Mutational analysis of the bacteriophage alpha 3 origin of complementary DNA synthesis: in vivo properties of mutants

Abstract

Bacteriophage alpha 3 origin of complementary strand DNA synthesis contains two potential secondary loop structures, I and II, which have been implicated in direct recognition sites for host Escherichia coli dnaG protein. To elucidate the function of the hairpin loops, we have introduced point mutations within the stem of the hairpin II so as to disturb its base-pairings. A mutant, oriAA, which had two point mutations in the region, formed minute plaques on E. coli host cells and its mean burst size at 37 degrees C was about 50, whereas that of wild-type was 250. In addition, the growth of oriAA at 42 degrees C was thermosensitive and the burst size was reduced to 5. From the oriAA, a revertant-like phage oriGA occurred spontaneously with a high-frequency of about 2.10(-2). It retained one point mutation and the plaque size and phage yield were nearly same as those of wild-type. These results are discussed with respect to the role of secondary structure as well as specific nucleotide sequence in the recognition site for the dnaG protein.