A nucleotide switch in the Escherichia coli DnaA protein initiates chromosomal replication: evidnece from a mutant DnaA protein defective in regulatory ATP hydrolysis in vitro and in vivo.
The ATP-bound DnaA protein opens duplex DNA at the Escherichia coli origin of replication, leading to a series of initiation reactions in vitro. When loaded on DNA, the DNA polymerase III sliding clamp stimulates hydrolysis of DnaA-bound ATP in the presence of the IdaB/Hda protein, thereby yielding ADP-DnaA, which is inactive for initiation in vitro. This negative feedback regulation of DnaA activity is proposed to play a crucial role in the replication cycle. We here report that the mutant protein DnaA R334A is inert to hydrolysis of bound ATP, although its affinities for ATP and ADP remain unaffected. The ATP-bound DnaA R334A protein, but not the ADP form, initiates minichromosomal replication in vitro at a level similar to that seen for wild-type DnaA. When expressed at moderate levels in vivo, DnaA R334A is predominantly in the ATP-bound form, unlike the wild-type and DnaA E204Q proteins, which in vitro hydrolyze ATP in a sliding clamp- and IdaB/Hda-dependent manner. Furthermore, DnaA R334A, but not the wild-type or the DnaA E204Q proteins, promotes overinitiation of chromosomal replication. These in vivo data support a crucial role for bound nucleotides in regulating the activity of DnaA during replication. Based on a homology modeling analysis, we suggest that the Arg-334 residue closely interacts with bound nucleotides.
Nishida
Satoshi
S
Department of Molecular Microbiology and the Department of Immunology, Kyushu University Graduate School of Pharmaceutical Sciences, Higashi-ku, Fukuoka 812-8582, Japan.
Fujimitsu
Kazuyuki
K
Sekimizu
Kazuhisa
K
Ohmura
Tadahiro
T
Ueda
Tadashi
T
Katayama
Tsutomu
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D002455:Cell Division; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; D009711:Nucleotides; D000255:Adenosine Triphosphate