Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome

Abstract

Three tandem repeats of a 13-mer in the AT-rich region are essential to the unique replication origin of E. coli and of remotely related Enterobacteriaceae. These iterated sequences are identified by deletion analysis and sensitivities to endonucleases as the site for initial duplex opening by the initiator dnaA protein. This "open complex" requires ATP and 38 degrees C for optimum formation and stability. The subsequent dnaC-dependent entry of dnaB helicase to form a "prepriming complex" stabilizes the open structure, blocks cleavages by a restriction endonuclease in the 13-mer region, and broadens the endonuclease cutting pattern. We propose that dnaA protein recognizes and successively opens the 13-mer sequences, thereby guiding the entry of dnaB helicase into the duplex preparatory to priming of replication.