Ten proteins required for conversion of phiX174 single-stranded DNA to duplex form in vitro. Resolution and reconstitution

Abstract

Protein requirements for conversion of phiX174 single-stranded DNA to a double-stranded replicative form with a small gap (RF II) have been determined by resolution and reconstitution of the multienzyme system from extracts of gently lysed Escherichia coli. Assays depended on: (a) complementation of extracts of thermosensitive mutants and (b) fractionation of extracts of wild type cells to divide essential components into groups, each of which was further resolved. These procedures have yielded eight proteins: dnaB protein, dnaC protein, proteins i and n (two novel proteins without a defined genetic locus), dnaG protein, DNA polymerase III holoenzyme (polymerase III and copolymerase III), and DNA unwinding protein; purification procedures for the first four are presented here. (Closure of RF 22 requires as with phage M13, DNA polymerase I and ligase.)