Novel replication mutant of microvirid phage alpha 3 deleted in the complementary strand origin

The bacteriophage alpha 3 origin of complementary strand DNA synthesis (-ori) contains two potential secondary loop structures (I and II), which have been implicated as direct recognition sites for host Escherichia coli DnaG protein. To elucidate to what extent such structures are essential, we introduced a nucleotide deletion within the -ori region, by nuclease digestion of alpha 3 replicative form DNA. A mutant, delB, thus constructed had a 121 nucleotide deletion within the -ori region and was completely lacking in the two putative hairpin loops, I and II. The delB mutant formed smaller plaques on the host E. coli C and had a longer latent period, but the mean burst size at 37 degrees C was almost the same (400 phages) as that of the wild type. In contrast to the parental phage, growth of the mutant depends on host dnaB and dnaC functions. These results indicate that the prototype secondary structures in the alpha 3 origin of complementary strand synthesis are dispensable for delB and that the alpha 3 mutant has an additional replication origin whose function is dependent on DnaB and DnaC proteins, rather than on DnaG protein alone.