Initiation of DNA replication at cloned origins of bacteriophage T7

Abstract

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (φ1.1A and φ1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions φOR, φ13 and φ6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.