Replication of bacteriophage M13: XIII. Structure and replication of cloned M13 miniphage

Restriction analysis of the duplex replicative forms of four cloned M13 miniphage indicates that all species examined contain a single copy of the intergenic space between genes II and IV plus one or more copies of a portion of the genome extending from within gene IV to a site in the HaeIII G fragment within the intergenic space. Both the viral and the complementary strand origins of replication have been localized previously within the 160 base-pair HaeIII G fragment. Since reiteration of a portion of the HaeIII G fragment could possibly lead to phages having multiple copies of the origin of replication, we have determined the location of the viral strand origin-terminus in M13 miniphage by mapping the position of the discontinuity(ies) in mini-RFII³ molecules isolated during asymmetric viral strand synthesis. Limited repair of late life-cycle mini-RFII molecules with DNA polymerase I in the presence of labeled deoxynucleoside triphosphates followed by restriction analysis demonstrates that the discontinuity in the RFII is contained at a unique site within the single HaeIII G fragment. The absence of a discontinuity in the reiterated DNA sequence containing only a portion of the HaeIII G fragment indicates that the reiterations of the origin region do not include the entire sequence specifying the viral strand origin-terminus.