Cloning of a functional replication origin of phage G4 into the genome of phage M13

Abstract

A chimeric single-stranded DNA phage, M13G ori1, has been formed as a result of the in vitro insertion of a 2216 base-pair HaeII fragment of bacteriophage G4 replicative form DNA into the replicative form DNA of bacteriophage M13. The inserted G4 DNA carries the dnaG-dependent origin for G4 complementary strand synthesis. The cloned G4 origin functions both in vivo and in vitro in the conversion of M13G ori1 single-stranded viral DNA to the duplex replicative form by a rifampicin-resistant mechanism. Labelling of the 3′ terminus of the single discontinuity in M13G ori1 replicative form II molecules synthesized in crude extracts and subsequent restriction analysis indicate that M13G ori1 complementary strand synthesis can be initiated at either the RNA polymeraseprimed M13 origin or at the dnaG-primed G4 origin. The M13G ori1 complementary strand initiated at the G4 origin terminates in the vicinity of the G4 origin after progressing around the circular template and traversing the M13 origin region, indicating the absence of a specific nucleotide sequence in the M13 origin for termination of the newly formed complementary strand. The ability of this chimeric phage to utilize the cloned G4 origin in vivo even in the presence of the presumed M13 pilot protein (gene 3 protein) indicate that the nucleotide sequence of the replication origin is sufficient for recognizing the appropriate initiation enzymes. Since decapsidation of M13 is tightly coupled to replicative form formation, initiation at the G4 origin, located over 1000 nucleotides from the M13 complementary strand origin, indicates that widely separated nucleotide sequences contained in the filamentous virion can be exposed to the cell cytoplasm during eclipse.