title: Localization of replication forks in wild-type and mukB mutant cells of Escherichia coli.
abstract: To examine the subcellular localization of the replication machinery in Escherichia coli, we have developed an immunofluorescence method that allows us to determine the subcellular location of newly synthesized DNA pulse-labeled with 5-bromo-2'-deoxyuridine (BrdU). Using this technique, we have analyzed growing cells. In wild-type cells that showed a single BrdU fluorescence signal, the focus was located in the middle of the cell; in cells with two signals, the foci were localized at positions equivalent to 1/4 and 3/4 of the cell length. The formation of BrdU foci was dependent upon ongoing chromosomal replication. A mutant lacking MukB, which is required for proper partitioning of sister chromosomes, failed to maintain the ordered localization of BrdU foci: (1) a single BrdU focus tended to be localized at a pole-proximal region of the nucleoid, and (2) a focus was often found to consist of two replicating chromosomes. Thus, the positioning of replication forks is affected by the disruption of the mukB gene.
authors.affiliation: Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Adachi
authors.forename: Masamichi
authors.initials: M
authors.lastname: Kohiyama
authors.forename: Toshinari
authors.initials: T
authors.lastname: Onogi
authors.forename: Sota
authors.initials: S
authors.lastname: Hiraga
mesh_terms: D001973:Bromodeoxyuridine; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics*; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D007150:Immunohistochemistry
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D029968:Escherichia coli Proteins; C067035:MukB protein, E coli; D004247:DNA; D001973:Bromodeoxyuridine