Mutational analysis of the bacteriophage φX174 replication origin*1

Bacteriophage φX174 mutants within the 30 base-pair replication origin were constructed using oligodeoxynucleotide-directed mutagenesis. A total of 18 viable base substitution mutants at 13 different positions within the origin region were obtained. The majority of these ori mutants have a plaque morphology and burst size comparable to that of wild-type φX174. Two φX174 ori mutants with a reduced growth ability spontaneously acquired additional mutations that enhanced the growth rate. The additional mutation was located at the same site as the original mutation or was located in the N-terminal part of the gene A protein. This latter secondary mutation is responsible for a better binding and/or recognition of the gene A protein to the mutated origin. In a Darwinian experiment wild-type φX174 outgrows all φX174 ori mutants, indicating the superiority of the wild-type ori sequence for the reproduction of bacteriophage φX174. Insertions and deletions were constructed at different positions within the φX174 replication origin cloned in a plasmid. Small insertions and deletions in the A + T-rich spacer region do not inhibit φX174 gene A protein cleavage in vitro, but severely impair packaging of single-stranded plasmid DNA in viral coats