DiaA, a novel DnaA-binding protein, ensures the timely initiation of Escherichia coli chromosome replication.
The DnaA protein is the initiator of Escherichia coli chromosomal replication. In this study, we identify a novel DnaA-associating protein, DiaA, that is required for the timely initiation of replication during the cell cycle. DiaA promotes the growth of specific temperature-sensitive dnaA mutants and ensures stable minichromosome maintenance, whereas DiaA does not decrease the cellular DnaA content. A diaA::Tn5 mutation suppresses the cold-sensitive growth of an overinitiation type dnaA mutant independently of SeqA, a negative modulator of initiation. Flow cytometry analyses revealed that the timing of replication initiation is disrupted in the diaA mutant cells as well as wild-type cells with pBR322 expressing the diaA gene. Gel filtration and chemical cross-linking experiments showed that purified DiaA forms a stable homodimer. Immunoblotting analysis indicated that a single cell contains about 280 DiaA dimers. DiaA stimulates minichromosome replication in an in vitro system especially when the level of DnaA included is limited. Moreover, specific and direct binding between DnaA and DiaA was observed, which requires a DnaA N-terminal region. DiaA binds to both ATP- and ADP-bound forms of DnaA with a similar affinity. Thus, we conclude that DiaA is a novel DnaA-associating factor that is crucial to ensure the timely initiation of chromosomal replication.
Ishida
Takuma
T
Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Akimitsu
Nobuyoshi
N
Kashioka
Tamami
T
Hatano
Masakazu
M
Kubota
Toshio
T
Ogata
Yasuyuki
Y
Sekimizu
Kazuhisa
K
Katayama
Tsutomu
T
D001425:Bacterial Outer Membrane Proteins / Q000502:physiology; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D002352:Carrier Proteins / Q000502:physiology*; D002876:Chromosomes, Bacterial; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000502:physiology; D018741:Replication Origin / Q000235:genetics*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D029968:Escherichia coli Proteins; C089746:SeqA protein, E coli