Protein-DNA interactions at the bacteriophage phi-X174 origin of replication

Abstract

The gene A protein of bacteriophage {dollar}\phi{dollar}X174 is a site-specific endonuclease-ligase required for the synthesis of {dollar}\phi{dollar}X single-strand circular (SS(c)) DNA from supercoiled {dollar}\phi{dollar}X RFI DNA. The {dollar}\phi{dollar}X A protein initiates this pathway by nicking {dollar}\phi{dollar}X RFI between nucleotide residues 4305 and 4306 of the viral strand (residues 7 and 8 of the 30 pb {dollar}\phi{dollar}X origin). In the process, it becomes linked to the 5{dollar}\sp\prime{dollar} terminus at residue 4306 and creates a 3{dollar}\sp\prime{dollar} hydroxyl terminus at residue 4305 which is utilized as a primer for DNA synthesis. Following a complete round of replication, the {dollar}\phi{dollar}X A protein circularizes the displaced strand and initiates a new round of synthesis.;The sequence requirements supporting the various activities of the {dollar}\phi{dollar}X A protein have been examined by in vitro analysis of a series of mutations in the 30 nucleotide {dollar}\phi{dollar}X origin. Mutations or deletions within the first three nucleotides at the 5{dollar}\sp\prime{dollar} end of the 30 nucleotide origin sequence did not alter the nicking reaction by the A protein, but resulted in reduced RF {dollar}\to{dollar} SS(c) synthesis due to poor reinitiation. A G {dollar}\to{dollar} A transition at residue 7 of the 30 bp {dollar}\phi{dollar}X origin (the upstream side of the nick site) prevented nicking, while an A {dollar}\to{dollar} G transition at residue 8 of the origin permitted efficient nicking and a reduced level of replication. Mutant origins with 3{dollar}\sp\prime{dollar} deletions of more than 4 nucleotides were deficient in both A protein nicking and replication activities, apparently due to deletion of a required A protein binding domain. Plasmids containing both a wild-type and a mutant origin of replication on the same strand, were used to determine whether origins defective for the initial nicking event could support termination. Initiation occurred at the wild-type origin in such constructs, as did termination, but no termination at origins defective for initiation could be detected. The presence of an initiation-defective origin with an intact 3{dollar}\sp\prime{dollar} end in these plasmids, however, resulted in diminished RF {dollar}\to{dollar} SS(c) synthesis, presumably due to {dollar}\phi{dollar}X A protein pausing at a site within the mutant origin. It thus appears that the {dollar}\phi{dollar}X origin region contains distinct binding and cleavage domains.