic region.
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