Localization of replication forks in wild-type and <Emphasis Type="Italic">mukB</Emphasis> mutant cells of <Emphasis Type="Italic">Escherichia coli</Emphasis> |
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Authors: | Shun?Adachi Masamichi?Kohiyama Toshinari?Onogi Email author" target="_blank">Sota?HiragaEmail author |
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Institution: | (1) Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan;(2) Institut Jacques Monod, Université Paris 6-7, CNRS, 2 Place Jussieu, 75251 Paris Cedex 05, France;(3) Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, 4-24-1 Kuhonji, Kumamoto 862-0976, Japan;(4) Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuou-ku, Chiba 260-8673, Japan |
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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. |
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Keywords: | MukB Nascent DNA Replication fork Chromosome partition Bromodeoxyuridine |
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