Isolation and Partial Characterization of Gene-II Protein from fd-infected Cells

Abstract

Propagation of the replicative-form (RF) DNA of phage fd is initiated by the action of phage-encoded gene-II protein. Defects in gene II prevent the formation of RFII molecules (circular duplexes in which at least one strand is not continuous) nicked specifically in the viral strand and thereby cause accumulation of RFI DNA (superhelical, covalently closed, circular duplexes) (Lin and Pratt 1972; Fidanián and Ray 1972). The protein isolated from membrane fractions of phage-infected cells migrates on SDS gels with an apparent molecular weight of 40,000 (Lin and Pratt 1974); when synthesized in vitro in a protein-synthesizing system it has a molecular weight of 40,000 (Model and Zinder 1974) or 46,000 (Konings et al. 1975). All previous attempts to demonstrate enzymatic activity of gene-II protein in vitro have failed. We report here the isolation of a gene-II protein that stimulates fd RFI replication in vitro and cleaves specifically fd RFI DNA.

Isolation of Gene-II Protein
Gene-II protein was isolated from Escherichia coli cells infected for 30 minutes with an fd am5 (gene V) mutant (multiplicity of infection [m.o.i.] = 5). Its activity was checked by incorporation of [α-³²P]dATP into fd RFI DNA, which was supplemented with partially purified extracts from uninfected cells, analogous to a system developed for the replication of ϕX174 RFI DNA (Eisenberg et al. 1976; Sumida-Yasumoto et al. 1976).

The infected cells were broken in an X-press, and the high-speed supernatant was precipitated with 0.25 g ammonium sulfate per ml. The pellet was dissolved in a small volume,...