Conversion of the M13 Viral Single Strand to the Double-stranded Replicative Forms by Purified Proteins

Abstract

Abstract Earlier studies showed that the M13 single-stranded circle is converted to duplex replicative forms (RF) in vivo and in vitro by a rifampicin-sensitive system that fails to act on φX174 DNA. In the absence of DNA polymerase I, the synthetic complementary strand produced by a crude cell extract contained a small gap at a unique position relative to the viral template strand. In these studies we show that the conversion of viral M13 DNA to the covalently closed duplex form requires the action of six purified proteins: RNA polymerase, DNA unwinding protein of Escherichia coli, DNA polymerase III*, copolymerase III*, DNA polymerase I, and DNA ligase. In the absence of polymerase I, the location of the gap in the RF produced by the reconstituted protein system is the same as that produced by the crude cell extract. The functions of the several proteins, either demonstrated or inferred, are as follows: RNA polymerase to form a priming fragment, DNA unwinding protein to mask the single-stranded DNA in all but the promoter region for RNA polymerase and to support polymerase III*-copolymerase III* in carrying out most of the DNA chain growth, polymerase I to fill the small remaining gap, including that created by its 5'→3' exonucleolytic excision of the remaining RNA fragment, and ligase to join the polynucleotide ends to complete the synthetic complementary circle. Essential functions of RNA polymerase, polymerase III*, and polymerase I in the crude system have been verified by studies of extracts of strains deficient in these proteins. The DNA unwinding protein coded by gene 5 of M13 and essential for synthesis of the viral strands later in infection, fails to replace the E. coli unwinding protein, but instead inhibits conversion of the single strand to RF.