THE MECHANISM OF SEQUENCE-SPECIFIC DNA CLEAVAGE AND STRAND TRANSFER BY PHI-X174 GENE-A-ASTERISK PROTEIN

We have examined the biological role and catalytic function of two jux taposed tyrosyl residues in the bacteriophage phiX174 gene A protein, Tyr-343 and Tyr-347, which have been implicated in the catalysis of se quence-specific DNA strand transfer. Site-directed mutagenesis changin g either tyrosine to phenylalanine abolishes phage viability. The bioc hemical basis of this inviability was studied using purified A protei n containing the carboxyl-terminal 341 amino acids of the A protein, a s well as purified A protein with a Y343F or Y347F mutation. All thre e proteins can cleave the phiX174 replication origin and perform stran d transfer between oligodeoxy-nucleotides bearing the recognition sequ ence of the A protein; however, both Tyr-343 and Tyr-347 appear to be required for coordinated DNA strand transfer by a single A protein mo lecule. The chirality of a phosphorothioate group at the site of stran d transfer in the DNA was found to be retained following the strand-tr ansfer reaction, which argues against transfer of Tyr-343-linked DNA t o Tyr-347 on the same protein molecule or vice versa. These results su pport the current model of gene A protein function in which the two ty rosines of a single protein molecule alternate in catalyzing DNA stran d transfer at the viral replication origin.