Reactivation of DnaA by DNA sequence-specific nucleotide exchange in vitro.
In Escherichia coli, ATP-bound DnaA protein can initiate chromosomal replication. After initiation, DnaA-ATP is hydrolyzed by interactions with a complex containing a replicase subunit to yield the inactive ADP-DnaA. However, the mechanisms which regenerate ATP-DnaA from ADP-DnaA are not well understood. We report here that a 70-bp DNA segment promotes exchange of the DnaA-bound nucleotide in a sequence-specific manner, thus reactivating the initiation function of DnaA in vitro. This segment contains a typical DnaA-binding 9-mer motif, the DnaA box, and two DnaA box-like sequences. The presence and precise composition of these three motifs are required for the DnaA-reactivating activity, which suggests that a highly ordered complex which includes multimeric DnaA molecules is formed for isomerization of DnaA. We named this DNA segment DARS, for DnaA-reactivating sequence. The role of DARS in regulation of DnaA function in vivo is discussed.
Fujimitsu
Kazuyuki
K
Department of Molecular Biology, Kyushu University Graduate School of Pharmaceutical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
Katayama
Tsutomu
T
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA