title: Sequential early stages in the in vitro initiation of replication at the origin of the Escherichia coli chromosome.
abstract: Complexes previously identified in the reconstitution of stages in the initiation of replication of plasmids (oriC) bearing the origin of the Escherichia coli chromosome have been examined further. These are: (i) an ATP complex of dnaA protein, (ii) an initial complex of ATP.dnaA protein with oriC DNA, (iii) an open complex in which a portion of the oriC duplex has been opened by dnaA protein action, (iv) a prepriming complex of the open complex with dnaB, dnaC, and HU proteins, and (v) a complex with a small bubble opened at oriC by dnaB helicase action and by coating with single strand-binding protein (SSB). Helicase and gyrase actions can enlarge the bubble; coupling to priming and replication propagates bidirectional fork movement. Formation and stability of these complexes are profoundly affected by ATP, Mg2+, and temperature, as well as the levels of the participating proteins, including HU and SSB. As examples, the open complex is stable to isolation at a temperature near 38 degrees C but not at 24 degrees C; the prepriming complex requires an elevated temperature and high ATP levels for its formation, but is maintained at a low temperature and is destabilized by Mg2+. These successive steps, subject to a variety of controls, are designed to open the supercoiled duplex for priming and bidirectional replication.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, California 94305.
authors.forename: K
authors.initials: K
authors.lastname: Sekimizu
authors.forename: D
authors.initials: D
authors.lastname: Bramhill
authors.forename: A
authors.initials: A
authors.lastname: Kornberg
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000502:physiology*; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008962:Models, Theoretical; D010957:Plasmids; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D000255:Adenosine Triphosphate; D008274:Magnesium