title: The mechanism of sequence-specific DNA cleavage and strand transfer by phi X174 gene A* protein.
abstract: We have examined the biological role and catalytic function of two juxtaposed tyrosyl residues in the bacteriophage phi X174 gene A protein, Tyr-343 and Tyr-347, which have been implicated in the catalysis of sequence-specific DNA strand transfer. Site-directed mutagenesis changing either tyrosine to phenylalanine abolishes phage viability. The biochemical basis of this inviability was studied using purified A* protein containing the carboxyl-terminal 341 amino acids of the A protein, as well as purified A* protein with a Y343F or Y347F mutation. All three proteins can cleave the phi X174 replication origin and perform strand transfer between oligodeoxynucleotides bearing the recognition sequence 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 molecule. The chirality of a phosphorothioate group at the site of strand transfer in the DNA was found to be retained following the strand-transfer reaction, which argues against transfer of Tyr-343-linked DNA to Tyr-347 on the same protein molecule or vice versa. These results support the current model of gene A protein function in which the two tyrosines of a single protein molecule alternate in catalyzing DNA strand transfer at the viral replication origin.
authors.affiliation: Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
authors.forename: R
authors.initials: R
authors.lastname: Hanai
authors.forename: J C
authors.initials: JC
authors.lastname: Wang
mesh_terms: D010584:Bacteriophage phi X 174 / Q000378:metabolism*; D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D013237:Stereoisomerism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins