Bacteriophage fd Gene-2 Protein. Processing of Phage fd Viral Straands Replicated by Phage T7 Enzymes

Abstract

Bacteriophage T7 gene 4 protein and DNA polymerase were used to study the viral strand synthesis of bacteriophage fd in vitro. Cleavage of supercoiled phage fd replicative form (RF) by fd gene 2 protein produced a nick at a specific site in the viral strand. The cleaved double-stranded DNA was unwound by T7 gene 4 protein and DNA polymerase and the 3'' end of the nicked strand extended simultaneously according to the rolling circle mechanism. After a complete round of DNA synthesis, fd gene 2 protein cleaved the viral strand, presumably at the same site where the endonuclease cuts fd RF 1, and subsequently sealed the single-stranded linear DNA into a circle. The reaction products were analyzed by velocity sedimentation, gel electrophoresis and EM. Most of the single-stranded DNA synthesized was circular. No host proteins were required for the formation of the single-stranded circles. Strand switching of the T7 DNA polymerase indicated by double-stranded tails of the rolling circle structures reduced the yield of viral single-stranded circles in this enzyme system.