8 Replicase of the Phage f2

Abstract

INTRODUCTION
Replicases of the bacteriophage group which includes f2, R17 and MS2 have proved difficult to purify and study. These replicases are quite unstable and appear to be strongly complexed with endogenous phage template RNA (Weissmann et al. 1963; August et al. 1963). Although template–enzyme complexes can be isolated from such cells rather readily, a method for obtaining reproducible, template-free preparations was not developed until recently (Fedoroff and Zinder 1971). Purification of these enzymes has been hampered, in part, by the lack of a simple, nonstringent, unambiguous assay for replicase activity. The phage-induced enzyme cannot be distinguished from bacterial enzymes on the basis of RNA-stimulated nucleotide polymerization alone, since RNA is used as template and primer, respectively, by the bacterial DNA-dependent RNA polymerase and polynucleotide phosphorylase enzymes (Fox et al. 1964; Robertson 1971; Melli and Pemberton 1972; Mii and Ochoa 1957; Kimhi and Littauer 1968). Template specificity, a highly characteristic property of all known RNA replicases (Miyaki et al. 1971), is a cumbersome assay, using multiple templates and requiring independent confirmation of the integrity of inactive template RNAs. Assuming that the replicases of this RNA phage group are comparable in complexity to the well-studied Qβ replicase, the routine use of viral (plus strand) RNA template is also an extremely stringent replicase assay, requiring the presence of all components of the replicase complex for activity (August et. al. 1968).

A simplified assay of the requisite specificity has been used in the purification of the Qβ replicase (Kamen 1970), capitalizing on...