Isolation and construction of mutants of the G4 minus strand origin: analysis of their in vivo activity

Abstract

An active, rifampicin-resistant primase-dependent bacteriophage G4 origin of complementary DNA strand synthesis has been cloned as a 274 bp fragment into the filamentous phase M13 and its secondary structure altered by deletion and insertion. It has been found that the entire 136 bp G4 intergenic region containing the secondary structure loops I and III is necessary for rifampicin-resistant conversion of SS----RF DNA in vivo. The secondary structures, however, can be widely separated by insertion between them of both random DNA sequences, and sequences that form strong additional secondary structure configurations and the origins still retain activity. Primase therefore probably recognises two DNA domains on loops I and III, the physical separation of which is not important.