title: Chromosomal location of the DnaA-reactivating sequence DARS2 is important to regulate timely initiation of DNA replication in Escherichia coli.
abstract: In Escherichia coli, the initiator protein ATP-DnaA promotes initiation of chromosome replication in a timely manner. After initiation, DnaA-bound ATP is hydrolyzed to yield ADP-DnaA, which is inactive in initiation. DnaA-reactivating sequences (DARS1 and DARS2) on the chromosome have predominant roles in catalysis of nucleotide exchange, producing ATP-DnaA from ADP-DnaA, which is prerequisite for timely initiation. Both DARS sequences have a core region containing a cluster of three DnaA-binding sites. DARS2 is more effective in vivo than DARS1, and timely activation of DARS2 depends on binding of two nucleoid-associated proteins, IHF and Fis. DARS2 is located centrally between the chromosomal replication origin oriC and the terminus region terC. We constructed mutants in which DARS2 was translocated to several chromosomal loci, including sites proximal to oriC and to terC. Replication initiation was inhibited in cells in which DARS2 was translocated to terC-proximal sites when the cells were grown at 42 °C, although overall binding efficiency of IHF and Fis to the translocated DARS2 was not affected. Inhibition was largely sustained even in cells lacking MatP, a DNA-binding protein responsible for terC-specific subchromosomal structure. These results suggest that functional regulation of DARS2 is correlated with its chromosomal location under certain conditions.
authors.affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Yukie
authors.initials: Y
authors.lastname: Inoue
authors.affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Tanaka
authors.affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Kasho
authors.affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan.
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Fujimitsu
authors.affiliation: Division of Genomics of Bacterial Cell Functions, Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, 630-0192, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Oshima
authors.affiliation: Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, 812-8582, Japan. katayama@phar.kyushu-u.ac.jp.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Katayama
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites; D002874:Chromosome Mapping; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate