Extensive unwinding of the plasmid template during staged enzymatic initiation of DNA replication from the origin of the Escherichia coli chromosome

Abstract

Early in the staged initiation of enzymatic replication of plasmids containing the unique origin of the E. coli chromosome (oriC), the plasmid is converted to a new topological form which is highly underwound, two to 15 times more than native supercoiled DNA. The underwinding reaction precedes priming of DNA synthesis and follows an initial complex formation, requiring ATP and proteins dnaA, dnaB, and dnaC; underwinding depends on the further addition of gyrase and SSB. DnaB protein as a helicase and gyrase as a topoisomerase drive the underwinding with the energy of ATP hydrolysis. The underwound template, extensively single-stranded and complexed with proteins, is an active form for priming by primase and elongation by DNA polymerase III holoenzyme.