title: DnaA, the initiator of Escherichia coli chromosomal replication, is located at the cell membrane.
abstract: Given the lack of a nucleus in prokaryotic cells, the significance of spatial organization in bacterial chromosome replication is only beginning to be fully appreciated. DnaA protein, the initiator of chromosomal replication in Escherichia coli, is purified as a soluble protein, and in vitro it efficiently initiates replication of minichromosomes in membrane-free DNA synthesis reactions. However, its conversion from a replicatively inactive to an active form in vitro occurs through its association with acidic phospholipids in a lipid bilayer. To determine whether the in situ residence of DnaA protein is cytoplasmic, membrane associated, or both, we examined the cellular location of DnaA using immunogold cryothin-section electron microscopy and immunofluorescence. Both of these methods revealed that DnaA is localized at the cell membrane, further suggesting that initiation of chromosomal replication in E. coli is a membrane-affiliated event.
authors.affiliation: Department of Biochemistry, Georgetown University Medical Center, Washington, D.C. 20007, USA.
authors.forename: G
authors.initials: G
authors.lastname: Newman
authors.forename: E
authors.initials: E
authors.lastname: Crooke
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002876:Chromosomes, Bacterial; D016616:Cryoultramicrotomy; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D019084:Fluorescent Antibody Technique, Indirect; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D016253:Microscopy, Immunoelectron / Q000379:methods; D011817:Rabbits; D018741:Replication Origin
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D007074:Immunoglobulin G