Replication of phase fd RF with fd gene 2 protein and phage T4 enzymes

Bacteriophage fd replicative form DNA with a nick in the viral strand serves as a template for DNa replication with purified bacteriophage T4 enzymes. As anticipated from previous in vitro studies carried out with this system (Morris, C. F., Sinha, N. K., and Alberts, B. M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4800-4804), DNA is synthesized by a rolling circle mechanism. We show here that the DNA strands synthesized are processed by the phage fd gene 2 protein into unit length products, providing that the gene 2 protein is present at the moment when this DNA is made. The products are mostly unit length linear single strands, indicating that the circularization step normally catalyzed by gene 2 protein subsequent to its site-specific cleavage of an fd DNA strand occurs only inefficiently in this system. The gene 2 protein reduces the level of DNA synthesis by 2-fold at low concentrations, even though it only cleaves the DNA products efficiently at higher levels of the enzyme. This indicates that there are at least two different effects of the fd gene 2 protein in processing of viral fd DNA.